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Am J Physiol Regul Integr Comp Physiol 279: R1685-R1694, 2000;
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Vol. 279, Issue 5, R1685-R1694, November 2000

Increased ganglionic responses to substance P in hypertensive rats due to upregulation of NK1 receptors

Robert V. Schoborg1, Donald B. Hoover2, John D. Tompkins2, and John C. Hancock2

Departments of 2 Pharmacology and 1 Microbiology, James H. Quillen College of Medicine, Johnson City, Tennessee 37614


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intravenous injection of substance P (SP) increases renal nerve firing and heart rate in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) by stimulating sympathetic ganglia. Blood pressure is increased in SHRs but lowered in WKYs. This study assesses the role of neurokinin-1 (NK1) receptors in mediating the ganglion actions of SP. Rats for functional studies were anesthetized and then treated with chlorisondamine. Renal nerve, blood pressure, and heart rate responses to intravenous injection of the NK1 receptor agonist GR-73632 were similar but less than those to equimolar doses of SP in SHRs. GR-73632 only slightly increased renal nerve firing and heart rate and lowered blood pressure in WKYs. The NK1 receptor antagonist GR-82334 (200 nmol/kg iv) blocked the ganglionic actions of GR-73632 and the pressor response to SP in SHRs. It reduced the renal nerve and heart rate responses by 52 and 35%. This suggests that the pressor response to SP is mediated by ganglionic NK1 receptors and that NK1 receptors also have a prominent role in mediating the renal nerve and heart rate responses to SP. Quantitative autoradiography showed that NK1 receptors are more abundant in the superior cervical ganglia of SHRs. RT-PCR showed increased abundance of NK1 receptor mRNA in SHRs as well. These observations suggest that the greater ganglionic stimulation caused by SP in SHRs is due to upregulation of NK1 receptors.

neurokinin-1 receptors; hypertension; sympathetic nervous system; inbred spontaneously hypertensive rats


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NEURONS IN SYMPATHETIC GANGLIA are surrounded by a dense network of substance P (SP)-immunoreactive fibers. These fibers are peripherally directed branches of sensory nerves projecting from dorsal root ganglia (3). When SP is applied to these neurons it causes a slow excitatory postsynaptic potential similar to that caused by stimulation of sensory collaterals to the ganglia (4, 22). Both potentials are blocked by SP antagonists (20, 21). These observations suggest that SP-containing sensory collaterals and efferent postganglionic sympathetic neurons constitute a peripheral reflex mechanism.

We have shown that stimulation of sympathetic ganglia by SP in situ can increase renal sympathetic nerve activity, blood pressure, and heart rate (12, 13). The effects of the ganglion stimulation on renal nerve firing and heart rate are greater in hypertensive than in normotensive rats. Blood pressure is increased in spontaneously hypertensive rats (SHRs) as a result of ganglion stimulation by SP. Blood pressure is lowered in Wistar-Kyoto rats (WKYs) (13). SP immunoreactivity is also greater in sympathetic ganglia of hypertensive than in normotensive rats (8). Because SP exerts excitatory influences on sympathetic neurons, this greater innervation may lead to an increase in the basal activity of sympathetic neurons in SHRs. This is consistent with the observations that postganglionic sympathetic nerve activity is enhanced in human essential hypertension and in SHRs (6, 26)

In addition to its action on sympathetic ganglia, SP has a direct action on endothelial NK1 receptors to cause vasodilation (23) by release of endothelium-derived relaxant factor (36). The vasodilation caused by SP and other endothelium-dependent vasodilators is less in SHRs than in WKYs (32). The intense ganglionic stimulation caused by SP appears to override the vasodilator response in SHRs with the result that SP increases blood pressure in this strain. In contrast, the stimulation of sympathetic ganglia by SP in WKYs does not appear to be intense enough to override the vasodilator action so SP only lowers blood pressure in WKYs. Thus the increase in blood pressure in SHRs results from impaired vasodilation and enhanced ganglion stimulation by SP.

SP is a member of the family of structurally related peptides named tachykinins. There are three tachykinin receptors that have been termed NK1, NK2, and NK3. Although SP can activate all three tachykinin receptors, its potency is greatest at NK1 receptors (17, 23, 31).

