© 1995 American Heart Association, Inc.
Articles |
Alterations in the Expression of the Genes Encoding Specific Muscarinic Receptor Subtypes in the Hypothalamus of Spontaneously Hypertensive Rats
From the Department of Pharmacology and Toxicology (J.J.B.) and the Department of Psychiatry and Health Behavior, Medical College of Georgia, Augusta, and the Department of Veterans Affairs Medical Center, Augusta, Ga.
Correspondence to Dr Jerry J. Buccafusco, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300.
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Abstract |
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Abstract A significant body of evidence exists that is consistent with the possibility that heightened cholinergic activity in certain brain regions, such as the hypothalamus, leads to increased sympathetic tone and subsequent hypertension. The increase in cholinergic activity is mediated at least in part through enhanced sensitivity of muscarinic receptors. In this study, we used the technique of reverse transcriptase–polymerase chain reaction to estimate the relative levels of mRNA encoding the five known subtypes of muscarinic receptors within the hypothalamus of spontaneously hypertensive rats (SHR), a genetic model of the disease, and their normotensive counterparts (Wistar-Kyoto rats). SHR exhibited a significant increase (40% to 50%) in the excitatory M1 subtype (confirmed by receptor binding) and a decrease in the inhibitory M4 subtype of muscarinic receptors before and during the establishment of hypertension. Such alterations may form part of the genotypic profile of inherited hypertension.
Key Words: muscarinic receptors • spontaneously hypertensive rats • transcription • blood pressure • hypothalamus
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Introduction |
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Essential hypertension continues to play a role in the morbidity and mortality associated with cardiovascular disease. At least one factor relates to the fact that the effects of enhanced central sympathetic drive to peripheral organs and the vasculature are not always abated by antihypertensive therapy. Various cardiotoxic sequelae have been attributed to exaggerated sympathetic activity, including chronic heart failure, atherosclerosis, angina, and cardiac arrhythmias.1 2 3 4 Although efferent sympathetic tone is constantly under baroreceptor control, heightened mental stress, particularly on a chronic basis, can lead to or exacerbate cardiovascular toxicity.5 6 Central autonomic dysfunction is also known to play an important role in the genesis and maintenance of hypertension in one of the most widely used genetically induced animal models of this disease, the spontaneously hypertensive rat (SHR). As with the humans, this strain is essentially normotensive at birth but gradually develops hypertension with adulthood.7 8 9 10 11
It has been appreciated since the 1930s that activation of central muscarinic receptors in several species, including humans, evokes a hypertensive response. Of particular relevance is the fact that the increase in blood pressure to central cholinergic stimulation is maintained predominantly by excessive sympathoadrenal tone.12 13 14 15 SHR are particularly sensitive in this regard, in that they exhibit pressor responses to central cholinergic receptor stimulation that are significantly greater in magnitude than the increases elicited from normotensive control rats12 13 14 15 (the most commonly used control is the Wistar-Kyoto [WKY] strain, from which the SHR strain was originally derived). The nature of the heightened sensitivity to brain muscarinic receptor stimulation in SHR is still being investigated, but both presynaptic and postsynaptic mechanisms have been suggested.12 13 14 15 16 17 Muscarinic receptor density has been reported to be increased in the brains of SHR compared with their normotensive controls.18 Moreover, selective blockade of brain muscarinic receptors results in a marked antihypertensive response in SHR.19 20 21 22
In the present study, we have focused on the cholinergic cardiovascular system located in the hypothalamus. Electrical stimulation of the posterior hypothalamus elicits a characteristic "defense" response, which is associated with an elevation in blood pressure.23 Chronic intermittent electrical stimulation of, or chronic infusion of, a muscarinic agonist within this region in normotensive rats leads to either a permanent or fulminant form of hypertension.24 25 26 Conversely, permanent destruction of cholinergic cells in the posterior hypothalamus after microinjection of the cholinergic neurotoxin AF64A resulted in a long-lasting antihypertensive response in SHR but not WKY rats.27 Moreover, transplantation of embryonic hypothalamic tissue from SHR to the medial hypothalamus of adult WKY rats resulted in sustained hypertension and left ventricular hypertrophy in the graft-bearing WKY rat.28
