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(Hypertension. 1995;26:1105-1110.)
© 1995 American Heart Association, Inc.

Articles

Thyrotropin-Releasing Hormone Hyperactivity in the Preoptic Area of Spontaneously Hypertensive Rats

S.I. Garcia; S.M. Dabsys; V.N. Martinez; A. Delorenzi; D. Santajuliana; V.E. Nahmod; S. Finkielman; C.J. Pirola

From the Departamento de Sustancias Vasoactivas, Instituto de Investigaciones Medicas "A. Lanari," Facultad de Medicina, Universidad de Buenos Aires (Argentina).

Correspondence to Carlos J. Pirola, PhD, Instituto de Investigaciones Medicas "A. Lanari," Donato Alvarez 3150, Buenos Aires 1427, Argentina.

	Abstract

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Abstract Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation through the activation of different neurotransmitter systems at distinct extrahypothalamic sites. To study possible alterations in the TRH system in the hypertensive state, we measured TRH concentration in cerebrospinal fluid and TRH content of the preoptic area in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) by radioimmunoassay. In addition, we also measured the density of the TRH receptor in this area by a rapid filtration technique using [³H]methyl-TRH. We found a significant increase in both the TRH content (634±61 versus 350±26 pg/mg protein, SHR versus WKY; P<.01, n=5) and density of TRH receptors without changes in affinity (B_max, 5.0±0.1 versus 3.3±0.1 fmol/mg protein, P<.01, n=4). An increase in TRH concentration was also found in the cerebrospinal fluid of SHR (30±3 versus 21±2 pg/mL, P<.01, n=5), suggesting increased TRH release in the central nervous system. Northern blot analysis indicated a threefold augmented abundance of TRH precursor mRNA in the preoptic area of SHR. A polyclonal antibody raised against TRH injected peripherally or intracerebroventricularly lowered arterial blood pressure in SHR but not in WKY. In addition, long-term treatment with enalapril (5 mg/kg twice daily), which was effective in inhibiting serum angiotensin-converting enzyme activity by more than 50%, decreased arterial blood pressure and preoptic area TRH content of SHR, whereas another vasodilator, diltiazem (10 mg/kg every 8 hours), failed to produce a similar change. These results point out an activation of the TRH system, with increased production of TRH and an upregulation of its receptors in this genetic model of hypertension.

Key Words: prothyroliberin • rats, inbred SHR • preoptic area

	Introduction

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The hormone TRH (pyro-Glu-His-Pro-amide) is widely distributed throughout the central nervous system and serves multiple physiological functions.^{1 2} Its presence in brain nuclei involved in cardiovascular regulation,³ such as the POA, suggests that the tripeptide may play a role in modulating cardiovascular function.⁴ The POA, which includes the anteroventral third ventricle region,⁵ is crucial in regulating arterial BP, dipsogenic behavior, antidiuretic hormone release, natremia, and blood volume.^{5 6 7} Its destruction avoids the development of different forms of hypertension, such as that produced by deoxycorticosterone acetate–salt, sinoaortic denervation, or lesion of the tractus solitarius nucleus.^{5 6 7} In fact, intracerebroventricular microinjections of TRH or injections into the POA produce dose-dependent pressor effects.^{8 9}

SHR are extensively used for the study of mechanisms in essential hypertension. An enhanced brain angiotensin system was detected in these rats that may be responsible for hypertension either by a direct action or through the activation of other hypertensive neurochemical mechanisms.¹⁰ We have recently reported that SHR display a central cholinergic muscarinic hyperactivity that could play a role in the development and/or maintenance of hypertension in these rats.¹¹ In addition, we have shown that TRH facilitates the pressor response to centrally infused acetylcholine, increasing the number of muscarinic receptors.¹² In turn, in vitro superfusion experiments with POA slices showed that cholinergic muscarinic stimulation evokes a specific TRH release.¹³ Therefore, we attempted to establish the participation of TRH in the pathogenesis of spontaneous hypertension. In the present study we show that SHR compared with the normotensive control strain (WKY) present (1) an increased TRH content and TRH precursor mRNA abundance in the POA, (2) a higher CSF TRH concentration, and (3) an augmented TRH receptor number in the POA. We also found that a polyclonal antibody raised against TRH infused peripherally or intracerebroventricularly significantly decreased arterial BP and that long-term enalapril treatment, which decreased arterial BP, reduced the abnormal levels of POA TRH content in the SHR, whereas another chronically administered vasodilator, diltiazem, failed to produce a similar change. These results point out that TRH may play a role in the maintenance of hypertension in SHR.

