Published online before print November 17, 2008, doi: 10.1161/HYPERTENSIONAHA.108.122341
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(Hypertension. 2009;53:97.)
© 2009 American Heart Association, Inc.
Original Articles |
Chronic Blockade of Phosphatidylinositol 3-Kinase in the Nucleus Tractus Solitarii Is Prohypertensive in the Spontaneously Hypertensive Rat
From the Department of Physiology and Pharmacology (J.Z., A.G., J.F.R.P.), Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol, United Kingdom; Department of Physiology and Functional Genomics (C.D.-F., M.K.R.), University of Florida, Gainesville; and the Department of Physiology (H.W.), University of Wakayama, Wakayama, Japan. Current address (J.Z.): Department of Integrative Physiology, Health Science Center, University of North Texas, Ft Worth.
Correspondence to Julian F.R. Paton, Department of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, United Kingdom. E-mail Julian.F.R.Paton{at}bris.ac.uk
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Abstract |
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Phosphatidylinositol 3-kinase (PI3K) within brain stem neurons has been implicated in hypertension in the spontaneously hypertensive rat (SHR). Previously, we demonstrated elevated expression of PI3K subunits in rostral ventrolateral medulla and paraventricular nucleus of SHRs compared with Wistar-Kyoto rats. Here, we considered expression levels of PI3K in the nucleus tractus solitarii, a pivotal region in reflex regulation of arterial pressure, and determined its functional role for arterial pressure homeostasis in SHRs and Wistar-Kyoto rats. We found elevated mRNA levels of p110β and p110
catalytic PI3K subunits in the nucleus tractus solitarii of adult (12 to 14 weeks old) SHRs relative to the age-matched Wistar-Kyoto rats (fold differences relative to β-actin: 1.7±0.2 versus 1.01±0.08 for p110β, n=6, P<0.05; 1.62±0.15 versus 1.02±0.1 for p110, n=6, P<0.05). After chronic blockade of PI3K signaling in the nucleus tractus solitarii by lentiviral-mediated expression of a mutant form of p85
, systolic pressure increased from 175±3 mm Hg to 191±6 mm Hg (P<0.01) in SHRs but not in Wistar-Kyoto rats. In addition, heart rate increased (from 331±6 to 342±6 bpm; P<0.05) and spontaneous baroreflex gain decreased (from 0.7±0.07 to 0.5±0.04 ms/mm Hg; P<0.001) in the SHRs. Thus, PI3K signaling in the nucleus tractus solitarii of SHR restrains arterial pressure in this animal model of neurogenic hypertension.
Key Words: hypertension • brain stem • NTS • PI3K • baroreflex control
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Introduction |
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The pathogenesis of essential hypertension is multifactorial. In a majority of hypertensive cases, the underlying causes remain unknown and are most probably a product of complex interactions between susceptibility genes and environmental factors influencing neural, humoral, cellular, and subcellular mechanisms.1–3 Both animal4–7 and human studies8–11 suggest that overactive sympathetic nerve activity participates in the pathogenesis of hypertension. In a rat model of genetic hypertension (the spontaneously hypertensive rat [SHR]), sympathetic nerve activity is significantly higher in comparison with its normotensive control, the Wistar-Kyoto (WKY) rat.6 Interestingly, this raised level of activity precedes the onset of hypertension in this animal model, supporting a causative role in the development of this pathology.11
However, the roles of brain regions and intracellular signaling pathways in the pathogenesis of chronic sympathetic overactivity and hypertension are not fully understood. Our previous studies have demonstrated an elevated phosphatidylinositol 3-kinase (PI3K) signaling in presympathetic brain regions of the SHR.12,13 We found that the mRNA levels of specific class I PI3K subunits (p85, p110, and p110) were elevated within the rostral ventrolateral medulla (RVLM) and paraventricular nucleus of the SHRs compared with the WKY rats, which was accompanied by increased PI3K activity in brain stem/hypothalamic neuronal cultures made from SHRs.13 In addition, acute PI3K inhibition within the RVLM decreased blood pressure (BP) in the anesthetized SHRs to levels similar to those of the WKY rats,14 confirming a functional role for PI3K signaling pathway in this brain region unique to the SHR.
