© 1995 American Heart Association, Inc.
Articles |
Antisense to Thyrotropin Releasing Hormone Receptor Reduces Arterial Blood Pressure in Spontaneously Hypertensive Rats
From the Department of Medicine and Centre for Neuroscience, Flinders Medical Centre, Adelaide, Australia.
Correspondence to Dr Paul Pilowsky, Department of Medicine, Flinders Medical Centre, Adelaide, South Australia 5042, Australia. E-mail paul.pilowsky@flinders.edu.au.
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Abstract |
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Abstract We report in the present study the effect of intrathecal treatment with antisense oligonucleotides complementary to thyrotropin releasing hormone (TRH) receptor mRNA on the pressor response to intrathecal administration of TRH and on resting arterial blood pressure in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). In 16-week-old male WKY rats, 18-base phosphodiester antisense or mismatch oligonucleotides to TRH receptor mRNA (100 µg per day) were injected intrathecally for 3 days. Twenty-four hours after the last injection, the magnitude of the pressor response to intrathecal TRH (10 µg) was significantly smaller in the antisense-treated group (n=7) compared with mismatch-treated controls (n=7) (change in mean arterial pressure, +20.3±3.0 versus +32.6±2.5 mm Hg, P<.01). No differences were observed in the pressor responses to injection of N-methyl-D-aspartic acid. Resting arterial blood pressure was unaffected by antisense treatment in WKY rats. In separate experiments, 16-week-old male SHR were treated with antisense (n=7) or mismatch (n=6) oligonucleotides for 3 days. Mean resting arterial blood pressure was significantly reduced by treatment with antisense oligonucleotides (from 157±4.8 to 119±8.8 mm Hg, P<.01), but no significant changes were observed in mismatch-treated animals. Our results suggest that the expression of TRH receptors in spinal sympathetic preganglionic neurons can be selectively reduced by intrathecal treatment with antisense oligonucleotides and that TRH projections to sympathetic preganglionic neurons play an important role in the elevation of arterial blood pressure in SHR.
Key Words: intrathecal • sympathetic preganglionic neurons • N-methyl-D-aspartic acid • oligonucleotides
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apart from its endocrine function, TRH also serves as a neurotransmitter in the central nervous system. TRH immunoreactivity is found in many areas of the central nervous system, including the brain stem and spinal cord.1 2 Retrograde tracing studies reveal that many TRH-containing neurons in the caudal raphe nuclei have spinal axons.3 TRH immunoreactivity is found within terminals in the IML of the spinal cord2 4 5 6 that are closely apposed to sympathetic preganglionic neurons.7 Intrathecal administration of TRH causes sympathoactivation and an increase in arterial blood pressure.8 9 Thus, there is strong evidence that TRH-containing bulbospinal neurons might be playing an important role in cardiovascular regulation. However, studies attempting to determine a physiological role for TRH in cardiovascular regulation are hampered by the lack of a selective antagonist to the TRH receptor.
Some studies have suggested a relation between TRH and hypertension. The density of TRH receptors is higher in brain tissues from SHR than from normotensive WKY rats.10 SHR also exhibit a supersensitivity to the hypertensive effects of exogenous TRH.11 However, a pathophysiological role for TRH in SHR is still not clear.
In the present study, we attempted to suppress the production of TRH receptors in the spinal cord by administration of antisense oligonucleotides directed against TRH receptor mRNA. Our aim was to assess the physiological role of spinal TRH receptors in cardiovascular regulation. First, we determined whether intrathecal treatment with an antisense oligonucleotide to TRH receptor mRNA could attenuate the pressor response to intrathecally injected TRH in WKY rats. Second, we investigated the role of spinal TRH receptors in the maintenance of hypertension in SHR. Preliminary results were reported to the Australian Neuroscience Society.12
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Materials and Methods |
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General Preparation
Male WKY rats (16 weeks old, 300 to 400 g) and age-matched SHR were anesthetized with sodium pentobarbital (60 mg/kg IP). A femoral artery and vein were cannulated with polyethylene tubing (inner diameter, 0.28 mm; outer diameter, 0.61 mm) for measurement of arterial pressure and for drug administration. Body temperature was maintained at
37°C with a heating pad.