	Methods

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Animals
Age-matched male SHR of the Okamoto-Aoki strain and normotensive WKY originally derived from Charles River Breeding Farm (Wilmington, Mass) were housed in a room with a controlled temperature (24±1°C) under a 12-hour light/dark schedule. Food and water were given ad libitum. Animal experimentation was approved by our Institutional Animal Care and Use Committee.

TRH Radioimmunoassay
Rats were killed by decapitation and brains rapidly removed. The POA of each rat was dissected from frozen brains with the aid of the stereotaxic atlas.¹⁴ To avoid the degradation of authentic TRH and the formation of TRH-like substances, we boiled samples (pool of three to four POAs) in acetic acid (2 mol/L)/HCl (100 mmol/L)¹⁵ for 20 minutes and then homogenized and centrifuged them at 10 000g for 10 minutes. The pellet was used for protein determination.¹⁶ The supernatants were lyophilized and residues dissolved in radioimmunoassay buffer. We confirmed that 90% of TRH-like immunoreactivity corresponds to authentic TRH with the use of a previously reported chromatographic method that consists of an SP-Sephadex C25 column¹⁵ and high-performance liquid chromatography.¹³

Radioimmunoassay for TRH has been described in detail.¹³ In brief, a polyclonal anti-TRH antibody was raised in New Zealand White rabbits immunized with TRH coupled to bovine serum albumin with the use of the bis-diazotized benzidine reaction.¹⁷ Standards or samples were incubated with ¹²⁵I-TRH and anti-TRH (1/10 000) at 4°C overnight. Free hormone was pelleted with carbon dextran T-70. All samples were assayed in duplicate. The minimum detectable amount was 5 to 10 pg. Intra-assay and interassay coefficients of variation were less than 7.0% and 14.0%, respectively.

CSF Extraction
Approximately 100 µL CSF was obtained by puncture of the cisterna magna from rats anesthetized with sodium pentobarbital (33 to 45 mg/kg) and placed in a stereotaxic apparatus (Scherr-Tumico, Inc). Samples were collected in 0.5 mL ice-cold 0.4 mol/L HClO₄ and centrifuged at 17 000g for 10 minutes. The supernatant was neutralized with 0.5 mol/L KOH, centrifuged, and lyophilized. The residue was redissolved in radioimmunoassay buffer.

[³H]Me-TRH Binding Assay
The binding assay was performed based on the method of Simasko and Horita.¹⁸ Ten to 12 POAs obtained from male SHR and WKY were homogenized in 10 vol of 0.32 mol/L sucrose with a glass-polytetrafluoroethylene homogenizer (Precytec; 20 seconds). The homogenate was centrifuged for 10 minutes at 1000g. The pellet (nuclear fraction, P₁) was discarded and the supernatant suspension centrifuged at 17 000g for 30 minutes (Ivan Sorvall Inc centrifuge). The pellet (P₂ fraction) was suspended in 20 mL of buffer (50 mmol/L PO₄H₂Na, pH 7.4), homogenized with a glass-polytetrafluoroethylene homogenizer, and used for the [³H]Me-TRH binding.

[³H]Me-TRH (NEN-DuPont; specific activity, 64.9 Ci/mmol), dissolved in buffer (50 mmol/L PO₄H₂Na, 0.1% bovine serum albumin, 0.1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L EDTA, 0.02% sodium azide, and 1 U/mL aprotinin, pH 7.4), was added to 200 µL of the homogenate at a concentration of 2 to 8 nmol/L. Incubation was carried out in triplicate in a shaking ice bath (New Brunswick Scientific) at 4°C for 5 hours. It was stopped by addition of 4 mL ice-cold saline to the incubation tubes. The contents were filtered through a glass fiber filter (GF/B) positioned over a vacuum. The filters were washed twice under vacuum with 2 mL ice-cold saline, dried, and transferred to liquid scintillation vials containing 10 mL of 30% Triton X-100/toluene/phosphor solution. The radioactivity in the samples was determined in a Packard Tri-Carb liquid scintillation spectrometer with an efficiency of 50%.

