Published online before print May 10, 2007, doi: 10.1161/CIRCRESAHA.107.153494
© 2007 American Heart Association, Inc.
Integrative Physiology |
Substance P Targets Sympathetic Control Neurons in the Paraventricular Nucleus
From the Veterinary Sciences Faculty, Brownlow Hill, University of Liverpool, UK.
Correspondence to Dr Richard Barrett-Jolley, University of Liverpool, Department of Preclinical Veterinary Sciences, Faculty of Veterinary Sciences, Liverpool L69 7ZJ, United Kingdom. E-mail RBJ{at}LIV.AC.UK
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Abstract |
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The paraventricular nucleus (PVN) contains spinally-projecting neurons implicated in fine-tuning the cardiovascular system. In vivo activity of "presympathetic" parvocellular neurons is suppressed by tonic inhibition from GABA-ergic inputs, inhibition of which increases sympathetic pressor activity and heart rate. Targeting of this specific neuronal population could potentially limit elevations of heart rate and blood pressure associated with disease. Here we show, for the first time, that "presympathetic" PVN neurons are disinhibited by the neuropeptide substance P (SP) acting via tachykinin NK1 receptor inhibition of GABAA currents. Application of SP to the paraventricular nucleus of rats increases heart rate and blood pressure. In in vitro brain slice experiments, in the presence of GABA, 1µmol/L SP increased action current frequency by a factor of 2.7±0.6 (n=5, P
0.05, ANOVA). Furthermore, 1µmol/L SP inhibited GABAA currents by 70±8% (n=8, P0.005 paired t test). These effects were abolished by NK1 antagonists, but not NK2 and NK3 antagonists. GABAA inhibition was not reproduced by NK2 or NK3 agonists. The inhibition of parvocellular GABAA currents by SP was also abolished by a protein kinase C (PKC) inhibitor peptide and mimicked by application of phorbol-12-myristate-13-acetate (PMA), implicating a PKC-dependent mechanism. Single-channel analysis indicates that SP acts through reduction of channel mean open-time (cmot): GABAA cmot being reduced by approximately 60% by SP (P0.05 ANOVA, Bonferroni). These data suggest that tachykinins mediate their pressor activity by increasing the excitability of spinally-projecting neurons and identifies NK1 receptors as potential targets for therapeutic modulation of the cardiovascular system.
Key Words: cardiovascular control • GABA • substance P
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Introduction |
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The parvocellular subdivision of the hypothalamic paraventricular nucleus (PVN) contains both hypothalamic-pituitary-adrenal axis control neurons, and spinally-projecting cardiovascular control neurons.1–6 Estimates suggest that up to 2000 neurons project directly from the PVN to the intermediolateralis (IML) of the spinal cord.7–9 These projecting parvocellular neurons have been implicated in a number of facets of cardiovascular control. For example, circadian regulation of blood pressure,10 blood-volume regulation,7,8 and the cardiovascular response to psychological stress.11 Furthermore, elevated sympathetic activity is associated with congestive heart failure observed in humans and animals,12–14 and rat models suggest that this is associated with a diminished GABA sensitivity of PVN neurons.13,15,16 As such, spinally-projecting PVN neurons are prime targets for therapeutic intervention with the cardiovascular system.
Although there have been, to date, few in vitro studies detailing cellular mechanisms potentially exploitable for therapeutic control of cardiovascular PVN neurons, there have been a number of, largely in vivo, studies investigating their basic control and physiological activity. Early in vivo electrophysiological studies on cats confirmed an anticipated monosynaptic connection between the PVN and spinal sympathetic motor area (the IML).17 Furthermore, stimulation (electrical or glutamate) of the PVN was shown to give rise to an extremely rapid rise of blood pressure and renal sympathetic outflow in conscious rats.18 Additional studies show that in vivo, the PVN receives tonic inhibition from GABA-ergic inputs and this maintains their activity at relatively low levels,19–21 despite the fact that these neurons also receive excitatory glutaminergic inputs.18 Strikingly, excitation of this nucleus by glutamate, or by blockade of GABAA receptors with the antagonist bicuculline, increases blood pressure and heart rate.18,22
Other physiological inputs to "presympathetic" parvocellular neurons have been, until now, unknown. Potentially, pharmacological inhibition of this neuronal population could limit sympathetically induced elevations of heart rate and blood pressure seen during heart failure or psychological stress.