In this study, we quantified the level of gene expression of specific class I PI3K subunits (p85, p85β, p110, p110β, p110, and p110
) in the nucleus tractus solitarii (NTS) of SHRs relative to WKY rats using real-time RT-PCR and assessed any functional role of PI3K signaling in this structure for chronic regulation of arterial pressure. The NTS is the principal termination site of baroreceptor afferents15,16 and, therefore, one of the key regulators of both baroreflex gain and the set point of arterial pressure.17–19 With accumulating evidence that the baroreceptor reflex plays a crucial role in the chronic regulation of arterial pressure,20 a reduction in its sensitivity may have long-lasting detrimental consequences for arterial pressure homeostasis. Here, we report differential expression of a subset of PI3K subunits in the NTS of SHRs versus WKY rats. Functionally, PI3K signaling in the NTS exerts a chronic restraining role on arterial pressure specific to the SHR.
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Methods |
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Procedures were carried out according to the United Kingdom Home Office Guidelines on Animals (Scientific Procedure) Act of 1986. The animal were housed individually, allowed normal rat chow and drinking water ad libitum, and kept on a 12-hour light/12-hour dark cycle.
Differential Gene Expression in the SHRs and WKY Rats
Extraction and purification of RNA were conducted from 13- to 16-week-old SHRs and WKY rats (n=6 per strain), as described previously.21–23 Transcript abundance of β-actin and class I PI3K subunits (p85, p85β, p110, p110β, p110, and p110) were measured using quantitative RT-PCR (see the data supplement at http://hyper.ahajournals.org for details of the primers and the real-time RT-PCR procedures performed). Real time RT-PCRs were carried out using a DNA Engine Opticon 2 system (MJ Research) and the QuantiTect SYBR Green RT-PCR kit (Qiagen), as described previously.19 Expression of target genes in each sample was assessed in relation to a housekeeping gene (β-actin) using the comparative (2–
CT) method.24 Fold differences against average values of WKY rats were calculated, as in Reference 19. Final products were confirmed to have correct sizes by gel electrophoresis.
Telemetric Recordings of Arterial Pressure
Male SHRs and WKY rats (13 to 16 weeks old; n=10 per strain) were anesthetized with ketamine (60 mg/kg) and medetomidine (250 µg/kg) intramuscularly. A radiotransmitter (TA11PAC40, Data Sciences International) was implanted to record arterial pressure (and from this, heart rate [HR] was derived) from the abdominal aorta, as described previously.25,26 Anesthesia was reversed with atipamezole (1 mg/kg). The rats were allowed to recover for 7 to 10 days before baseline telemetric measurements were taken. A full spectral analysis was performed on the BP signal to reveal potential mechanisms (see the data supplement for details and results of spectral analysis).
Cloning and Lentiviral Vector Production
DNp85 construct27 was cloned in a lentiviral (LV) vector driven by the human elongation factor 1 (EF1) promoter, as described previously28 (see the data supplement for details of our reasoning for the choice of the construct to reduce PI3K signaling). LV-expressing enhanced green fluorescent protein (LV-eGFP) driven by the same promoter was used as a control. The typical LV titers of the control LV-eGFP and LV-DNp85 vectors used were comparable, ranging from 1 to 2x1010 transducing units per milliliter. This titer range was shown to be efficient in central neuronal transduction.28 Confirmation of the dominant-negative action of LV-EF1-DNp85-IRES-eGFP (LV-DNp85) in vitro is described in the online supplement (see the data supplement).
LV Microinjections into NTS
After control recordings of arterial pressure were taken for 24 hours, animals were reanesthetized. Three 200- to 300-nL volumes of either LV-DNp85 or LV-eGFP were microinjected bilaterally into the NTS at separate sites spanning ±500 µm rostrocaudal to calamus scriptorius, 350 to 500 µm from the midline, and 500 to 600 µm ventral to the dorsal surface. The animals were allowed to recover for 1 week before further arterial pressure measurements were taken. The site of the microinjection, the extent of the LV transduction, and protein expression were all confirmed by posthoc immunohistochemistry (see the data supplement for details of the immunohistochemical procedures).
Data and Statistical Analysis
Group data were expressed as mean±SEM 2-way repeated-measures ANOVAs, and the posthoc (Bonferroni) test was used to allow multiple comparisons of cardiovascular variables across time and between different groups. Paired/unpaired Student t test was used for further comparisons between 2 groups where applicable, with P<0.05 considered significant.