The specific binding, defined as the difference between the binding obtained in the absence or presence of 3-Me-TRH (10 µmol/L), accounted for 60% to 65% of the total binding.

To calculate the concentration of TRH receptors (B_max) and dissociation constant (K_d) we analyzed the binding data for [³H]Me-TRH by the nonlinear least-squares curve-fitting procedure using a generalized model for ligand-receptor systems. The amount of [³H]Me-TRH specifically bound was expressed as femtomoles of ligand bound per milligram of protein. The protein content was determined by the method of Lowry et al.¹⁶

Northern Blot Analysis
Total RNA was extracted from a pool of three to four POAs of adult male SHR and WKY immediately after decapitation. In some experiments we used hypothalamus as a positive control. Approximately 60 to 80 µg of total RNA of the POA was extracted with the use of the single phenol extraction step method.¹⁹ UV spectrophotometry at 260 nm was used for quantitation of total RNA. The integrity and accuracy of the RNA quantitation were confirmed by 5-µg aliquots of each sample undergoing 1% agarose-formaldehyde gel electrophoresis with ethidium bromide staining. Only undegraded samples with intact 28S/18S ribosomal RNA and A_260/280 ratios higher than 1.9 were processed.

For Northern blot analysis 40 µg total RNA underwent gel electrophoresis on 1% agarose gels containing 2.2 mol/L formaldehyde and was transferred to a nylon membrane support (Magma, MSI) and baked for 2 hours at 80°C in a vacuum oven. The filters were hybridized for 24 hours at 42°C in a buffer containing 50% formamide, 5x SSPE (43.8 g/L NaCl, 6.9 g/L NaH₂PO₄-H₂O, 1.85 g/L EDTA), 5x Denhardt's solution (1 g/L polyvinylpyrrolidone, 1 g/L bovine serum albumin, 1 g/L Ficoll 400), 0.1% SDS, and 200 µg/mL salmon sperm DNA, with probes labeled with [-³²P]dCTP (NEN-DuPont) using the random primer technique following the manufacturer's protocol (GIBCO-BRL).

For TRH precursor mRNA quantitation we used a 396-bp cDNA fragment produced by polymerase chain reaction from PLW_4-2 TRH cDNA kindly donated by Dr M.R. Lechan (Tufts University, New England Medical Center, Boston, Mass) with the use of the following primers: upper, 5'-GCCTTGCCTTGCACAGATGGGAAAAC-3'; lower, 5'-GAAGAGTGCAAACTGGCTGGGTAGAG-3'. In addition, we measured the expression of the housekeeping gene, cyclophilin, as a control for loading.²⁰

After hybridization the blots were washed twice with 2x SSC and 0.1% SDS at room temperature and twice with 0.5x SSC and 0.1% SDS at 50°C for TRH and 60°C for cyclophilin. Membranes were exposed to Kodak X-Omat-AR films at -70°C, which were developed after 24 to 48 hours for TRH and 6 to 12 hours for cyclophilin. Films were scanned by a laser densitometer.

In Vivo Experiments
Long-term Treatment
Male WKY and SHR were treated with saline (vehicle group), enalapril (5 mg/kg per 12 hours), or diltiazem (10 mg/kg every 8 hours) intraperitoneally during 20 days. Rats were killed by decapitation. The brain tissue was processed as described for POA TRH content determination, and blood was collected for measurement of ACE activity as described below. Systolic BP and HR were recorded weekly by a plethysmographic tail-cuff method.

Measurement of ACE Activity
The biochemical assay was performed in rat serum. ACE activity was assayed by a modification of Friedland and Silverstein's method.²¹ Each serum (10 µL) was added to 100 µL substrate buffer solution, 6.26 mol/L hippuryl-L-histidyl-L-leucine (pH 8.3), and phosphate-buffered saline (0.1 mol/L K₂HPO₄, 0.3 mol/L NaCl), and the mixture was incubated at 37°C for 30 minutes. The reaction was stopped by addition of 1 mL of 0.4 mol/L HClO₄ and centrifuged 15 minutes at 2500 rpm.