Arterial pressure and heart rate were recorded with a
Grass polygraph. All experiments were conducted in accordance with National Health and Medical Research Council of Australia guidelines.
Implantation of Intrathecal Catheters
Ten days before starting the injection of
oligonucleotides, a catheter (clear vinyl; inner
diameter, 0.28 mm; outer diameter, 0.61 mm) was implanted through a
hole in the atlanto-occipital membrane into the
subarachnoid space, with the tip at approximately the 10th
thoracic segment as previously described.13 Sodium
methohexital (80 mg/kg IP) was used to obtain short-lasting
anesthesia. Briefly, the rats were secured in a
stereotaxic frame, and the neck was flexed 40° to 50°.
After skin incision, muscles were retracted, and the
atlanto-occipital membrane was exposed. A catheter was introduced
6.5 cm into the subarachnoid space through a small hole. The
catheter was secured to the occipital bone with cyanoacrylate adhesive,
and muscle and skin were sutured. The catheter (inner volume, 5 µL)
was filled with sterile saline, and the outer tip was heat-sealed.
Correct placement of the catheter was checked by aspiration of
cerebrospinal fluid from the catheter at the end of surgery and by dye
injection at the end of the experiment. Dye spread was usually
restricted to the thoracic segments of the spinal cord. Occasionally,
dye reached lumbar or cervical levels but never reached the brain
stem.
Injection of Oligonucleotide and Other
Drugs
Oligonucleotides (Bresatec) were diluted in
sterile saline (0.9% sodium chloride, pH 6.7). Phosphodiester
antisense (5' GAC GGT TTC ATT CTC CAT 3') or four mismatch (5'
GAT GGT CTC ACT CTT CAT
3') oligonucleotides to TRH receptor mRNA (100 µg in
5 µL with a 10 µL saline flush) were injected through the
intrathecal catheter once daily for 3 days. Preliminary
experiments revealed this to be the minimum dose that effectively
attenuated the response to intrathecal injection of TRH.
The injection of oligonucleotides had no acute
behavioral effects, and no anesthesia was required. The
antisense oligonucleotide was complementary to the
first 18 bases downstream from the initiation codon of the rat TRH
receptor mRNA sequence.14 The mismatch sequence used
showed no significant complementarity to any part of the TRH receptor
mRNA. Neither oligonucleotide showed any significant
complementarity to any other gene sequence in the GenBank database.
TRH (pyroglutamyl-histidyl-proline amide, Auspep) and NMDA (Sigma Chemical Co) were diluted in 0.02 mol/L phosphate buffer (pH 7.4) containing 160 mmol/L sodium chloride (PBS). Each dose of TRH (0.1, 1.0, and 10 µg) or NMDA (0.5, 5.0, and 50 µg) was given in 5 µL of PBS, followed by a 10 µL PBS flush through the intrathecal catheter at the time of the experiment.
Experimental Protocols
Effect on Cardiovascular Response to
Intrathecal TRH in WKY Rats
Twenty-four hours after the last injection of antisense
(n=19) or mismatch (n=19) oligonucleotides or saline
vehicle (15 µL each day, n=7), rats were anesthetized with
sodium pentobarbital (60 mg/kg IP). The trachea was intubated with a
14-gauge polytetrafluoroethylene (Teflon)
catheter (Jelco), and rats were mechanically ventilated (Harvard 608
ventilator) with room air. Arterial pressure was monitored,
and TRH or NMDA was injected intrathecally. Three doses of
TRH or NMDA were tested in antisense- or mismatch-treated animals
(n=6 to 7 rats for each dose). Because tachyphylaxis to repeated doses
of intrathecal TRH was observed and has been reported by
others,9 only one dose of TRH and NMDA was given to each
rat. In saline-treated animals, only the response to the highest
dose of TRH (10 µg) was determined. The arterial pressure
and heart rate responses to intrathecal TRH and NMDA were
determined at the time of the peak pressor response.