Five muscarinic receptor genes (M1 through M5) that encode distinct muscarinic cholinergic receptors have been cloned.29 30 Because of their structural homology and pharmacological similarity, the pharmacological ligands presently available do not clearly distinguish the five subtypes. Recently, we used reverse transcriptase (RT)–polymerase chain reaction (PCR) methodology to detect low abundance mRNA for the five muscarinic receptor subtypes. PCR products were quantified by using weak anion exchange high-performance liquid chromatography (HPLC).31 32 In the case of muscarinic receptors, it has been demonstrated that a wide variety of chemical stimuli and disease states that are known to alter the expression of the receptor protein are reflected in changes in animal behavior or physiology, which are mediated by the respective cholinergic pathways.33 34 35 36 37 38 39 The mechanism for such changes in mRNA levels has been attributed both to altered transcription rates and altered stability of transcribed mRNA, depending on the subtype being studied. In either case, relatively small changes in mRNA levels or muscarinic receptor protein (less than onefold increases or decreases) are sufficient to alter behavior or physiological function.
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Materials and Methods |
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Animals
Male SHR and age-matched normotensive WKY rats were obtained from Taconic Farms, Germantown, NY. The animals were housed in our animal care facilities for 1 week before experimentation. They had access to standard laboratory rodent chow and tap water on an unlimited basis. A 12-hour light-dark cycle was maintained. Taconic farms was chosen as the supplier because their SHR and WKY rats are maintained as outbred strains that share many common phenotypes except for hypertension.40 Thus, although an outbred strain may allow for more variability than an inbred strain with regard to genetic studies, there will be a greater likelihood that the genotypic differences we may find are related to the hypertension phenotype. Procedures involving animals were reviewed by the institutional committee for animal use in research and education before work was undertaken.
Measurement of Blood Pressure
The unrestrained animals were prewarmed in an environment
maintained at 35°C for 5 to 10 minutes before blood pressure
measurement to facilitate tail blood flow. Rats were then restrained in
a Plexiglas chamber, which allows access to the tail. Systolic pressure
was measured by an electrosphygmomanometer with a pneumatic pulse
transducer. Tail blood flow was occluded by a tubular cuff (8 and 11 mm
in length for 8- and 12-week-old rats, respectively) that was inflated
by an automatic cycling cuff pump, which deflates the cuff at 
20
mm Hg/s.
Quantitative RT-PCR and HPLC
On the day after blood pressure measurement, the rats were
killed by decapitation, the brains were removed, the hypothalamus was
isolated, and total RNA was extracted for the RT-PCR methodology as
previously described.31 Both the hypothalamus and cerebral
cortex were dissected out over ice. Undegraded total RNA was isolated
from each sample by using the RNAzol kit developed by Biotex
Laboratories, Inc. Pelleted RNA fractions were suspended in water and
stored at -80°C before the next stage. Extracted RNA samples were
first treated for 30 minutes with 2 U of RNase-free DNase (Bethesda
Research Laboratories) at 37°C to remove any residual genomic DNA.
M-MLV RT (United States Biochemical) and priming with random hexamers
(2.5 µmol/L, Promega) were used to synthesize cDNA in a reaction
volume of 20 µL. The reaction volume also contained (mmol/L)
MgCl2 5, KCl 50, Tris HCl 10, and dNTP precursors 1 at pH
8.3. The resulting cDNAs were amplified (33 cycles of 95°C for 1
minute and 62°C for 1 minute) by using the GeneAmp RNA PCR kit
(Perkin-Elmer) and a Perkin-Elmer Cetus model 480 thermal cycler.
Aliquots of the DNA samples (10 µL) were loaded onto agarose gels
(1.8% agarose), where one lane in each gel contained a molecular
weight standard. Each gel was then stained with ethidium bromide, and
the reaction products were visualized with fluorescent illumination and
photographed. In our earlier experiments,31 the amount of
product increased in a log-linear fashion for up to 35 cycles. At 40
cycles, each of the curves showed evidence of reaching a plateau.