Two hundred and fifty microliters of 0.38 mol/L NaHO was added to 50 µL supernatant, followed by 25 µL of 2% (wt/vol) o-phthalaldehyde in methanol, followed 10 minutes later by 30 µL of 2.5 mol/L PO₄H₃. Fluorescence was measured in a spectrophotofluorometer (Waters, Millipore Corp) at 360/500 nm. Enzyme activity was expressed as nanomoles L-histidyl-L-leucine released per milliliter of serum per minute of incubation.

Peripheral and Intracerebroventricular Injection of Anti-TRH
Adult male SHR and WKY were anesthetized with pentobarbital (33 to 45 mg/kg) and chronically instrumented. MABP and HR were recorded in conscious rats throughout the experiment by a polyethylene cannula previously inserted into the left carotid artery and connected to a Statham transducer coupled to a polygraph (Grass Instrument Co). A 25-gauge stainless steel cannula was directed to the third ventricle through a burr hole in the skull for antibody infusion. Coordinates for implantation were 0.8 mm posterior to bregma on the midline and 6.6 mm below dura. At the end of each experiment the position of the cannula was assessed by injection of 2 µL of 2% bromphenol blue solution. Only data collected from experiments in which the correct insertion of the cannula was verified are reported. In some experiments the jugular vein was cannulated for intravenous antibody infusion. The antibody was a purified rabbit IgG from the same antiserum raised against TRH used for the above-mentioned TRH radioimmunoassay. The antibody purification required a chromatographic step in DEAE Sephadex. The purity of the resulting IgG was more than 95% as assessed by isoelectric focusing, bovine serum albumin being the main contaminant. Nonimmune rabbit IgG was used as a control.

Five microliters of IgG dissolved in phosphate-buffered saline (17.2 mg/mL) was delivered by a 15-second intracerebroventricular injection. We used 150 µL of the antibody solution for intravenous injection.

Statistical Analysis
Results are expressed as mean±SE from separate experiments. Statistical studies were performed with the use of ANOVA and the Tukey test for individual differences.

	Results

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As can be seen in the Table, 16-week-old SHR presented a significant twofold increase in POA TRH content with respect to age-matched WKY. This difference was not observed among young (21-day-old) rats of both strains (WKY, 456±8; SHR, 450±21 pg/mg protein).

View this table: [in this window] [in a new window]   Table 1. TRH Content of the POA, TRH Concentration in CSF, and TRH Receptor Parameters in SHR and WKY

To further investigate whether this higher POA TRH content may reflect an increase in TRH precursor synthesis, we determined TRH precursor mRNA abundance in the POA of hypertensive and normotensive control rats. Northern blot analysis indicated that there was a threefold increase in TRH precursor mRNA abundance in the POA of adult and young SHR compared with age-matched WKY. In fact, in WKY there was an apparently developmentally related decrease in POA TRH precursor mRNA level that was not seen in SHR (Fig 1). These results pointed out a probable increase in POA TRH synthesis in hypertensive rats.

View larger version (51K): [in this window] [in a new window]   Figure 1. Northern blot analysis of TRH precursor mRNA in the POA of 21-day-old (21d) and 16-week-old (16w) SHR and WKY. Forty micrograms total RNA was sequentially hybridized with 32P-labeled cDNA probes for TRH and cyclophilin. Top, 1.6-kb TRH transcript; bottom, 0.7-kb cyclophilin mRNA as a control for loading.

To address the question of whether central TRH release could also change, we determined TRH concentration in CSF. SHR showed a significantly elevated TRH concentration in CSF compared with WKY (Table).

Fig 2 shows that the number of TRH binding sites in the POA of SHR was increased compared with that in WKY, indicating a possible postsynaptic hypersensitivity to TRH in this central nucleus of the hypertensive strain. We did not observe any significant change in the affinity of the TRH receptor (Table).

View larger version (14K): [in this window] [in a new window]   Figure 2. Plot shows Scatchard analysis of specific binding of [3H]Me-TRH to synaptosomes of the POA of normotensive WKY () and SHR (). Each point corresponds to the mean of triplicate samples in one representative experiment of four.