Our purpose in this study was to assess the role of NK1 receptors in mediating the ganglionic action of SP and to determine whether differences in ganglionic responses to SP in SHRs and WKYs are due to an increase in NK1 receptor expression. Multifiber renal nerve activity, blood pressure, and heart rate were recorded to quantify the effects of SP and the NK1 receptor agonist GR-73632 on sympathetic ganglia mediating those responses. Ganglionic nicotinic receptors were blocked to reduce the possibility that the effects of the tachykinins were due to actions involving the central nervous system. The relative abundance of NK1 receptors in ganglia of SHRs and WKYs was determined by quantitative receptor autoradiography. NK1 receptor mRNA expression was determined by RT-PCR.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In Vivo Studies with NK1 Receptor Agonists and Antagonists

Male SHRs and WKYs (300-400 g, aged 3-4 mo) obtained from Harlan (Indianapolis, IN) were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and pentobarbital sodium (35 mg/kg). The trachea was cannulated and opened to room air to facilitate respiration. The right jugular vein was cannulated for intravenous injection of drugs. Systemic arterial pressure was monitored from the femoral artery on a Gould 2400S recorder. Mean arterial pressure (blood pressure) was calculated from the pulse pressure. Heart rate was determined from the pulse pressure by a Gould electrocardiogram/Biotach amplifier. Body temperature was maintained with a heat lamp and monitored with a rectal Yellow Springs Instruments tele-thermometer probe (Yellow Springs, OH). This study was reviewed and approved by the University Animal Care Committee.

Renal nerve recording. The left kidney was exposed via a retroperitoneal flank incision, and a 10-mm section of one renal nerve was placed over 32-gauge bipolar platinum electrodes and covered with mineral oil. Renal sympathetic nerve activity was amplified (×20,000) by a Grass P511 series A-C preamplifier. The output was sent to an oscilloscope (Tektronix 5110) and a Gould Universal preamplifier on the Gould recorder for visualization and amplification. From the recorder, the output passed to a Zenith 433Dh computer. Renal nerve activity was stored in digital format at a sampling rate of 2,000 Hz and analyzed using the RC Electronics Computerscope Acquisition and Analysis System software package (Santa Barbara, CA). Permanent records were made on an inkjet printer and the Gould recorder. Total power (µV2/s) was obtained by summing voltages contained in all frequencies of 2-s segments of unrectified neurograms. Five 2-s segments were used to obtain the average power before the injection of drugs. Two 2-s segments were used to obtain the average power during the maximal response to drugs. This interval was selected because the values during this period are directly proportional to the level of nerve activity obtained by integration. Drug-induced changes in firing are expressed as the difference between the average power before the injection of drugs and the average power during the maximal response minus the basal noise level. Basal noise is the activity remaining after reflex inhibition of the renal nerve activity produced by intravenous injection of phenylephrine or the level of firing 20 to 30 min after death.

Experiments with chlorisondamine. Chlorisondamine (10.5 µmol/kg) was administered by intravenous injection 30 min after completion of the surgery to block nicotinic ganglionic transmission. This reduced the possibility that the effects of SP and GR-73632 were due to actions on the central nervous system. Blockade of ganglion transmission also prevented reflex inhibition of sympathetic nerve activity by the pressor responses to SP and GR-73632 in SHRs. Chlorisondamine was administered by slow intravenous injection in divided doses of 0.5 µmol/kg followed by 10 µmol/kg in volumes of 100 µl to minimize the lowering of blood pressure. The effectiveness of chlorisondamine was verified by blockade of the response to 0.56 µmol/kg iv 1,1-dimethyl-4-phenylpiperazinium (DMPP) and of the reflex increase in renal nerve firing caused by injection of 2 µmol/kg iv isoproterenol or carotid occlusion. Nicotinic receptor blockade persisted for the duration of the experiments. Chlorisondamine lowered renal nerve firing from 148 ± 22 to 27 ± 6 µV2/s, mean blood pressure from 163 ± 10 to 83 ± 6 mmHg, and heart rate from 284 ± 10 to 185 ± 10 mmHg in SHRs (n = 21). Chlorisondamine lowered renal nerve firing from 18 ± 2 to 11 ± 1 µV2/s, mean blood pressure from 96 ± 3 to 64 ± 3 mmHg, and heart rate from 206 ± 12 to 128 ± 5 in WKYs (n = 12). Previous work has shown that ganglion blockade does not qualitatively affect the responses to SP in SHRs and WKYs (12, 13).