Least-squares regression lines were plotted through the data points
encompassing 25 to 35 cycles. The following values were obtained for
the slopes: M1=0.231, M2=0.217, M3=0.231, M4=0.211, and M5=0.212. Since
the initial slopes of each curve were similar, it was concluded that
the respective fragments were amplified with approximately the same
efficiency. Primer sequences, corresponding base sites, the size of the
PCR product, and sequence number (Genbank) are indicated as
follows: M1: 5'-GCA CAG GCA CCC ACC AAG CAG-3'
(sense; base position, 1073) and 5'AGA GCA GCA GCA GGC GGA ACG-3'
(antisense; base position, 1425) (PCR product, 373 bp; Genbank sequence
number, M 16406) M2: 5'-CAC GAA ACC TCT GAC CTA CCC-3' (sense;
base position, 826) and 5'-TCT GAC CCG ACG ACC CAA CTA-3' (antisense;
base position, 1488) (PCR product, 686 bp; Genbank sequence number, J
03025) M3: 5'-GTC TGG CTT GGG TCA TCT CCT-3' (sense; base
position, 606) and 5'GCT GCT GCT GTG GTC TTG GTC-3' (antisense; base
position, 1019) (PCR product, 434 bp; Genbank sequence number, M
16407) M4: 5'-TGG GTC TTG GCC TTT GTG CTC-3' (sense; base
position, 461) and 5'-TTC ATT GCC TGT CTG CTT TGT TA-3' (antisense;
base position, 1026) (PCR product, 588 bp; Genbank sequence number, M
16409) M5: 5'-CTG GTC TCC TTC ATC CTC TGG-3' (sense; base
position, 1436) and 5'-CCT GGG TTG TCT TTC CTG TTG-3' (antisense; base
position, 1809) (PCR product, 394 bp; Genbank sequence number, M
22926)
PCR-amplified products (cDNA oligos) representing all five subtypes of muscarinic receptors were quantified (mRNA encoding glyceraldehyde-3-phosphate dehydrogenase [G3PDH] was used as the internal standard-control gene). The PCR products were separated by using standard electrophoretic gels and visualized by ethidium bromide staining.
Aliquots (5 µL) of PCR reaction products were used for HPLC analysis without further purification. The HPLC system used in the present study consisted of Bio-Rad model 1350 HPLC pumps and a model 1706 UV-visible monitor. Pumping rate and gradient production, as well as peak identification and quantification, were controlled from an on-line computer by using the Bio-Rad series 800 HRLC system (version 2.30.1a) HPLC software package. The analytical column (TSK DEAE-NPR, Perkin-Elmer) was packed with 2.5-nm particles of hydrophilic resin bonded with DEAE groups. The mobile phase consisted of reservoir A, containing 1 mol/L NaCl and 25 mmol/L Tris HCl at pH 9.0, and reservoir B, containing 25 mmol/L Tris HCl at pH 9.0. The gradient used was as follows: 46% to 54% A for 0.1 minute, 54% to 60% A for 3.9 minutes, 60% to 75% A for 1 minutes, 75% to 100% A for 5 minutes, and 100% A to 46% A for 3 minutes. The column was operated at a flow rate of 1 mL/min at room temperature, and the UV detector set at 260 nm. The relative amount of PCR products was determined as the area under the peak in arbitrary units.
Saturation Binding to Brain Muscarinic Receptors
Animals were killed by decapitation, the brains were quickly
removed, and the hypothalamus and cerebral cortex were dissected out
over ice. The tissue was homogenized in 50 mmol/L Tris-HCl, pH 7.4,
containing 2 mmol/L MgCl2 and centrifuged at
20 000g for 20 minutes. The membranes were then washed with
fresh buffer, resuspended, and recentrifuged. After two washes, the
membranes were resuspended and refrigerated, and the protein
concentration was determined with the Bio-Rad protein assay system.