To further study whether the alteration in TRH is an epiphenomenon unrelated to hypertension, we treated SHR and WKY with enalapril for 20 days. We tested the effectiveness of the long-term treatment by measuring serum ACE from the blood collected at the time of death. ACE activity was inhibited by more than 50% in both strains. In addition, enalapril treatment significantly (P<.05, n=10) diminished systolic BP in both SHR and WKY (34±6 and 14±5 mm Hg, respectively). Interestingly, the ACE inhibitor also brought down the elevated values of POA TRH content seen in SHR to match those observed in WKY, whereas it did not induce any significant change in POA TRH content of WKY (Fig 3). Another vasodilator, the calcium channel blocker diltiazem injected in a dose effective to decrease BP in SHR (35±7 mm Hg) but not in WKY (8±4 mm Hg), failed to reduce the elevated POA TRH content observed in SHR versus WKY (WKY vehicle, 378±38 versus diltiazem, 342±43 pg/mg protein; SHR vehicle, 770±76 versus diltiazem, 926±85; n=4).

View larger version (23K): [in this window] [in a new window]   Figure 3. Bar graph shows TRH content in the POA of SHR and WKY treated chronically with enalapril; black bars represent basal vehicle-treated rats; hatched bars, enalapril-treated rats. *P<.05 with respect to vehicle-treated WKY; #P<.05 with respect to vehicle-treated SHR.

Our results indicating an overall hyperactivity of the TRH system in the POA nucleus of this model of genetic hypertension prompted us to study the effect of blockade of the central TRH system. We followed two approaches. One involved intravenous infusion of a semipurified rabbit polyclonal IgG raised against TRH. This maneuver resulted in significant (P<.01, n=4) decreases of 17±3 mm Hg and 26±9 bpm in MABP and HR, respectively, only in SHR, with a maximal effect after 15 to 20 minutes of infusion. MABP and HR changes in WKY were not significant (2±3 mm Hg and -5±6 bpm, respectively). The other approach was to infuse the antibody into the third ventricle. In this case, the significant (P<.01, n=5) decreases in MABP and HR, observed only in SHR, were 23±4 mm Hg and 35±12 bpm, respectively. Again, the antibody effects on MABP and HR of WKY were not significant (MABP and HR changes, 5±4 mm Hg and 12±7 bpm, respectively). In either case, the injection of nonimmune IgG produced no effect.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Several studies point out a modulatory role for TRH in cardiovascular and sympathetic functions.⁸ Hypertensive responses to centrally administered TRH have been reported in anesthetized rats.^{9 22 23} This effect was blocked in pithed rats in which the autonomic pathways were destroyed, indicating that the action of TRH is mediated via sympathetic activity. Central TRH actions seem to involve the modulation of classic neurotransmitter systems.

In the SHR model, many neurochemical abnormalities have been described, in particular involving the cholinergic system.¹¹ Since a TRH-acetylcholine interaction exists in several central nuclei,^{12 13} we decided to explore the endogenous activity of the TRH system in one of the central areas of cardiovascular regulation, the POA.^{5 6 7 8}

Our study showed for the first time that SHR have a twofold increase in TRH content of the POA with respect to the normotensive WKY control strain. Two possibilities could explain these results: a reduced TRH release or an enhanced synthesis. Northern blot analysis indicated that the TRH precursor mRNA is more abundant in the POA of SHR than in age-matched WKY, pointing out a probable increase in TRH synthesis. The difference was more apparent in adult rats, which have developed hypertension, but it was also seen in rats during the prehypertensive state. Our conclusion agrees with other studies which show that changes in TRH content parallel changes in precursor synthesis, as demonstrated in postnatal development, circadian rhythm, and hypothyroidism, in which increases in TRH content accompany increases in TRH precursor mRNA abundance.²⁴ Interestingly, we also found a developmental TRH pattern showing an increase in TRH precursor mRNA abundance and TRH content of the POA in young WKY that was absent or attenuated in SHR. This developmental TRH pattern was also seen in Wistar rats by Covarrubias et al.²⁴ Since SHR show a significant increase in TRH concentration of the CSF, we postulate that the TRH release may also be elevated in this hypertensive condition, although a reduced degradation cannot be discarded as a possibility. It should be taken into account that the CSF is a pool in which extreme and opposite variations in tissue TRH release are attenuated, and therefore, precise changes in TRH presynaptic activity of particular nuclei may not be reflected. In any case, the elevated TRH concentration in SHR indicated an enhanced overall central TRH hyperactivity.