Experiments with SP and GR-73632. Studies on the effects of GR-73632 or SP were begun 30 min after injection of chlorisondamine. Dose-response curves were obtained for the effects of 1.0, 3.2, 10, and 32 nmol/kg of SP and GR-73632 on blood pressure, heart rate, and renal nerve activity in separate groups of rats. Both drugs were administered from low to high dose by rapid intravenous injection in a volume of 50 µl. Doses of SP were given at 30-min intervals. Reproducible responses were obtained with four injections of either 10 or 32 nmol/kg of SP at 15-min intervals (n = 3), indicating that tolerance did not develop to this regimen. Doses of 1, 3.2, and 10 nmol/kg of GR-73632 were separated by 30 min. For higher doses of GR-73632, a 45-min interval was required to avoid tolerance (n = 3). SP and GR-73632 were dissolved in sterile, distilled water, and 50-µl aliquots (200 nmol) were stored at -80°C. The peptides were then diluted with saline before use.

Experiments with the NK1 receptor antagonist GR-82334. GR-73632 or SP (32 nmol/kg) was administered by intravenous injection in separate rats treated with chlorisondamine. For experiments to determine the effect of GR-82334 on responses to GR-73632, GR-73632 was administered 45 min before and 5 min after 200 µmol/kg of GR-82334. SP was administered 30 min before and 5 min after either 200 or 600 µmol/kg of GR-82334. GR-82334 was dissolved in sterile distilled water, and 100-µl aliquots (240 nmol) were stored at -80°C. GR-82334 was diluted in saline and injected intravenously in a volume of 100 µl.

Experiments with reserpine. Reserpine was administered to deplete catecholamines from adrenergic nerve terminals and thus verify that the pressor and tachycardic responses to GR-73632 were not due to a direct action on blood vessels or the heart. Depletion of catecholamines was accomplished by intraperitoneal injection of reserpine (5 mg/kg) 24 h before the experiment. Depletion of catecholamines was verified by intravenous injection of tyramine (3 µmol/kg). Reserpine stock solution was prepared by dissolving 100 mg reserpine in 100 ml distilled water containing 2.098 g benzyl alcohol, 250 mg citric acid, and 10.8 g Tween 80. Rats for this study were anesthetized and treated with chlorisondamine before the administration of SP or GR-73632 as described previously.

Quantification of NK1 Receptors by Autoradiography

SHRs and WKYs were anesthetized with pentobarbital sodium (50 mg/kg ip) to remove the left superior cervical ganglion and a portion of attached nerve. A silk suture was attached to the nerve to facilitate handling. Ganglia were frozen horizontally on specimen plates using saline as the mounting medium. Saline was applied to the plate and partially frozen on powdered dry ice before transfer of the ganglia. After the tissues were frozen, the samples were stored in 50 ml polypropylene tubes (Corning) at -80°C.

Serial 20-µm sections were cut through the ganglia at -20°C using an IEC microtome cryostat. The sections were thaw mounted onto three groups of chrome alum-gelatin coated slides. Sections on the first group of slides were stained with hematoxylin and eosin. The remaining slides were stored at -80°C for autoradiography.

Established methods were followed for labeling and autoradiographic detection of NK1 receptors (1, 25). Slides were brought to room temperature and preincubated for 10-min in 50 mM Tris · HCl (pH 7.4) containing 0.005% polyethylenimine. After a brief rinse in 50 mM Tris · HCl buffer and draining, slides with adjacent sections were transferred to separate incubating solutions for total and nonspecific binding. The incubation buffer for total binding contained 50 mM Tris · HCl (pH 7.4), 3 mM MnCl2, 0.02% bovine serum albumin, peptidase inhibitors (40 mg/l bacitracin, 2 mg/l chymostatin, and 4 mg/l leupeptin), and 0.1 nM 125I-labeled Bolton-Hunter SP (125I-BHSP; 2,200 Ci/mmol). The buffer for nonspecific binding contained the same components plus 1 µM [Sar9,Met(O2)11]SP, an NK1 agonist. After incubation of slides for 1 h at room temperature, they were drained on a piece of absorbent bench paper; washed four times for 2-min each in 50 mM Tris · HCl buffer at 4°C; dipped in ice-cold sterile, distilled water; and dried with an electric fan. Dried slides were loaded into an X-ray cassette with 125I microscales and a sheet of Hyperfilm 3H (Amersham, Arlington Heights, IL). The film was processed by standard methods after 8 days exposure at 4°C. A microcomputer-assisted imaging device (Imaging Research, Ontario, Canada) was used for quantitative evaluation of the film autoradiograms. For each ganglion, mean values for total and nonspecific binding were determined from relative optical density readings for five to ten sections.