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Quantitative Comparison of Transcript Abundances of Different PI3K Subunits in the NTS of Adult SHRs and WKY Rats
Using 40 cycles of real-time RT-PCR, we observed significantly higher transcript levels of both p110β and p110
catalytic subunits of PI3K in the NTS of adult SHRs compared with the WKY rats (Figure 1). Specifically, the level of p110β mRNA transcript was significantly elevated in the SHRs compared with age-matched (adult) WKY rats (1.7±0.2 versus 1.01±0.08, respectively, normalized to β-actin; n=6 per strain; P<0.05; Figure 1). Similarly, in SHRs, the level of p110 mRNA transcript was significantly higher (1.62±0.15 versus 1.02±0.1, respectively; n=6 per strain; P<0.05; Figure 1). In contrast, we failed to observe any differences in the NTS transcript abundances of p85, p85β, p110, and p110 subunits of PI3K in the adult SHRs versus WKY rats relative to β-actin transcript levels (see Table S2 in the online data supplement). We confirmed the RNA quality by constructing melting curves for β-actin in the samples from both SHRs and WKY rats (see Figure S1).
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Effects of DNp85 Expression in the NTS
Arterial BP
Considering that p110β and p110 catalytic subunits of PI3K were upregulated in the NTS of SHRs (compared with WKY rats), we tested the functional impact of chronic PI3K blockade in the NTS on arterial pressure in both rat strains by LV-mediated expression of a dominant-negative for PI3K.
Because mature adult SHRs and WKY rats were used in the present study, with stable BP levels, we found no significant interaction between the time-related BP changes (ie, BP progression) and rat species (ie, WKY rats and SHRs). Over a period of 4 weeks postinjection, systolic BP (SBP) rose significantly in the SHRs injected with LV-DNp85 from a control of 175±3 mm Hg (day 0) to 191±6 mm Hg at day 28 compared with no change in SBP in SHRs injected with the control LV (LV-eGFP; n=5 per group; P<0.01, Figure 2A). In contrast, there were no significant differences in SBP between the WKY rats injected with either LV-DNp85 or LV-eGFP at any time point over the 4 weeks of monitoring (n=5 per group; Figure 2). Similarly, we observed significant increases in both the diastolic BP (DBP; from 130±2 to 157±5 mm Hg at day 28; P<0.05; Figure 2B) and the mean BP (MBP; from 145±2 to 165±5 mm Hg at day 28; P<0.05; Figure 2C) in the LV-DNp85–treated SHRs. No significant changes were detected in DBP or MBP of WKY rats injected with either virus (Figure 2B and 2C). A significant rise in SBP, DBP, and MBP values in the LV-DNp85 SHR was first observed as early as 7 days after the viral injection (data not shown). Changes in night versus day BPs were also assessed in the LV-eGFP- and LV-DNp85–treated SHRs (Figure S3). We found that the SBP, DBP, and MBP of SHRs significantly rose after the LV-DNp85 injection during both the light and dark phases compared with the LV-eGFP–treated SHRs, the values of which remained unchanged (Figure S3). These results suggest that increased PI3K signaling in the NTS of SHRs exerts a chronic restraining role on arterial pressure in the SHRs.
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P<0.05,
HR and Spontaneous Baroreceptor Reflex Gain
Over the period of 28 days of monitoring, the HR values significantly rose from 331±6 to 342±6 bpm (n=5 per group; P<0.05; Figure 2D) in the SHRs injected with LV-DNp85 compared with the LV-eGFP–treated SHRs, in which the HR values showed a significant decline over the time (from 346±7 to 322±6 bpm at day 28; P<0.05; Figure 2D). Similar to the LV-eGFP–treated SHRs, we found that the HR values in both the LV-DNp85- and LV-eGFP–treated WKY groups also significantly decreased over time to a similar extent (Figure 2D). We attributed this to the age-related decreases in the HR values reported previously in mature rats.29–32 We also observed a significant decrease in values of the spontaneous baroreceptor reflex gain (sBRG; from 0.7±0.07 to 0.5±0.04 ms/mm Hg; n=5; P<0.001; Figure 2E). Changes in the night (dark phase) versus day (light phase) HR and sBRG values in LV-DNp85- and LV-eGFP–treated SHRs followed a similar pattern in both phases (Figure S3). These results suggest a role for PI3K in the chronic modulation of the cardiac component of the baroreflex and regulation of HR in the NTS of the SHR.