Increasing evidence from in vivo experiments also suggest that the substance P (SP) family of neuropeptide neurotransmitters (tachykinins) play a major role in cardiovascular control and the expression of a cardiovascular stress response.23 For example, SP is found at high levels within the PVN.24 Studies also show that central tachykinins increase efferent sympathetic nerve activity,25 and injection of tachykinin agonists into the hypothalamus leads to rapid elevation of heart rate and blood pressure.23,26 Furthermore, activation of PVN tachykinin receptors increase blood pressure and heart rate.27,28 Crucially, central administration of tachykinin antagonists reduce the elevation of blood pressure and heart rate resulting from psychological stress of conscious rats.29 We therefore sought to investigate whether tachykinins activated PVN spinally-projecting neurons, and if so, by what receptor and mechanism.
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Materials and Methods |
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All procedures were performed humanely in accordance with the UK Home Office Regulations.
Physiological Experiments
Male Wistar rats (University of Liverpool Biomedical Service Unit) 200 to 300 g were anesthetized with urethane (1.3 to 1.5g/kg ip), temperature was monitored by thermocouple and infrared thermometer (Infrascan). The head was placed in a stereotactic frame and a small craniotomy made near to bregma to allow micropipette insertion. The tip of the micropipette was located in the PVN following the coordinates of Paxinos and Watson30 (1.3 mm posterior, and 0.4 mm lateral to bregma and 7.8 mm below the dura). Post-hoc confirmation of injection site was made either by inclusion of FITC-SP, or by following SP injection by injection of triarylmethane dye. SP, vehicle (ACSF), or bicuculline were applied unilaterally via a Hamilton syringe (100 nL of 5 mmol/L). Order of application was randomized. Blood pressure (MAP) and heart rate (HR) were measured via femoral artery cannulae. To measure renal sympathetic nerve activity (RSNA), the left kidney was exposed retroperitoneally and the nerve to the kidney placed on bipolar silver wire. The area was coated with silicone grease and the wire connected to a Neurolog amplifier. Low and high-pass filtering was 1KHz and 100Hz, respectively. Data acquired and analyzed by a calibrated PC interface (CED). Injection accuracy was assessed by post-hoc microscopy.
Brain Slice Preparation
Spinally-projecting parvocellular neurons can be readily identified in thin fixed brain slices from aged rats, but patch clamp recording from relatively thicker, living slices of aged rats is not practical. Therefore we used Wistar P14-30 rats in which visualized patch clamp is more feasible. Studies of age related receptor expression in the diencephalon show that most GABAA subunit expression occurs between E6 and P10. There are smaller changes in expression of 
1 to 2/3 in P10-P16.31 Furthermore, in vivo studies of tachykinin influence on cardiovascular control used aged rats.32 Therefore, available evidence suggests that our findings would be similar in experiments on older animals. Brain slice (250 µm) preparation and identification of spinally-projecting neurons by the retrograde-labeling technique has been described previously.33 Individual parvocellular neurons were identified under near-infrared DIC, with retrogradely labeled neurons being visualized through a G2A filter block (Ex510-560/DM575/BA590 nm) on a Nikon E600FN microscope.
Electrophysiology
Our methods for whole-cell recording, action-current frequency (cell-attached-patch), and single-channel (cell-attached-patch) experiments of PVN slices have all been described recently.34 Slices were superfused with a modified artificial CSF (ACSF) and pipettes filled with an appropriate physiological saline (supplemental Table I, available online at http:/?circres.ahajournals.org). We used an Axopatch 200B and acquired data at 10 kHz with an Axon DigiData 1200, analogue prefiltering appropriately.