NK1 Receptor mRNA Expression

Tissues were removed from anesthetized SHRs and WKYs, collected in polypropylene tubes, and frozen on dry ice.

Isolation of total RNA. Frozen ganglia were homogenized in a glass mortar and pestle containing 500 µl RNAzol B (TelTest) and the homogenate transferred to 1.5 ml polypropylene tubes. The mortar and pestle were rinsed twice with 250 µl of RNAzol B, and the rinses were added to the homogenate. One microliter of pellet paint (Novagen) was added to each 1-ml sample, and the RNA was purified according to the manufacturer's instructions. RNA pellets were dried under vacuum for 5 min and resuspended in 200 µl 10 mM Tris-1 mM EDTA, pH 8.0. Twenty microliters of 3 M NaOAc, pH 5.2, and 440 µl of absolute ethanol were added to each tube and samples were stored as ethanol precipitates at -70°C.

Reverse transcription of RNA. One third (220 µl) of each ethanol precipitated RNA sample was centrifuged at 12,000 g for 15 min. RNA pellets were washed with 70% ethanol and dried for 5 min. Each pellet was resuspended in 22 µl double-distilled water, combined with 2 µl of 1 mg/ml random primers (Pharmacia Biotech, NJ), incubated at 70°C for 10 min, quenched on ice, and divided into two equal aliquots. One aliquot was reverse transcribed with 200 units of Maloney Murine Leukemia Virus reverse transcriptase (Promega Biotech, WI) using the manufacturer's protocol (RT(+) reactions). As a negative control, an equal volume of double-distilled water was added to the other aliquot of sample and primers (RT(-) reactions). Each sample was diluted one to ten with double distilled water before PCR amplification.

PCR amplification. The sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and NK1 specific oligonucleotides used for PCR amplification have been published (5, 18, 34). Both primer sets have been shown to specifically amplify the sequences of interest (18, 34). PCR amplification reactions were performed at a final concentration of 2 mM MgCl2, 0.8 mM dNTPs, 1 µM each primer, 2 µl of diluted cDNA, and 1 unit each of AmpliTaq Gold and AmpliTaq (Perkin/Elmer). NK1 reactions were cycled as follows: 94°C for 9 min-94°C for 20 s and 70°C for 40 s (40 cycles)-72°C for 5 min. GAPDH reactions were cycled as follows: 94°C for 9 min-94°C for 20 s, 63°C for 30 s, and 72°C for 30 s (30 cycles)-72°C for 5 min.

Gel electrophoresis and amplimer optical density analysis. Ten microliters of each RT-PCR reaction was electrophoresed on 1.4% agarose-0.5 × Tris-borate-EDTA (TBE) gels. Gels were stained in TBE containing 0.125 µg/ml ethidium bromide for 30 min and destained in double-distilled and filtered water for 3 h. The gels were photographed on an ultraviolet transilluminator using Polaroid type 665 film. Negatives were scanned on a microcomputer-assisted imaging device (Imaging Research, Ontario, Canada) to determine the relative optical densities (OD) of each band. The final OD for each GAPDH and NK1 receptor band was obtained by subtracting the OD value of a similar block in the RT(-) control lane from the initial OD value of each band. The NK1 receptor amplimer OD values were then normalized to the amount of GAPDH amplimer present in each sample to control for differences in the amount of RNA used in each cDNA synthesis.

Drugs

All drugs were diluted in saline. Rapid intravenous injection of 50 µl of saline did not affect renal nerve firing, blood pressure, or heart rate. The drugs used for in vivo studies and their sources were substance P (Bachem); GR-73632 and GR-82334 (RBI, MA); DMPP, isoproterenol, phenylephrine, tyramine, and reserpine (Sigma Chemical); ketamine (Fort Dodge Laboratories); pentobarbital sodium (Abbot Laboratories); and chlorisondamine (CIBA Pharmaceutical). The drugs used for receptor autoradiography and their sources were: [Sar9,Met(O2)11]SP (Peninsula Laboratory, CA) and 125I-BHSP (NEN Life Science Products, MA).