In addition, we found that the POA TRH receptor number is significantly increased in SHR with respect to normotensive rats, indicating that TRH sensitivity may also be augmented. These results agree with those of Bansinath et al²³ and Bhargava et al,²⁵ who have reported a greater magnitude of TRH effects on arterial BP and body temperature in SHR compared with WKY that can be related to an increase in the TRH receptor number in the hypothalamus and striatum of 6-week-old SHR compared with age-matched WKY.

Although our study was not focused on the TRH activity of the hypothalamic-pituitary axis, our data show an increased basal plasma thyrotropin level and a greater thyrotropin response to intraperitoneally administered TRH in SHR than in WKY but similar plasmatic triiodothyronine and thyroxine levels (unpublished data, 1995). These findings indicate that in addition to alterations in POA TRH activity, SHR may have abnormalities of this peptide likely related to thyroid-pituitary-brain regulation, as shown by Trippodo and Frohlich.²⁶ In addition, juvenile surgical thyroidectomy and radiothyroidectomy performed in the prehypertensive state prevented development of hypertension in SHR.²⁷

Presynaptic and postsynaptic TRH hyperactivity in the POA of SHR may be either a cause of or an adaptive biochemical change consequent upon a rise in arterial BP. The latter possibility is supported by our data indicating that POA TRH content is similar to that of WKY in SHR in the prehypertensive state, such as 21-day-old rats. However, the higher abundance of TRH precursor mRNA in the POA of 4-week-old SHR compared with that of age-matched WKY and the hypotensive effect of a polyclonal semipurified anti-TRH IgG infused either peripherally or intracerebroventricularly in SHR argue in favor of a pathogenic role of TRH in this condition. The action of the antibody was modest and transient, probably because of the presence of neutralizing endogenous TRH and the difficulty of big molecules in reaching the synaptic space. The POA is considered to be outside of the blood-brain barrier. This fact may in part explain the hypotensive effect of the intravenously injected TRH antibody.

Finally, long-term treatment of rats with enalapril, a specific ACE inhibitor, produced a significant decrease of arterial BP in both rat strains.²⁸ This treatment produced a significant decrease of POA TRH content in SHR, bringing the values down to those seen in normotensive rats. These data indicate that enalapril, by a nonspecific effect such as enhancement of protease activities or through the inhibition of humoral or local renin-angiotensin systems, directly decreased POA TRH hyperactivity, suggesting some effect of angiotensin on the synthesis and/or release of TRH. Whether or not the last possibility proves to be true, a highly interacting mechanism involving several neurotransmitters and neuropeptides may be responsible for the high arterial BP in SHR. The possibility that the decrease of POA TRH content induced by enalapril in SHR was due to the BP lowering seems unlikely because another vasodilator, the well-known peripherally acting L-type calcium channel blocker diltiazem,²⁹ did not reduce POA TRH content in either rat strain.

To conclude, 70% of the TRH content of the brain is extrahypothalamic.³⁰ Therefore, an elevated tone of this system in those nuclei important for cardiovascular regulation, such as the POA, may play an important role in the pathogenesis of rat spontaneous hypertension.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme BP = blood pressure bpm = beats per minute CSF = cerebrospinal fluid [3H]Me-TRH = 3-([3H]methyl)-thyrotropin-releasing hormone HR = heart rate MABP = mean arterial blood pressure POA = preoptic area SDS = sodium dodecyl sulfate SHR = spontaneously hypertensive rat(s) TRH = thyrotropin-releasing hormone WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and Universidad de Buenos Aires (UBA). S.I.G., V.M., S.F., and C.J.P. are members of CONICET and V.E.N. of UBA.

Received June 18, 1995; first decision September 16, 1995; accepted October 6, 1995.

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