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Confirmation of the LV-Mediated Gene Expression in NTS
Posthoc examination of the sites of LV injections revealed abundant eGFP fluorescence in numerous cells of the NTS of all animal groups. Some eGFP-positive staining was also found in the dorsal vagal motor nucleus, because of the inevitable unsolicited viral spread in this area, which is in very close proximity to NTS and contains dendrites from dorsal vagal motor nucleus neurons. No eGFP-positive staining was found in any other medullary nuclei known to control the cardiovascular system, indicative of an absence of both retrograde and anterograde transport of this vector. Immunostaining for neuronal nuclei (marker) (NeuN) and double-fluorescence labeling revealed that a proportion (
45%) of the eGFP-positive cells was double labeled and, therefore, neuronal (Figure 3). Comparable regions of the NTS were transduced with both viral vectors in all of the animal groups that spanned regions from –13.80 to –14.60 mm relative to bregma, 300 to 800 µm from midline, and 400 to 800 µm below the dorsal surface of the medulla (Figure 3).
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Discussion |
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The most significant finding of the present study is that chronic and specific suppression of PI3K signaling in the NTS of the SHR causes arterial pressure to increase. This chronic effect, together with increases in HR and depression of the spontaneous baroreflex gain, are exclusive to the SHR and not seen in the WKY strain. These observations suggest that PI3K signaling is more active in the NTS of the SHR than the WKY rat and that this pathway could act to restrain the hypertension in the SHR.
PI3K Expression in the NTS of the SHR Versus WKY Rat
We provide the first direct evidence for increased PI3K transcript abundance within the NTS of the SHR relative to a normotensive rat strain. Specifically, we demonstrate increased gene transcript abundances of p110β and p110 catalytic subunits of PI3K in the NTS of SHR compared with the WKY rat. This is consistent with our previous study, which also showed an elevated mRNA level of p110 in both the paraventricular nucleus and RVLM neurons of the SHR and was similarly accompanied by increased PI3K activity.13 These latter regions also showed alterations in p110 and p85 subunits.13 Therefore, the elevated mRNA levels of the catalytic p110β and PI3K subunits in the NTS of SHR seen in the present study may also be accompanied by elevated PI3K activity in these rats. We recognize that a caveat of the present study is that, for technical limitations (see below), we did not show directly increased PI3K activity in NTS of the SHR versus the WKY rat. However, an effective and robust way to test whether PI3K protein activity was elevated was to block the activity of p110β and p110 catalytic subunits simultaneously by antagonizing the regulatory subunit p85 on which their catalytic activity depends. This would reveal whether the activity of PI3K was upregulated in the SHR and, importantly, whether this had any functional implications for arterial pressure control in the SHR.
We hypothesized that the elevated PI3K mRNA in the NTS may be of neuronal origin. This is supported by abundant transgene expression in NTS neurons, as revealed by the expression of eGFP in NeuN (a specific neuronal marker) immunopositive cells (in Figure 3), consistent with the known neuronal tropism of the lentivirus in the NTS.33 However, a limitation of the present study is that it fails to pinpoint the exact neuronal phenotype in which this elevation of PI3K signaling is occurring. Considering that the differences in mRNA levels of p110β and p110 are relatively small between SHRs and WKY rats (1.7-fold and 1.6-fold, respectively), this may reflect a highly select and cell type–specific PI3K elevation in NTS neurons exerting a powerful effect on cardiovascular autonomic activity in the SHR. We discuss putative neuronal phenotypes below. Although we have not demonstrated reduced PI3K activity in vivo, which would be technically difficult, in our previous studies we have demonstrated the capability of the DNp85 construct to reduce the PI3K activity.13 Here we also demonstrate expression of the DNp85 protein in vivo, as evidenced from the expression of eGFP using the internal ribosome entry site construct (Figure 3). All told, our data support the presumption that our viral-induced intervention reduced PI3K activity in NTS in vivo.