Effect on Resting Arterial Pressure and Heart Rate
of SHR
Antisense (n=7) or mismatch (n=6)
oligonucleotides were injected once per day for 3 days
(days 0, 1, and 2). No injection of any substance was made in a further
four rats because the outer ends of the intrathecal
catheters became lost in the wound, and treatment was not possible
(no-treatment group). Before injection of
oligonucleotides (day 0) and 24 hours after the last
injection of oligonucleotides (day 3), rats were
anesthetized with sodium pentobarbital (60 mg/kg IP), and
arterial pressure and heart rate were monitored.
Arterial pressure and heart rate were also determined in
four of the antisense-treated and three of the mismatch-treated
animals 7 days after the last injection of
oligonucleotides (day 9) under pentobarbital
anesthesia. Resting values were obtained exactly 2 hours
after injection of sodium pentobarbital. Samples of plasma were
collected from some rats at the end of the experiment on day 3 (n=3 for
each group) and were analyzed for free T4
concentrations by using a commercial enzyme-linked immunosorbent
assay system (Enzymun-Test FT4, Boehringer Mannheim).
Statistical Analysis
Values are expressed as mean±SEM. Statistical significance was
determined by ANOVA for the dose-response data in WKY rats.
Differences between the effects of treatments on resting values were
determined by repeated-measures ANOVA in SHR. Where pairwise
comparisons were made after ANOVA or repeated-measures ANOVA, the
Bonferroni adjustment was used to control type I error.15
A value of P
.05 was considered to be statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of Intrathecal Oligonucleotide Administration on Arterial Pressure and Heart Rate Responses to Intrathecal TRH and NMDA in WKY Rats
The magnitude of the pressor response at the highest dose of TRH (10 µg) was significantly smaller in antisense-treated rats compared with mismatch-treated control rats and saline-treated rats (change in mean arterial pressure, +20.3±3.0 versus +32.6±2.5 and +32.3±1.9 mm Hg, respectively; P<.01; Fig 1
). No difference was observed in response to lower
doses of TRH (0.1 and 1 µg, Fig 1). Treatment with
oligonucleotides had no effect on the pressor responses
to NMDA (Fig 2). No difference was observed in the mean
resting arterial pressure between antisense-, mismatch-,
and saline-treated rats (99±4.7, 95±5.5, and 95±2.2 mm Hg,
respectively).
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No difference was observed in the resting heart rate among antisense-,
mismatch-, and vehicle (saline)–treated rats (315±16, 317±15, and
297±12 bpm, respectively). However, the magnitude of the increase in
heart rate at the highest dose of TRH (10 µg) was significantly
smaller in antisense-treated rats compared with
mismatch-treated control and saline-treated rats (change in
heart rate, +35.7±5.7 versus +55.7±7.2 and +58.6±5.9 mm Hg,
respectively; P<.05; Fig 1
). No difference was observed in
the heart rate responses to lower doses of TRH (0.1 and 1 µg, Fig 1).
Oligonucleotides had no effect on the increases in
heart rate caused by injection of NMDA (Fig 2).
Effect of Intrathecal Oligonucleotides
on Resting Arterial Pressure and Heart Rate of
SHR
No difference was observed in the mean resting
arterial pressure between antisense-treated,
mismatch-treated, and no-treatment rats (157±4.8, 160±6.9,
and 153±3.4 mm Hg, respectively) before starting injection at day 0.