Analysis of Data

Dose response curves for the effects of SP and GR-73632 on blood pressure, heart rate, and renal nerve activity in SHRs and WKYs were compared with each other by repeated-measures ANOVA (3-factor ANOVA). Comparisons at each dose between strains were made by the least significant difference multiple comparison test. The effect of GR-82334 on responses to GR-73632 and SP within strains were compared by the paired t-test. Control responses to SP and GR-73632 between strains were compared by the Students t-test. Data are expressed as means ± SE. A P <=  0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In Vivo Studies

Renal nerve responses. Renal nerve responses to intravenous injection of GR-73632 were qualitatively similar to those evoked by SP in SHRs (Fig. 1). Both compounds cause a delayed-onset long-duration increase in renal nerve firing. The responses differed in that the magnitude of the response to GR-73632 was significantly less than the response to SP (Fig. 3A). SP also caused a delayed-onset long-duration increase in renal nerve firing in WKYs (Fig. 2). In contrast, GR-73632 had almost no effect on renal nerve firing in this strain of normotensive rats (Figs. 2 and 3A). Figure 4A provides a dose-response comparison of the responses to GR-73632 in SHRs and WKYs. The threshold for eliciting the renal nerve responses to GR-73632 in SHRs was 3.2 nmol/kg. Whereas the renal nerve response to GR-73632 was minimal in WKYs, the increase was different from baseline [F(4,20) = 9.622 (P < 0.002)]. Administration of reserpine (5 mg/kg) did not affect the renal nerve response to 32 nmol/kg of GR-73632.


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  		Fig. 1.   Recorder tracings showing the effects of intravenous injection of 32 nmol/kg of substance P (SP) and GR-73632 on renal sympathetic nerve firing, blood pressure, and heart rate in spontaneously hypertensive rats (SHRs). Rats were treated with chlorisondamine (10.5 µmol/kg) to block nicotinic receptors in autonomic ganglia. Arrows indicate drug injection.



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  		Fig. 2.   Recorder tracings showing the effects of intravenous injection of 32 nmol/kg of SP and GR-73632 on renal sympathetic nerve firing, blood pressure, and heart rate in Wistar-Kyoto rats (WKYs). Rats were treated with chlorisondamine (10.5 µmol/kg) to block nicotinic receptors in autonomic ganglia. Arrows indicate drug injection.



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  		Fig. 3.   Comparison of renal nerve (A), mean blood pressure (B), and heart rate (C) responses to intravenous injection of 32 nmol/kg of SP or GR-73632 in SHRs and WKYs treated with chlorisondamine (10.5 µmol/kg) to block nicotine receptors in autonomic ganglia. GR-73632 was administered at 30-min intervals. * Indicates significant difference (P <=  0.05, paired t-test) for the effect of GR-73632 from the response to SP in the corresponding strain. # Indicates significant difference (P <=  0.05, unpaired t-test) in the responses to SP or GR-73632 in WKYs from the response in SHRs.



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  		Fig. 4.   Dose-response graphs for effect of GR-73632 injected intravenously on renal nerve firing (A), mean blood pressure (B), and heart rate (C) in SHRs and WKYs treated with chlorisondamine (10.5 µmol/kg) to block nicotine receptors in autonomic ganglia. Doses of peptides were administered at 30- to 45-min intervals. Symbols indicate means, and vertical lines indicate the SE. The small magnitude of the renal nerve [F(4,20) = 9.622 (P < 0.002)] and heart rate [F(4,30) = 26.4 (P < 0.0001)] increases were different from baseline values (repeated-measures ANOVA). Other responses to GR-73632 were also significant, P <=  0.05. * Indicates significant difference from the response to the same dose in WKYs (3-factor ANOVA, P <=  0.05).

Blockade of NK1 receptors with 200 nmol/kg of GR-82334 prevented the increase in renal nerve firing after injection of 32 nmol/kg GR-73632 in SHRs but only reduced the response to 32 nmol/kg of SP by 52 ± 9% (Fig. 5A). Increasing the concentration of GR-82334 to 600 nmol/kg (n = 4) did not cause a greater block of the response to SP. This dose of GR-82334 decreased the response to SP by 55 ± 13%. Antagonism of the responses to GR-73632 and SP was maximal within 10 min after administration of GR-82334 and persisted for >2 h. GR-82334 did not affect the baseline level of renal nerve firing.


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  		Fig. 5.   Renal nerve (A), mean blood pressure (B), and heart rate (C) responses to intravenous injection of SP (n = 8) and the neurokinin-1 (NK1) receptor agonist GR-73632 (n = 7) before and after selective blockade of NK1 receptors by GR-82334 in SHRs. GR-73632 was administered 30 min before and 5 min after administration of GR-82334. Bars represent the mean, and vertical lines show the SE. * Indicates a significant block of the responses to SP or GR-73632 by GR-82334 (P <=  0.05, paired t-test). # Indicates significant difference between the control responses to GR-73632 and SP (P <=  0.05, unpaired t-test).