Cardiovascular Functional Role of PI3K in the NTS of SHRs
Dysregulation within the NTS has been linked to the development of hypertension in animal models, including the SHR.17,19,21,26 To test for an overall functional role of PI3K in the NTS for regulating arterial pressure, we chronically blocked PI3K activity by expressing a dominant-negative of the regulatory p85 PI3K subunit using LV gene transfer (see online supplement and below for further discussion), because this provided the means to block the catalytic activity of the 2 subunits (p110β and p110) that we found to be overexpressed in the NTS of the SHR. We found a chronic and sustained elevation of arterial pressure (SBP, DBP, and MBP) in the SHR but no change in normotensive WKY rats. In the SHR, this was accompanied by an increase in very low frequency, very low frequency+low frequency (Figure S4), and HR and decreases in the sBRG and high frequency of HR variability (Figure S4). Increases in both very low frequency and very low frequency+low frequency could imply alterations in the sympathetic and hormonal controls of arterial pressure, whereas reductions in the high frequency of HR variability indicate loss of vagal tone.34–36 This suggests that PI3K activity in the NTS of the SHR controls autonomic activity destined for both the heart and vasculature. Moreover, suppression of the sBRG after PI3K blockade is consistent with the reduced cardiac vagal activity (high frequency of pulse interval).37 We recognize the limitations of our analysis in that we are unable to provide a full baroreceptor reflex function curve. However, the sBRG values obtained here are of physiological relevance, because they fall around the operating point of this reflex in an unrestrained conscious rat.38 Whether the gain of the sympathetic vasoconstrictor component has also been modified remains unknown. Because both sBRG and SBP are affected by PI3K blockade in the SHR, it is unclear whether the increase in arterial pressure is caused by a depression of the baroreceptor reflex and/or direct action on neurons determining the set point of arterial pressure.
Others have shown that acute blockade of PI3K activity in other regions of the brain stem can affect arterial pressure. In the RVLM, wortmannin (a PI3K antagonist) decreased arterial pressure in anesthetized SHRs but not WKY rats.14 Our data are consistent with the holistic viewpoint that PI3K activity in the SHR is increased in key regions of the medulla and hypothalamus known to control vasomotor tone (eg, RVLM and paraventricular nucleus).12–14 However, the question of the signaling downstream of PI3K remains uncertain. It is generally believed that the main downstream effector of PI3K activation is the protein kinase B(PKB)/Akt pathway.39,40 We have confirmed in the present study in vitro that overexpression of DNp85 blocks PI3K activity13 to reduce activation of PKB/Akt signaling (see online supplement for details). However, this does not mean that PKB/Akt is involved in NTS signaling in the SHR in vivo. Indeed, others have shown in other animal models of hypertension that increased levels of PI3K subunits and PI3K activity did not necessarily lead to elevation of PKB/Akt signaling,41 suggesting the presence of a PI3K-dependent but a PKB/Akt-independent signaling pathway. A recent study also revealed that baseline phosphorylated (but not total) PKB/Akt levels are lower in the NTS of SHRs compared with the WKY rats.42 These authors also showed that injection of phosphoinositide(3,4,5)P3 (a phospholipids second messenger produced by PI3K, thereby mimicking activation of PI3K) in the NTS of the SHR failed to activate the downstream PKB/Akt but produced cardiovascular responses that are in full agreement with the findings of our present study (ie, depressor action). Therefore, although we are confident in the ability of our construct to reduce PI3K activity, the relevance of this to the downstream PKB/Akt in vivo remains uncertain.
Because angiotensin II potentiates PI3K signaling and release of catecholamines from brain stem SHR neurons,12,13 the A2 neurons in NTS may mediate the hypertensive effect after chronic PI3K blockade in the NTS of SHRs. Although we did not assess the proportion of A2 neurons that were transfected by LV-DNp85 in the present study, it is likely that many A2 neurons were transduced, because their location in NTS included neurons that had been transduced. The A2 noradrenergic cells are involved in BP homeostasis. Indeed, several studies reported that selective destruction43,44 or electric "silencing"26 of A2 neurons causes either lability43,44 or increased BP being more pronounced in the SHR.26 This last study also emphasized the putative homeostatic role of A2 neurons in SHRs in restraining BP in this model of hypertension. The contribution of A2 neurons to baroreflex regulation is not well established and remains controversial.45,46 However, cardiac baroreceptor reflex was attenuated after the A2 neurons were lesioned.44 In addition, vagal sensory afferents could be found in close proximity to the NTS catecholamine neurons.47 Therefore, the A2 neuronal population, or at least the portion that regulates cardiovascular homeostasis, seems to be sympathoinhibitory.
Finally, as the dendrites of dorsal vagal motor nucleus project up into the NTS,48 some of these were also transduced with the LV-DNp85. Thus, there is a possibility that a component of the cardiovascular effects seen in the present study is because of the blockade of PI3K in this nucleus, especially because some of these motoneurons have chronotropic influences.49
Elevated PI3K in the NTS of SHR: Cause or Consequence of Hypertension?