Analysis: Whole Cell
Whole-cell current-voltage data were fitted with the Goldman-Huxley-Katz (GHK) current equation: equation
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Where Vm is the command potential, pCl is the chloride permeability. R, T, z, and F have their usual meanings.
Single-Channel Analysis
Membrane potential for cell-attach-patch mode was calculated from: equation
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Where Vm is the potential across the patch, RMP is the resting membrane potential, and Hp is the holding potential. Predicted reversal potential (Vrev) was calculated by combining Equation 2 with the Nernst equation: equation
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Where RMP was measured for parvocellular neurons by ourselves as a part of this study (–62±1mV, n=4), [Cl]out is the extracellular chloride concentration, and [Cl]in is the estimated intracellular chloride concentration. Kinetic analysis followed previous methods.35 In the discussion, we assume whole-cell ion channel current to be comparable to the product of number of channels, open probability, and unitary amplitude. For any given holding potential (Hp), unitary current is proportional to unitary conductance. In this study we calculate mean open times, rather than Po specifically.
Software
Software for acquisition and analysis were non-commercial. Details (including suppliers) available on our website (http://pcwww.liv.ac.uk/
rbj/RBJ/software.htm).
Statistics
Statistics were performed with StatsDirect. We used Student paired t tests for single comparisons between means, or, for multiple comparisons, ANOVA with Bonferroni post-hoc analysis as appropriate (stated in the text). Means are given ±SEM, and n is the number of experiments.
Chemicals
FITC-SP was obtained from Molecular Probes. Drugs supplied by Sigma-Aldrich, UK, except PMA (Calbiochem), okadaic acid (Calbiochem), SB222200, and MEN10376 (Tocris). In terms of antagonist selectivity, spantide shows >10x selectivity for NK1 over NK2/3,36 MEN10376 shows >20 fold selectivity for NK2 over NK1/3,37 and SB222200 shows >1000 fold selectivity for NK3 over NK1/2.38
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Results |
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Intra-PVN SP Increases Blood Pressure and Heart Rate
Unilateral injection of SP/FITC-SP into the PVN resulted in a 10 mm Hg rise in MAP (from 77±6 to 87±4mmHg, n=7, P<0.05, paired t test) and 14 BPM rise in HR (from 462±12 to 486±9BPM, n=7, P<0.05, paired t test; Figure 1). Post-hoc epi-fluorescence analysis of the PVN shows that a proportion of parvocellular neurons took up FITC-SP (indicative of SP-receptor activity39).
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Intra-PVN SP Increases RSNA
One possible way in which SP could increase HR and MAP would be by increasing sympathetic outflow. To investigate this, we applied SP to the PVN while recording RSNA (Figure 2A). This resulted in a significant increase in RSNA (Figure 2B).
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SP Increases Activity of GABA Inhibited Parvocellular Neurons
To investigate the mechanism of action of SP, we switched to in vitro brain slide recording. We began by measuring the effect of SP on spontaneous action-current frequency. In the presence of ACSF alone, SP had little effect (Figure 3A and 3E). Importantly, however, SP significantly increased frequency against a background of submaximal GABA inhibition. This activation was abolished by the highly selective NK1 antagonist L703606 (Figure 3D and 3H).
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SP Inhibits GABAA Whole-Cell Currents in Parvocellular Neurons
Pressure application of 300µmol/L GABA evokes large inward currents in these neurons (0.1 to 5nA, –60mV). These are inhibited by bath applied SP. Figure 4A illustrates such a reversible inhibition in a retrogradely labeled spinally-projecting parvocellular neuron. On average currents were reduced to 30±8% (n=8) of the control current (P0.05 paired t test). To confirm the identity of the GABA-evoked currents we constructed current-voltage curves (Figure 4B). These reversed close to the calculated chloride equilibrium potential of 35mV (Figure 4D) and were powerfully inhibited by the selective GABAA receptor antagonist bicuculline (10µmol/L, Figure 4C and 4E).
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SP-GABAA Inhibition Is Mediated by the NK1 Receptor
Selective NK2 (NKA(4–10)) and NK3 (senktide) agonists and the endogenous NK3 agonist NKB (all 1µmol/L) failed to inhibit GABA currents (Figure 5A and 5C).