Equilibrium binding of [3H]methyl scopolamine was
accomplished by using standard filtration procedures. An aliquot of
tissue (30 to 50 µg protein) was incubated with one of at least eight
concentrations of [3H]methyl scopolamine in buffer (50
mmol/L Tris-HCl, pH 7.4, and 2 mmol/L MgCl2). The
suspensions were then filtered through glass-fiber filters (Whatman
GF/B) by using a Brandel filtration manifold. The incubation tubes and
filters were washed, and the radioactive content of the filters was
determined by liquid scintillation spectroscopy. Nonspecific binding
was determined by incubation of the membranes in the presence of 10
µmol/L atropine. Binding parameters were determined by nonlinear
regression analysis using a mass action expression for ligand
binding to a single population of noninteracting sites:
B=Bmax*C/(C+Kd), where B is the
bound fraction of label, C is the concentration of ligand, and
Kd is the dissociation constant.
Statistics
Data are presented as mean±SEM. A paired Student's
t test was used to determine which group means differed.
Means were considered to be statistically significant at
P<.05.
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Results |
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Blood Pressure in Hypertensive Rats
For the six 4-week-old WKY rats and five 4-week-old SHR, the systolic pressures averaged 95.8±6.1 and 102±4.3 mm Hg, respectively. There was no significant difference between the two groups. For the four 12-week old WKY rats and four 12-week-old SHR, the systolic pressures averaged 146.6±1.5 and 197.0±2.6 mm Hg, respectively; and the means were significantly different (t=16.9, P<.01).
Relative Levels of Hypothalamic mRNA in SHR and WKY Rats
The specificity of the five PCR probes and the identity of the
respective cDNA products were first determined by gel electrophoresis.
A typical gel electrophoresis pattern for the PCR products obtained for
all five subtypes of the muscarinic receptor is depicted in Fig 1
. All PCR products migrated in the gel according to
their expected molecular weights (see "Materials and Methods").
In our earlier study31 in which we used the same primers,
we reported that no amplified products were present in gel lanes
where reverse transcription was omitted, demonstrating the absence of
contaminating DNA. Also, it may be observed that only one band is
present for each subtype. By visual inspection, the densities of
the bands corresponding to the M1 and M4 products in the SHR appeared
to be respectively higher and lower than for the WKY rat. In a further
effort to confirm that the PCR products for these two subtypes were
derived from the M1 and M4 genes, we subjected the products to
digestion by the restriction enzymes Pvu II and
Xho I, respectively. On the basis of known restriction sites
(DNA Strider, version 1.1) within the expected PCR products, each
enzyme was expected to produce two new fragments of known molecular
weights (111 and 262 bp for M1 and 319 and 269 bp for M4). When the
products were run on electrophoretic gels after digestion, only two new
fragments were detected for each subtype, which ran exactly at the
expected molecular weights (data not shown).
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More quantitative results were obtained when PCR products were measured
by HPLC. In Fig 2, data are presented as the peak
area ratio of subtype product to G3PDH product. The internal standard
controlled for differences in RT efficiencies among samples. In fact,
we observed no significant differences between the hypothalamic mRNA
encoding the internal standard gene between SHR and WKY rats. The
average levels (peak area) of G3PDH message derived from 4-week-old WKY
and SHR samples were 787.3±34.8 and 777.3±18.8, respectively. For
12-week-old rats, the respective values were 874.2±41.0 and
897.6±26.0. In the prehypertensive SHR, the levels of mRNA encoding
the M1 and M4 subtypes were altered. The level of M1 mRNA was
significantly increased by 53%, and the level of M4 mRNA was
significantly decreased by 15% in the SHR. Similar differences were
observed in samples derived from the 12-week-old rats; however, the
decrease in M4 mRNA levels observed in SHR was greater in magnitude
(54%) in the older hypertensive rats. There were no differences
between the strains for the other subtypes derived from the
hypothalamus; however, in a separate experiment using cerebral cortex
from 12-week-old rats, we found no differences among the five subtypes
between strains (data not shown). Thus, at the transcriptional level,
there exists a difference in message that suggests overexpression of a
subtype (M1) that is linked to excitatory synaptic transmission and an
underexpression of a subtype (M4) that is linked to inhibitory
transmission.29 30
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Estimation of Muscarinic Receptor Density
Since changes in mRNA levels encoding selective subtypes of
muscarinic receptors merely infer changes in receptor protein levels,
it was necessary to determine whether we could indeed measure changes
in receptor numbers by using standard ligand binding techniques. As a
first approximation, we used the nonselective ligand
[3H]methyl scopolamine, recognizing that we were
minimizing any changes due to dilution with nonchanging receptors or
receptors that may change in alternate directions as observed for the
M4 mRNA. Nevertheless, using saturation binding techniques, we
determined that SHR do express increased levels of muscarinic receptors
in the hypothalamus for both age groups (Table).