Because the present study compared the adult SHRs (with fully developed hypertension) with their age-matched WKY controls, we cannot conclude whether the elevated PI3K signaling in the NTS is a cause or consequence of the hypertensive phenotype. However, we showed previously that the PI3K signaling was enhanced in cultured brain stem/hypothalamic neurons from the neonatal SHR,12,13 indicating that that the raised PI3K signaling in the brain stem is a congenital feature of the SHR. Future studies could assess whether chronic blockade of PI3K signaling in NTS in the developing SHR has any effect on the hypertension developed with maturation. Holistically, in the SHR, PI3K activity appears to be elevated in all of the cardiovascular control regions studied to date (RVLM, paraventricular nucleus, and now NTS, shown herein) and that PI3K activity is presumed to exert an excitatory effect on cardiovascular neurons directly or when driven by an endogenous ligand.50 Because we centered our LV injections to a sympathoinhibitory region of the NTS, its blockade resulted in a pressor effect. All told, it would appear that PI3K activity within the brain of the SHR plays a major role in the maintenance of the precise level of the hypertension by acting at regions exerting excitatory or inhibitory influences on sympathetic activity.
Future Perspectives
This study reveals a novel role for the PI3K signaling pathway in the NTS of SHRs in restraining BP in this animal model of human hypertension. Future studies should seek to determine the genomic and/or modulatory factors that cause this pathway to predominate in the NTS of the SHR, as well as to identify which specific neuronal types are involved. Such information might allow the development of new tools for specific activation of this signaling pathway in NTS, which may provide a powerful intervention for lowering arterial pressure chronically.
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Acknowledgments |
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Sources of Funding
The financial support of the National Institutes of Health (HL033610) and the British Heart Foundation is acknowledged. J.F.R.P. was in receipt of a Royal Society Wolfson Research Merit Award.
Disclosures
None.
Received August 29, 2008; first decision September 24, 2008; accepted October 13, 2008.
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References |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Carretero OA, Oparil S. Essential hypertension. Part I: definition and etiology. Circulation. 2000; 101: 329–335.
2. Touyz RM. Molecular and cellular mechanisms regulating vascular function and structure–implications in the pathogenesis of hypertension. Can J Cardiol. 2000; 16: 1137–1146.[Medline] [Order article via Infotrieve]
3. Oudit GY, Crackower MA, Backx PH, Penninger JM. The role of ACE2 in cardiovascular physiology. Trends Cardiovasc Med. 2003; 13: 93–101.[CrossRef][Medline] [Order article via Infotrieve]
4. Takeda K, Bunag RD. Chronic propranolol treatment inhibits sympathetic nerve activity and keeps blood pressure from rising in spontaneously hypertensive rats. Hypertension. 1980; 2: 228–235.
5. Arribas SM, Alonso MJ, Marin J, Fernandes F, Llergo JL, Sanchez-Ferrer CF, Salaices M. Noradrenergic transmission in the tail artery of hypertensive rats transgenic for the mouse renin gene Ren-2. J Auton Pharmacol. 1996; 16: 69–77.[Medline] [Order article via Infotrieve]
6. Cabassi A, Vinci S, Cantoni AM, Quartieri F, Moschini L, Cavazzini S, Cavatorta A, Borghetti A. Sympathetic activation in adipose tissue and skeletal muscle of hypertensive rats. Hypertension. 2002; 39: 656–661.
7. Leenen FH, Ruzicka M, Huang BS. The brain and salt-sensitive hypertension. Curr Hypertens Rep. 2002; 4: 129–135.[Medline] [Order article via Infotrieve]
8. Goldstein DS. Plasma norepinephrine in essential hypertension. A study of the studies. Hypertension. 1981; 3: 48–52.
9. Anderson EA, Sinkey CA, Lawton WJ, Mark AL. Elevated sympathetic nerve activity in borderline hypertensive humans. Evidence from direct intraneural recordings. Hypertension. 1989; 14: 177–183.
10. Grassi G, Cattaneo BM, Seravalle G, Lanfranchi A, Mancia G. Baroreflex control of sympathetic nerve activity in essential and secondary hypertension. Hypertension. 1998; 31: 68–72.
11. Grassi G, Seravalle G, Trevano FQ, Dell'oro R, Bolla G, Cuspidi C, Arenare F, Mancia G. Neurogenic abnormalities in masked hypertension. Hypertension. 2007; 50: 537–542.
12. Yang H, Raizada MK. Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat. J Neurosci. 1999; 19: 2413–2423.
13. Veerasingham SJ, Yamazato M, Berecek KH, Wyss JM, Raizada MK. Increased PI3-kinase in presympathetic brain areas of the spontaneously hypertensive rat. Circ Res. 2005; 96: 277–279.