After 3 days of treatment with antisense
oligonucleotides to TRH receptor mRNA, the mean resting
arterial pressure of SHR was significantly reduced when
measured again at day 3 (from 157±4.8 to 119±8.8 mm Hg,
P<.01). Mean resting arterial pressure returned
to control levels 7 days after the last injection of antisense
oligonucleotides (Fig 3). There were no
significant changes in mean resting arterial pressure in
mismatch-treated rats (from 160±6.9 to 162±6.8 mm Hg) and the
no-treatment rats (from 153±3.4 to 154±6.6 mm Hg) after 3 days
(Fig 3).
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No difference was observed in the resting heart rate between
antisense-treated, mismatch-treated, and no-treatment rats
(344±16, 342±25, and 328±14 bpm, respectively) before starting
injection at day 0. There were no significant changes in resting heart
rate in SHR in antisense-treated (from 344±16 to 305±23 bpm),
mismatch-treated (from 342±25 to 342±23 bpm), and
no-treatment rats (from 328±14 to 348±10 bpm) (Fig 3).
There were no significant differences in plasma-free T4 concentrations at day 3 among antisense-treated, mismatch-treated, and no-treatment groups (21.0±1.7, 19.7±2.7, and 23.7±3.2 pmol/L, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major findings of the present study are as follows: First, the cardiovascular response to intrathecal TRH can be selectively attenuated by 3 days of intrathecal treatment with an antisense oligonucleotide to TRH receptor mRNA. Second, 3 days of intrathecal treatment with antisense oligonucleotide to TRH receptor mRNA decreases the resting arterial blood pressure of SHR but does not affect that of WKY rats. Resting arterial blood pressure in SHR returns to control levels after the injection of oligonucleotides is stopped.
Attenuation of TRH Receptor Gene Expression
TRH is localized to cell bodies and nerve terminals in several
areas of the central nervous system, including the spinal
cord,2 where the density of TRH immunoreactive nerve
terminals is particularly high in the IML2 4 5 6 and
morphologically identifiable synapses are seen.16 TRH
immunoreactive terminals are often closely apposed to sympathetic
preganglionic neurons.7 TRH binding sites17
and TRH receptor mRNA18 are also distributed throughout
the central nervous system, including the IML of the spinal cord. In
physiological studies, intrathecal
administration of TRH causes sympathoactivation8 9 and an
increase in arterial blood pressure and heart
rate.9 Direct iontophoretic application of TRH increases
the firing rate of identified sympathetic preganglionic
neurons.19
Although no specific antagonist exists for the TRH receptor, attenuation of receptor gene expression offers an alternative approach. Recent attempts to selectively reduce gene expression with short antisense oligonucleotides have met with some success.20 21 22 By attenuating gene expression, antisense techniques permit the antagonism of specific receptor-mediated responses.23 24 To date, several examples have been reported in vivo, including attenuation of behavioral and physiological responses to administration of dopamine D2, gamma-, mu-, and delta 2–opioid receptor agonists.25 26 27 28 A reduction in receptor binding to neuropeptide Y-Y1, NMDA, dopamine D2, and gamma opioid receptors21 22 25 26 and reductions in progesterone receptor immunoreactivity29 have also been achieved by using antisense oligonucleotides.
In the present study, we observed that pretreatment with antisense oligonucleotides that are complementary to TRH receptor mRNA attenuates the pressor response and the increase in heart rate that follows the intrathecal injection of TRH. Although we have not presented evidence here to prove that the antisense oligonucleotide used caused a decrease in the amount of TRH receptor protein, several features of the present study suggest that this is the likely mechanism of action. First, the observed effect of the antisense oligonucleotides is sequence specific and not due to a nonspecific toxicity of oligonucleotides, since treatment with mismatch oligonucleotides, which had the same base composition in a slightly different sequence, did not change the response to TRH (10 µg) when compared with saline vehicle–treated rats. Second, the responses to NMDA, which also excites sympathetic preganglionic neurons,30 were not affected by this treatment. Finally, the oligonucleotides used did not cause any behavioral or neurological sequelae.