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Next, we measured the inhibition of GABA responses by SP in the presence of selective tachykinin antagonists. Antagonists were added to the superfusing ACSF 10 minutes before the addition of SP. Spantide (1µmol/L), a selective NK1 antagonist, prevented the SP activity (Figure 5Bi and 5D), whereas neither selective NK2 (MEN10376, 1µmol/L) nor NK3 (SB222200, 1µmol/L) antagonists prevented the SP effect (Figure 5Bii,iii, and 5D).
SP-GABAA Inhibition Is PKC Dependent
The NK1 receptor is a classical 7-transmembrane (7-TM) domain G protein–coupled receptor (GPCR) and, although there are exceptions, typically couples to pertussis toxin insensitive G proteins such as Gq/11.40 The GABAA ß and 
-subunits are, in turn, targets for a number of protein kinases, such as PKC.41 Furthermore, the SP-GABAA receptor coupling in frog dorsal root ganglia (DRG)42 was mediated by PKC. We therefore investigated whether the PKC activator PMA mimicked the whole-cell effects of SP. Figure 6 (A and C) shows 100nmol/L PMA bath applied to retrogradely labeled parvocellular neurons significantly inhibits whole-cell GABA currents. Furthermore, when included in the patch pipette, a selective PKC inhibitor peptide (PKC,19–31 1µmol/L) prevents the effects of SP (Figure 6B and 6D).
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SP-GABAA Inhibition Involves Reduction of GABAA Channel Open Time
To further elucidate mechanisms by which SP reduces these whole-cell GABAA currents we used cell-attached-patch single-channel recording of parvocellular neurons in the presence and absence of 1µmol/L extracellular SP. To analyze whether SP alters GABAA channel conductance, we constructed all-point-amplitude histograms of GABAA channel events (Figure 7A through 7D). This was done at a range of potentials (with 3 GABA concentrations), with and without SP. Slope conductances were all in the range of 14 to 18pS (Figure 7E and 7F), and there was no difference between any of these, even in the presence of SP (Figure 7F; P>0.05, ANOVA, Bonferroni multi-comparison).
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As there is no change in GABAA channel conductance, but a striking reduction of whole-cell GABAA current, ion channel dogma states that there must be a change in either the number of active channels, or of channel kinetic properties. To analyze which of these mechanisms underlies SP inhibition of GABAA current, we undertook kinetic analysis of GABAA channel inhibition by SP, again in cell-attached-patch mode. We recorded channel activity at –100mV (Hp) with 10 µmol/L or 100 µmol/L GABA with and without 1µmol/L SP. Dwell times were fit as described in the methods (Figure 8A and 8B). Full kinetic analysis revealed that in the presence of 100 µmol/L GABA there was a significant decrease in burst length (from 63.0±20ms, n=3 to 29.4±9 ms n=6, P<0.05) and corrected mean open time (from 3.7±0.3 ms, n=3 to 1.6±0.2 ms, n=6, P<0.05; Figure 8D), compared with those in the presence of 1 µmol/L SP. None of the other parameters tested were significantly altered by SP (Figure 8C and 8D; ANOVA, Bonferroni multi-comparison analysis for all kinetic parameters).
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Discussion |
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In this work we show tachykinins to activate parvocellular neurons in the rat PVN. We show this to be mediated by an interaction between GABAA and tachykinin NK1 receptors via a PKC mechanism. Kinetic analysis shows this to be associated with a reduction of single-channel cmot, but not conductance.