Blood pressure responses. Intravenous injection of GR-73632 and SP produced pressor responses in SHRs (Fig. 1). The pressor responses were preceded or followed by a depressor response in six of the eight SHRs for each agent. The magnitude of the pressor response to GR-73632 was significantly less than the response to SP (Fig. 3B). In contrast to their effect in SHRs, GR-73632 and SP lowered blood pressure in WKYs (Figs. 2 and 3B). Figure 4B provides a dose-response comparison of the responses to GR-73632 in SHRs and WKYs. The threshold dose for the pressor response to GR-73632 was 10 nmol/kg. The pressor response to 32 nmol/kg of GR-73632 did not occur in rats pretreated with reserpine (5 mg/kg). Instead, GR-73632 decreased blood pressure by 18 ± 4 mmHg (data not shown). Mean blood pressure was 82 ± 3 mmHg (n = 4) in SHRs treated with reserpine and 82 ± 3 in a separate group (n = 9) of control SHRs.

The NK1 receptor blocking agent GR-82334 (200 nmol/kg iv) eliminated the pressor response to 32 nmol/kg of GR-73632 in SHRs and a small depressor response (-6 ± 2 mmHg) occurred in its place (Fig. 5B). GR-82334 also blocked the pressor response to SP (Fig. 5B). Blockade of responses to SP and GR-73632 was maximal 10 min after administration of GR-82334 and persisted for >2 h. GR-82334 (200-600 nmol/kg) did not affect resting blood pressure.

Heart rate responses. Intravenous injection of GR-73632 caused an increase in heart rate in SHRs similar to the increase caused by SP but of smaller magnitude (Figs. 1 and 3C). SP also increased heart rate in WKYs, whereas GR-73632 had almost no effect on rate in this strain of normotensive rats (Figs. 2 and 3C). Figure 4C provides a dose-response comparison of the chronotropic response to GR-73632 in SHRs and WKYs. The threshold for the tachycardic responses to GR-73632 and SP in SHRs was 3.2 nmol/kg. Although the heart rate response to GR-73632 was minimal in WKYs, the increase was different from baseline [F(4,30) = 26.4 (P < 0.0001)]. The tachycardic response to 32 nmol/kg of GR-73632 was prevented in rats pretreated with reserpine (5 mg/kg). Baseline heart rate was 156 ± 7 beats/min (n = 4) in SHRs treated with reserpine and 156 ± 7 beats/min in a separate group (n = 9) of control SHRs.

The NK1 receptor blocking agent GR-82334 (200 nmol/kg iv) inhibited the tachycardic response to 32 nmol/kg of GR-73632 in SHRs but only reduced the response to SP by 35 ± 10% (Fig. 5C). Attenuation of the tachycardic response to SP was not significantly greater when a higher dose of GR-82334 was used (600 nmol/kg; 55 ± 13% decrease, n = 4; P = 0.2598). Blockade of the responses was maximal within 10 min after administration of GR-82334 and persisted for >2 h. GR-82334 did not affect baseline heart rate.

Receptor Autoradiography

Binding sites for 125I-BHSP were detected throughout the superior cervical ganglion (Fig. 6). Nonspecific binding was 36% of total binding for WKYs and 23% for SHRs (0.324 ± 0.35 vs. 0.345 ± 0.024 fmol 125I-BHSP bound/mg; unpaired t-test, P = 0.647; n = 6). Specific binding of 125I-BHSP was approximately threefold higher in ganglia from SHRs compared with WKYs (1.564 ± 0.202 vs. 0.571 ± 0.054 fmol 125I-BHSP bound/mg; unpaired t-test, P = 0.001; n = 6).


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  		Fig. 6.   Photographs demonstrating the presence of specific 125I-labeled Bolton-Hunter SP (125I-BHSP) binding sites in a superior cervical ganglion from SHRs. A and B: photos of film autoradiograms for total (A) and nonspecific (B) binding. C and D: photomicrographs of an adjacent section that was stained with hematoxylin and eosin. Scale bar = 1 mm in A-C and 200 µm in D.