Standifer et al26 reported that in mice, oligonucleotides can survive for up to 48 hours after intrathecal injection and that three injections every 48 hours for 5 days more effectively reduce opioid receptor–mediated analgesia than does a daily injection over 3 days. In the present study, however, we obtained a sufficient effect with injections every 24 hours for 3 days. In preliminary experiments, we could not obtain a significant effect using a three-dose–over–5-day protocol (data not shown). The reasons for the differences in effective protocols for antisense administration are not clear, but species differences (mouse versus rat), differences in mode of administration (direct injection versus implanted catheter), and differences in the targeted mRNA (opioid receptor versus TRH receptor) might be responsible. Clearly, the manner, dosage, and timing of oligonucleotide administration must be determined separately in each case.23 24
Role of Spinal TRH Receptors in SHR
SHR are known to have enhanced sympathoadrenal responses to
various stressful stimuli,31 32 and some studies suggest a
relation between TRH and hypertension. The density of TRH receptors is
higher in brain tissue from SHR than from normotensive rats, and the
development of hypertension parallels increases in the brain TRH
receptor numbers.10 SHR exhibit a supersensitivity to the
hypertensive effects of exogenous TRH,11 and
intracerebroventricular injection of
antiserum to TRH reduces arterial blood pressure in
SHR.33 However, the role of spinal TRH receptors in the
pathogenesis of hypertension is still not clear, again because of the
lack of a selective antagonist to the TRH receptor.
In the present study, we observed a significant reduction in resting mean arterial blood pressure after 3 days of treatment with antisense oligonucleotides to TRH receptor mRNA. This phenomenon was sequence specific and not due to a toxic effect of oligonucleotides, since the arterial pressure of SHR treated with mismatch control oligonucleotides did not change and showed no difference from the no-treatment group.
It is not clear why a reduction in TRH receptor number caused a fall in resting arterial blood pressure in SHR but not in WKY rats. One possibility is that in the resting condition, a fall in sympathetic activity produces more obvious effects in SHR because of their higher resting sympathetic tone.34 In WKY rats, which have lower levels of sympathetic activity, the effects of reducing TRH receptor numbers may be less obvious. Similarly, the fall in blood pressure after ganglion blockade34 or after activation of depressor sites in the caudal ventrolateral medulla35 is greater in SHR than in WKY rats. It is also possible that bulbospinal TRH-containing neurons are more active in SHR than in WKY rats, and this difference contributes to the pathogenesis of hypertension in SHR.
Another possible site of action of antisense oligonucleotides to TRH receptor mRNA is in the hypothalamus, where alterations in the number of TRH receptors might affect thyroid function. However, plasma free T4 concentrations were identical in both mismatch- and antisense-treated rats, so that a change in thyroid status does not seem to be the cause of the depressor effect of antisense oligonucleotides in SHR. The concentrations of plasma free T4 seen in the present study are similar to those found by previous workers.36
In summary, we report in the present study that the pressor responses to an intrathecal injection of TRH can be selectively reduced by 3 days of treatment with an antisense oligonucleotide to TRH receptor mRNA. This treatment also decreases the resting arterial blood pressure of SHR, an effect that is reversible after cessation of treatment. Therefore, we conclude that the number of TRH receptors on sympathetic preganglionic neurons can be selectively and reversibly reduced by intrathecal treatment with antisense oligonucleotides. Furthermore, spinal TRH receptors appear to play an important role in the maintenance of the high resting arterial blood pressure seen in SHR.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the National Health and Medical Research Council, the National Heart Foundation, and the National Sudden Infant Death Council of Australia. Dr Suzuki was supported by the Japan Heart Foundation and Bayer Yakuhin Research Grant Abroad. The authors thank Michelle Anderson and Claudine Frisby for their excellent technical assistance. We are grateful to Dr Graham White, Department of Biochemistry and Chemical Pathology, Flinders Medical Centre, for assay of plasma free T4 concentrations.
Received March 3, 1995; accepted July 5, 1995.
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References |
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