14. Seyedabadi M, Goodchild AK, Pilowsky PM. Differential role of kinases in brain stem of hypertensive and normotensive rats. Hypertension. 2001; 38: 1087–1092.
15. Ciriello J. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci Lett. 1983; 36: 37–42.[CrossRef][Medline] [Order article via Infotrieve]
16. Pilowsky PM, Goodchild AK. Baroreceptor reflex pathways and neurotransmitters: 10 years on. J Hypertens. 2002; 20: 1675–1688.[CrossRef][Medline] [Order article via Infotrieve]
17. Doba N, Reis DJ. Acute fulminating neurogenic hypertension produced by brainstem lesions in the rat. Circ Res. 1973; 32: 584–593.
18. Talman WT, Perrone MH, Scher P, Kwo S, Reis DJ. Antagonism of the baroreceptor reflex by glutamate diethyl ester, an antagonist to L-glutamate. Brain Res. 1981; 217: 186–191.[CrossRef][Medline] [Order article via Infotrieve]
19. Waki H, Murphy D, Yao ST, Kasparov S, Paton JF. Endothelial NO synthase activity in nucleus tractus solitarii contributes to hypertension in spontaneously hypertensive rats. Hypertension. 2006; 48: 644–650.
20. Thrasher TN. Arterial baroreceptor input contributes to long-term control of blood pressure. Curr Hypertens Rep. 2006; 8: 249–254.[CrossRef][Medline] [Order article via Infotrieve]
21. Waki H, Liu B, Miyake M, Katahira K, Murphy D, Kasparov S, Paton JF. Junctional adhesion molecule-1 is upregulated in spontaneously hypertensive rats: evidence for a prohypertensive role within the brain stem. Hypertension. 2007; 49: 1321–1327.
22. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162: 156–159.[Medline] [Order article via Infotrieve]
23. Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006; 1: 581–585.[CrossRef][Medline] [Order article via Infotrieve]
24. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25: 402–408.[CrossRef][Medline] [Order article via Infotrieve]
25. Waki H, Kasparov S, Wong LF, Murphy D, Shimizu T, Paton JF. Chronic inhibition of endothelial nitric oxide synthase activity in nucleus tractus solitarii enhances baroreceptor reflex in conscious rats. J Physiol. 2003; 546: 233–242.
26. Duale H, Waki H, Howorth P, Kasparov S, Teschemacher AG, Paton JF. Restraining influence of A2 neurons in chronic control of arterial pressure in spontaneously hypertensive rats. Cardiovasc Res. 2007; 76: 184–193.
27. Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y, Sakaue H, Ando A. Chavanieu A, Calas B, Grigorescu F, Nishiyama M, Waterfield MD, Kasuge M. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. Embo J. 1994; 13: 2313–2321.[Medline] [Order article via Infotrieve]
28. Coleman JE, Huentelman MJ, Kasparov S, Metcalfe BL, Paton JF, Katovich MJ, Semple-Rowland SL, Raizada MK. Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo. Physiol Genomics. 2003; 12: 221–228.
29. Struyker-Boudier HA, Evenwel RT, Smits JF, Van Essen H. Baroreflex sensitivity during the development of spontaneous hypertension in rats. Clin Sci (Lond). 1982; 62: 589–594.[Medline] [Order article via Infotrieve]
30. Head GA, Adams MA. Time course of changes in baroreceptor reflex control of heart rate in conscious SHR and WKY: contribution of the cardiac vagus and sympathetic nerves. Clin Exp Pharmacol Physiol. 1988; 15: 289–292.[Medline] [Order article via Infotrieve]
31. Munakata M, Imai Y, Minami N, Sasaki S, Ichijyo T, Yoshizawa M, Sekino H, Abe K, Yoshinaga K. Cosinor analysis of changes in circadian blood pressure rhythm with aging in spontaneously hypertensive rats. Tohoku J Exp Med. 1990; 161: 55–64.[Medline] [Order article via Infotrieve]
32. Head GA, Adams MA. Characterization of the baroreceptor heart rate reflex during development in spontaneously hypertensive rats. Clin Exp Pharmacol Physiol. 1992; 19: 587–597.[Medline] [Order article via Infotrieve]
33. Teschemacher AG, Paton JF, Kasparov S. Imaging living central neurones using viral gene transfer. Adv Drug Deliv Rev. 2005; 57: 79–93.[CrossRef][Medline] [Order article via Infotrieve]
34. Cerutti C, Gustin MP, Paultre CZ, Lo M, Julien C, Vincent M, Sassard J. Autonomic nervous system and cardiovascular variability in rats: a spectral analysis approach. Am J Physiol. 1991; 261: 1292–1299.