NK1 receptor RNA Expression

Amplification of NK1 receptor and GAPDH RNAs produced single products of the expected sizes from rat brain cDNA (Fig. 7, lane 2). The identity of each amplifier was confirmed by purification, cloning, and sequencing of the GAPDH and NK1 receptor bands (Fig. 7, lane 2). Both the NK1 receptor and the GAPDH products contained the expected internal sequences. Reactions that did not contain template DNA did not produce amplified product (Fig. 7, lane 3). Also, PCR analysis of RT(-) reactions did not produce visible amplified DNAs. Amplification of GAPDH messages from the superior cervical ganglion of SHRs and WKYs produced approximately the same amount of product in each animal, indicating that similar amounts of RNA were used in each cDNA synthesis reaction (Fig. 7, top, lanes 4-11). In contrast, the amount of NK1 receptor-specific product was consistently lower in the WKYs compared with the SHRs (Fig. 7, bottom, lanes 4-11). The NK1 receptor mRNA level was approximately threefold higher in ganglia of SHRs compared with WKYs (relative optical density of 0.603 ± 0.008 vs. 0.250 ± 0.036; unpaired t-test, P < 0.05; n = 4).


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  		Fig. 7.   RT-PCR analysis of NK1 receptor mRNA expression in SHR and WKY superior cervical ganglion. RT(+) cDNA reactions were PCR amplified and electrophoresed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific amplification reaction products were loaded in the top set of lanes; the NK1 receptor-specific amplification products were loaded in the bottom set. Lane 1 contains a 100-bp molecular weight ladder (Promega). Lane 2 contains PCR amplified rat brain (+) control cDNA. Lane 3 contains an identical PCR reaction in which double-distilled (dd) water was substituted for template DNA. Lanes 4-7 contain PCR amplified cDNA from the superior cervical ganglion of 4 SHRs. Lanes 8-11 contain amplified superior cervical ganglion cDNA from 4 WKYs. The positions of the GAPDH (510 bp) and NK1 receptor (537 bp) amplified products are indicated to the right. The sizes of the pertinent molecular weight marker DNAs are indicated to the left.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stimulation of NK1 receptors on sympathetic ganglia by GR-73632 caused a prominent increase in blood pressure, renal nerve firing, and heart rate in SHRs. In contrast, GR-73632 lowered blood pressure and caused only slight increases in renal nerve firing and heart rate in WKYs. These effects were similar to those caused by SP but of smaller magnitude. Autoradiographic studies showed that NK1 receptor density is higher in superior cervical ganglia of SHRs compared with WKYs. Results from RT-PCR experiments suggest that the increased density of NK1 receptors in SHRs may be due to increased transcription of the NK1 receptor gene. These observations support the conclusion that the greater effects of SP to increase renal nerve firing, blood pressure, and heart rate in SHRs compared with WKYs can be attributed to an upregulation of ganglionic NK1 receptors in this hypertensive strain.

We previously showed that SP increases renal nerve firing, blood pressure, and heart rate by stimulation of sympathetic ganglia (12, 13). Pretreatment with reserpine to deplete norepinephrine from sympathetic nerves eliminated pressor and tachycardic responses to GR-73632 in the present study without affecting renal nerve responses. Accordingly, the NK1 agonist likewise evoked these responses by activating efferent neurons in sympathetic ganglia.

The present data show that the pressor response to SP in SHRs is mediated by NK1 receptors because the NK1 receptor agonist GR-73632 mimicked the response to SP and the NK1 receptor antagonist GR-82334 (9) totally blocked the response. The receptors mediating the renal nerve and heart rate responses are less clear. NK1 receptors appear to be involved, because GR-73632 mimicked the renal nerve and heart rate responses to the ganglion action of SP and GR-82334 partially blocked these responses. The observation that attenuation of renal nerve and heart rate responses was not greater when the dose of GR-82334 was increased from 200 to 600 nmol/kg suggests that a complete block of NK1 receptors was obtained with these doses. These doses were selected because in vivo responses to NK1 receptor agonists have been shown to be blocked by 200 nmol/kg of GR-82334 (30), and 200 nmol/kg blocked responses to GR-73632 in this study. Other studies have shown that NK1 and NK2 receptor agonists accelerate heart rate by activation of neurokinin receptors on sympathetic ganglia of the rat (2). There is also evidence that NK3 receptors mediate the response of postganglionic sympathetic neurons to SP in the rat (35) and guinea pig (35, 37). Our unpublished observations indicate that GR-64349, an NK2 receptor agonist, and senktide, an NK3 receptor agonist, have effects on renal nerve firing and heart rate similar to those caused by SP or GR-73632 but of smaller magnitude (14, 15). These findings raise the possibility that all three neurokinin receptors are present on cells of origin of postganglionic renal and cardiac sympathetic nerves and that activation of NK2 and NK3 receptors accounts for the renal nerve and tachycardic responses to SP that are not blocked by GR-82334. In the dorsal nucleus of the vagus and the nucleus of the solitary tract, GR-82334 blocked depolarizations of neurons by GR-73632 but only reduced responses to SP (27). GR-82334 similarly blocked field depolarization of the rat superior cervical ganglia by GR-73632 but only reduced the depolarization caused by SP (16). These findings raise the possibility of multiple sites for action of agonists and antagonists on NK1 receptors or that the ganglion receptor mediating these responses is an atypical NK1 receptor (27).