Bmax increased by 41% and 57% in the SHR for the
prehypertensive and hypertensive groups, respectively. Coincidentally,
the percent increase in M1 mRNA correlates very well with the increase
in Bmax between the two strains. Note that there was no
difference in receptor numbers (Bmax) between
strains for the cerebral cortex, a region that has not been implicated
in cholinergic control of blood pressure. Also, there was no difference
in the apparent affinity for the ligand (Kd)
between the strains for any of the three brain regions. The increased
Bmax measured in the hypothalamus most likely
represents the M1 subtype, since the mRNA encoding the M4 subtype
actually decreased and the other three subtypes remained unchanged.
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Discussion |
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In recent years, several groups have attempted to determine the nature of hereditary transfer of genes responsible for hypertension in the SHR by backcrossing SHR with normotensive control rats. Estimates have ranged from a single gene with simple additive allelic effects to as few as six major genes that determine high blood pressure (see Reference 4141 ). Application of biometric genetic analysis to this problem has suggested that for SHR reared under normal laboratory conditions, the level of blood pressure fits an additive-dominance model of inheritance in which alleles decreasing blood pressure are partially dominant.41 This finding is particularly interesting and relevant since it has often been considered that hypertension could be related to factors increasing blood pressure that are overexpressed. As suggested in the latter study, however, altered expression of factors decreasing blood pressure may prove to be the underlying mechanism. For example, the cardiovascular response to startle in SHR is different from that in WKY rats. A characteristic transient bradycardia (inhibitory response) before tachycardia that was observed in WKY rats as a response to startle does not occur in SHR. This transient bradycardia is vagally mediated and represents a phenotype that cosegregates with hypertension in crosses between SHR and WKY rats.42 43 The involvement of parasympathetic tone in the heart is particularly interesting, since the target receptors are almost exclusively of the M2 (inhibitory) subtype.
In the present study, hypothalamic M4 mRNA levels decreased with age, paralleling the development of hypertension. Since the M4 subtype is linked to inhibitory synaptic responses, it is possible that this gene alteration associated with hypertension may reflect, in part, the reduced expression of factors decreasing blood pressure as mentioned above. However, since both M1 and M4 mRNAs were altered in SHR, potentially both receptors may interact and play a role in enhanced sympathetic outflow in this strain. The selectivity of alterations in receptor regulation in the SHR is underscored by the observations that (1) the level of the control gene G3PDH was not different between strains of either age group; (2) the levels of the other subtypes, M2, M3, and M5, were similar between strains; and (3) the density of muscarinic receptors in the cerebral cortex was similar between the strains. Although the magnitude of the alterations in hypothalamic muscarinic receptor mRNA levels obtained are sufficient to account for changes in the binding parameters and in physiological function,33 34 35 36 37 38 39 they should be considered minimum values, since it is more likely that there was some dilution owing to the size of the tissue studied. The fact that these receptor changes, both at the mRNA and protein level, occurred before the onset of significant hypertension suggests that if these factors are involved, they play an initiation role as well as a maintenance role in hypertension.
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Acknowledgments |
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This study was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
Received May 27, 1994; accepted September 27, 1994.
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