"Presympathetic" Parvocellular Neurons
The PVN of the hypothalamus coordinates a number of hormonal and autonomic physiological responses.5,6 Indeed, Loewy5 referred to the PVN as being the "autonomic master controller". In terms of cardiovascular control, it appears that much of the sympathetic drive is mediated by direct projection of parvocellular vasopressinergic neurons to the IML of the spinal cord.43–45 Injection of retrograde tracer into the IML has been shown in several studies, to label the spinally-projecting "presympathetic" vasopressinergic neurons, emanating mainly from the ventral medial and lateral parvocellular subdivisions, but also to a lesser degree from the dorsal parvocellular subdivision.44,46,47 Stimulation of the PVN with either glutamate or the GABAA antagonist bicuculline increases both heart rate and blood pressure,18,19,22 and this effect can be reduced by the injection of a vasopressin antagonist into the spinal cord.45 Furthermore, elevated activity of PVN "presympathetic " neurons has been linked to heart failure in rat models.13,15 Modulation of these parvocellular neurons at the level of the PVN is therefore of potentially profound importance to the fine control of the cardiovascular system. In vivo, these neurons are tonically inhibited by GABA.19–21 We show here that these neurons are activated by SP and, interestingly, this is GABA dependent.
GABAA Receptors in Parvocellular Neurons
Despite the great importance of parvocellular "presympathetic" neurons, there have been relatively few studies directly investigating their pharmacological control. A number of studies have demonstrated the presence of GABAA receptors in the PVN.22,35,48–52 More recently, studies by others have shown spinally-projecting parvocellular neurons to also be inhibited by GABA,10,53 as predicted from earlier in vivo work.22 In this study we found retrogradely labeled spinally-projecting neurons to elicit inward currents in response to pressure application of 300 µmol/L GABA. GABAA receptors are, however, ubiquitous throughout the central nervous system, and before these parvocellular neurons could be targeted in potential cardiovascular therapies, one would need to identify a more specific pharmacological profile. In previous studies, researchers have investigated whether either orexin54 or angiotensin II55 may be direct pharmacological regulators of spinally-projecting parvocellular neurons. Although both neuropeptides have receptors expressed in the PVN56–58 and, indeed, angiotensin II directly modulates "medulla-projecting" PVN neurons,59 neither modulates spinally-projecting neurons themselves.54,55 In this study, however, we show that SP acts within the PVN to increase HR, MAP, and RSNA, and we present first direct evidence that spinally-projecting PVN neurons are modulated SP.
Inhibition of GABAA Ion Channels by SP
The tachykinins are a family of neuropeptides (SP; neurokinin A, NKA; and neurokinin B, NKB), members of which have been implicated in central cardiovascular control.23 Several studies have reported high levels of tachykinin receptor expression within the PVN.60–62 All 3 of the principal mammalian tachykinin receptors (NK1, NK2, and NK3) are 7-TM GPCRs, and they act through a range of intracellular pathways, generally via pertussis toxin independent G proteins.40,63 In particular, NK1 receptors activate PKC.40 This is particularly interesting, because PKC dependent phosphorylation of recombinant GABAA channel subunits, in turn, results in reduction of GABAA current in cultured cells.64 We knew of no previous reports of SP acting by this mechanism in brain slices, however a similar mechanism was proposed for the SP inhibition of GABAA current in frog DRG neurons.42 We therefore sought to investigate whether tachykinins interacted with GABAA receptors of PVN parvocellular neurons. We find that SP does indeed powerfully inhibit GABAA receptors. This effect was prevented by 10 minutes pretreatment with the SP antagonist spantide. Although both SP and spantide are relatively selective for tachykinin NK1 receptors, we also performed experiments with additional highly selective tachykinin receptor ligands. NK2 and NK3 receptor agonists (NKA4–10 and senktide, respectively) did not mimic SP, furthermore, selective NK2 and NK3 antagonists (MEN10376 and SB222200, respectively) failed to inhibit SP action. Together these data strongly suggest that the tachykinin receptor mediating the inhibition of GABAA receptors in parvocellular neurons is of the NK1 subtype.
Mechanism of Action of SP
We approached this question from two perspectives. (1) What is the intracellular signaling pathway? (2) What are the changes in individual single channel properties, in the presence of SP?