At equimolar doses, the pressor response evoked by GR-73632 was less than the response to SP in SHRs. The reason for this difference is not clear, because several studies have shown that GR-73632 has a greater potency than SP on NK1 receptors and that both compounds are competitive full agonists and have equal maximum responses (10, 11).

Autoradiographic studies reported here and from other laboratories have shown specific binding of 125I-BHSP in rat and guinea pig superior cervical ganglia (19, 24, 28). These observations show that NK1 receptors are present in sympathetic ganglia of rats, because 125I-BHSP preferentially binds to NK1 receptors at the concentrations used (29, 33). In the present study we further showed that the binding of 125I-BHSP was to NK1 receptors by using a selective NK1 receptor agonist to define nonspecific binding. Receptor autoradiography using 125I-BHSP was performed to determine if NK1 receptors are upregulated in the superior cervical ganglia of SHRs. There were approximately threefold more NK1 receptor sites in ganglia of SHRs than in those of WKYs. This suggests that the enhanced ganglionic response in SHRs is due to a greater number of NK1 receptors in sympathetic ganglia of SHRs compared with WKYs. Our RT-PCR studies using NK1 receptor-specific primers show that NK1 receptor mRNA is expressed in the rat superior cervical ganglia. RT-PCR analysis was performed on total superior cervical mRNA to determine if NK1 receptor expression was upregulated in SHRs at the transcriptional level. The superior cervical ganglia of SHRs contained approximately threefold more NK1 receptor mRNA than did those from WKYs. Taken together, these data indicate that the superior cervical ganglia from SHRs express more NK1 receptors than do those from WKYs and that this difference is primarily due to increased accumulation of NK1 receptor transcripts in ganglia of SHRs.

There are several possible explanations for greater NK1 mRNA expression in SHRs compared with WKYs. One possibility is that the genes responsible for the increased receptor expression are cosegregated with the genes for hypertension and contribute to hypertension by enhancing postganglionic sympathetic nerve activity. This scenario is consistent with the postulate advanced by Folkow (6) that hypertension occurs as a result of the enhanced sympathetic nerve activity, which is characteristic of clinical hypertension and this animal model of hypertension (6, 26). It is also possible that the genes responsible for the increase in NK1 receptor expression are cosegregated with the genes responsible for hypertension in SHRs but are unrelated to hypertension. Another possibility is that increased transcription of the receptor gene in SHRs is induced by unidentified factors associated with hypertension. This scenario raises the interesting possibility that factors associated with hypertension may induce receptor gene expression and is consistent with the observation that cytokines can enhance SP expression in superior cervical ganglion neurons in vitro (7).

Perspectives

Although many factors are involved in the development of hypertension, a primary causative factor is increased sympathetic nerve activity. This effect is prominent in both human essential hypertension and in SHRs. In 1975, Folkow (6) postulated that increased sympathetic nerve activity was responsible for the decrease in lumen-to-wall ratio and increased vascular resistance that is characteristic of hypertension (5, 26). The cause of the increase in sympathetic nerve activity is not fully understood. Because SP is localized to sensory motor neurons (3, 8), sensory nerve stimulation may cause the release of endogenous SP to activate sympathetic neurons and increase blood pressure by an axon reflex mechanism. This mechanism would be present in both normotensive and hypertensive rats and function as a local modulator of sympathetic nerve activity. Because the SP innervation (8) and NK1 receptor number are increased in ganglia of hypertensive rats, the effects of SP would be greater in this strain and may be an important factor leading to the increased nerve activity.


    ACKNOWLEDGEMENTS

Technical assistance was provided by Rebecca Doman and Sharon Stover.


    FOOTNOTES

This project was supported by National Heart, Lung, and Blood Institute Grant HL-54268.

Address for reprint requests and other correspondence: J. C. Hancock, Dept. of Pharmacology, College of Medicine, East Tennessee State Univ., Johnson City, TN 37614-0577 (E-mail: hancock{at}etsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 October 1999; accepted in final form 9 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 279(5):R1685-R1694
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