35. Cerutti C, Barres C, Paultre C. Baroreflex modulation of blood pressure and heart rate variabilities in rats: assessment by spectral analysis. Am J Physiol. 1994; 266: 1993–2000.
36. Chen RF, Tseng WT, Yen CT, Tsai ML. Frequency response characteristic of sympathetic mediated low-frequency blood pressure fluctuations in conscious rats. Auton Neurosci. 2006; 128: 48–52.[Medline] [Order article via Infotrieve]
37. Pagani M, Lombardi F, Guzzetti S, Rimoldi O, Furlan R, Pizzinelli P, Sandrone G, Pagani M, Lombardi F, Guzzetti S, Rimoldi O, Furlan R, Pizzinelli P, Sandrone G, Malfatto G, Dell'Orto S, Piccaluga E. Power spectral analysis of heart rate and arterial pressure variabilities as a marker of sympatho-vagal interaction in man and conscious dog. Circ Res. 1986; 59: 178–193.
38. Waki H, Katahira K, Polson JW, Kasparov S, Murphy D, Paton JF. Automation of analysis of cardiovascular autonomic function from chronic measurements of arterial pressure in conscious rats. Exp Physiol. 2006; 91: 201–213.
39. Plas DR, Thompson CB. Akt-dependent transformation: there is more to growth than just surviving. Oncogene. 2005; 24: 7435–7442.[CrossRef][Medline] [Order article via Infotrieve]
40. Sale EM, Sale GJ. Protein kinase B: signaling roles and therapeutic targeting. Cell Mol Life Sci. 2008; 65: 113–127.[CrossRef][Medline] [Order article via Infotrieve]
41. Northcott CA, Poy MN, Najjar SM, Watts SW. Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats. Circ Res. 2002; 91: 360–369.
42. Hsiao M, Lu PJ, Huang HN, Lo WC, Ho WY, Lai TC, Chiang HT, Tseng CJ. Defective phosphatidylinositol 3-kinase signaling in central control of cardiovascular effects in the nucleus tractus solitarii of spontaneously hypertensive rats. Hypertens Res. 2008; 31: 1209–1218.[Medline] [Order article via Infotrieve]
43. Reis DJ, Doba N, Snyder DW, Nathan MA. Brain lesions and hypertension: chronic lability and elevation of arterial pressure produced by electrolytic lesions and 6-hydroxydopamine treatment of nucleus tractus solitarii (NTS) in rat and cat. Prog Brain Res. 1977; 47: 169–188.[Medline] [Order article via Infotrieve]
44. Talman WT, Snyder D, Reis DJ. Chronic lability of arterial pressure produced by destruction of A2 catecholaminergic neurons in rat brainstem. Circ Res. 1980; 46: 842–853.
45. Moore SD, Guyenet PG. Effect of blood pressure on A2 noradrenergic neurons. Brain Res. 1985; 338: 169–172.[CrossRef][Medline] [Order article via Infotrieve]
46. Tanaka J, Seto K. Neurons in the nucleus of the solitary tract with ascending projections to the subfornical organ in the rat. Neurosci Lett. 1988; 89: 152–155.[CrossRef][Medline] [Order article via Infotrieve]
47. Sumal KK, Blessing WW, Joh TH, Reis DJ, Pickel VM. Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius. Brain Res. 1983; 277: 31–40.[CrossRef][Medline] [Order article via Infotrieve]
48. Deuchars J, Li YW, Kasparov S, Paton JF. Morphological and electrophysiological properties of neurones in the dorsal vagal complex of the rat activated by arterial baroreceptors. J Comp Neurol. 2000; 417: 233–249.[CrossRef][Medline] [Order article via Infotrieve]
49. Cheng Z, Zhang H, Guo SZ, Wurster R, Gozal D. Differential control over postganglionic neurons in rat cardiac ganglia by NA and DmnX neurons: anatomical evidence. Am J Physiol Regul Integr Comp Physiol. 2004; 286: 625–633.
50. Sun C, Du J, Sumners C, Raizada MK. PI3K inhibitors abolish the enhanced chronotropic effects of angiotensin II in spontaneously hypertensive rat brain neurons. J Neurophysiol. 2003; 90: 3155–3160.
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