- (1) The NK1 receptor frequently couples to PKC via Gq (reviewed40), and PKC phosphorylation of GABAA channel ß-subunits (conserved serine-409 and 410 residues) reduces GABAA whole-cell current in cultured cortical neurons.41 In this study, we found that the PKC inhibitor peptide (PKC(19–31)) prevented the SP inhibition of parvocellular GABAA responses. Furthermore, the SP inhibition was mimicked by PMA (a diacylglycerol mimetic), lending further support to the hypothesis that SP inhibits GABAA channels via PKC.
- (2) Reduction of whole-cell current generally results from either a reduction of the unitary current (i), channel membrane expression (n), or the open time/probability (cmot/Po). Recent studies have also shown that in some preparations, GABAA channels have an increasing unitary conductance with increasing concentrations of GABA65 and the archetypal GABAA antagonist bicuculline decreases GABAA conductance.65 Here, we found no change of GABAA channel conductance in the presence of SP. This implies that either the number of active GABAA channels (n) or the open time is reduced by SP. In fact both of these mechanisms have been shown to mediate GABAA inhibition via PKC phosphorylation previously.41,64 In this study, we observed a decrease in corrected mean open time (cmot) in the presence of SP. This decrease in cmot would be large enough to contribute to a large proportion of the inhibition seen in whole-cell experiments. These data also argue against a mechanism solely involving an increase in receptor desensitization, such as that observed by Si et al (SP inhibition of GABA responses in DRG),66 or indeed the PKC driven reduction of the GABAA receptor surface expression.64 Rather, the mechanism appears to involve an allosteric reduction in channel activity, which would be consistent with the inhibitory pathway described by Brandon et al41 and involving phosphorylation of the conserved serine-409 and 410 residues of the ß-subunit. The fact that SP inhibition of GABAA current is maintained in cell-attached experiments also demonstrates, unequivocally, that the effect of SP is directly on the target neuron, rather than by causing the release of some intermediary factor from a neighboring neuron. This is because SP exposure is restricted to the area of membrane directly underneath the patch pipette tip.
- (2) Reduction of whole-cell current generally results from either a reduction of the unitary current (i), channel membrane expression (n), or the open time/probability (cmot/Po). Recent studies have also shown that in some preparations, GABAA channels have an increasing unitary conductance with increasing concentrations of GABA65 and the archetypal GABAA antagonist bicuculline decreases GABAA conductance.65 Here, we found no change of GABAA channel conductance in the presence of SP. This implies that either the number of active GABAA channels (n) or the open time is reduced by SP. In fact both of these mechanisms have been shown to mediate GABAA inhibition via PKC phosphorylation previously.41,64 In this study, we observed a decrease in corrected mean open time (cmot) in the presence of SP. This decrease in cmot would be large enough to contribute to a large proportion of the inhibition seen in whole-cell experiments. These data also argue against a mechanism solely involving an increase in receptor desensitization, such as that observed by Si et al (SP inhibition of GABA responses in DRG),66 or indeed the PKC driven reduction of the GABAA receptor surface expression.64 Rather, the mechanism appears to involve an allosteric reduction in channel activity, which would be consistent with the inhibitory pathway described by Brandon et al41 and involving phosphorylation of the conserved serine-409 and 410 residues of the ß-subunit. The fact that SP inhibition of GABAA current is maintained in cell-attached experiments also demonstrates, unequivocally, that the effect of SP is directly on the target neuron, rather than by causing the release of some intermediary factor from a neighboring neuron. This is because SP exposure is restricted to the area of membrane directly underneath the patch pipette tip.
Significance of SP-GABAA Interaction to Cardiovascular Control
Accumulating evidence shows that spinally-projecting parvocellular neurons exert a powerful modulatory influence on the cardiovascular system. These neurons may be involved with volume regulation,7 circadian regulation of the cardiovascular system,10 the stress response,11 or heart failure.13,15 The current work presents the SP NK1 receptor as a putative target for pharmacological control of the cardiovascular system.
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Acknowledgments |
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Source of Funding
This work was funded by the British Heart Foundation, UK.
Disclosures
R.B.-J. is PI on the BHF Research Grant.
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Footnotes |
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Original received December 21, 2005; resubmission received November 28, 2006; revised resubmission received April 4, 2007; accepted April 26, 2007.
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