Donate Help Contact The AHA Sign In Home
American Heart Association
Hypertension
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Hypertension. 2008;52:351-358
Published online before print July 7, 2008, doi: 10.1161/HYPERTENSIONAHA.108.114371
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
52/2/351    most recent
HYPERTENSIONAHA.108.114371v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Teschemacher, A. G.
Right arrow Articles by Kasparov, S.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Teschemacher, A. G.
Right arrow Articles by Kasparov, S.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*2-PHENYLETHYLAMINE
*LOSARTAN POTASSIUM
Medline Plus Health Information
*High Blood Pressure
Related Collections
Right arrow ACE/Angiotension receptors
Right arrow Animal models of human disease
Right arrow Other hypertension
Right arrow Hypertension - basic studies
Right arrow Autonomic, reflex, and neurohumoral control of circulation

(Hypertension. 2008;52:351.)
© 2008 American Heart Association, Inc.

Original Articles

Area-Specific Differences in Transmitter Release in Central Catecholaminergic Neurons of Spontaneously Hypertensive Rats

Anja G. Teschemacher; Sheng Wang; Mohan K. Raizada; Julian F.R. Paton; Sergey Kasparov

From the Department of Physiology and Pharmacology (A.G.T., J.F.R.P., S.K.), School of Medical Sciences, University of Bristol, Bristol, United Kingdom; Department of Neurosciences (S.W.), School of Medicine, Case Western Reserve University, Cleveland, Ohio; and the Department of Physiology (M.K.R.), University of Florida, Gainesville.

Correspondence to Sergey Kasparov, Department of Physiology and Pharmacology, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United Kingdom. E-mail sergey.kasparov{at}bristol.ac.uk


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The link among blood pressure, sympathetic output, and brain neurons producing catecholamines is well documented. Nevertheless, their intrinsic properties and any alterations in signaling characteristics between normotensive and hypertensive phenotypes remain unknown. Here, we directly compared neurophysiological properties of catecholamine release of C1 and A2 neurons of the spontaneously hypertensive rat and Wistar rat in organotypic slices. C1 and A2 areas were studied because both are widely implicated in the pathophysiology of hypertension. Catecholaminergic neurons were visualized using viral vectors to express green fluorescent protein. Microamperometry revealed that C1 axonal varicosities of spontaneously hypertensive but not normotensive Wistar rats release a transmitter predominantly ({approx}86%) in very large quanta, comparable in catecholamine load to adrenal chromaffin granules. Because quantal size affects the spread of transmitter in the extracellular space, this may enhance the impact of C1 varicosities on their downstream targets and increase sympathetic drive in the hypertensive rat. Electrophysiological properties and Ca2+ handling were studied using patch clamp and confocal imaging. Although overall electrophysiological characteristics of C1 and A2 neurons were comparable between strains, the characteristic angiotensin-II–induced Ca2+ mobilization was reduced in A2 neurons of the spontaneously hypertensive rat. Because A2 neurons are a part of a homeostatic antihypertensive circuit, this could reduce their restraining influence on blood pressure. Thus, we have revealed an increased quantal size in C1 varicosities and a reduced responsiveness of A2 neurons of the spontaneously hypertensive rat to angiotensin II. Both effects could contribute to elevated sympathetic activity and blood pressure in the spontaneously hypertensive rat.


Key Words: norepinephrine • epinephrine • medulla • hypertension • blood pressure • neurotransmitter release • patch clamp


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Essential hypertension in both human patients1 and an animal model of this disease (the spontaneously hypertensive rat [SHR]) is accompanied characteristically by increased central sympathetic drive.2–4 The link among brain stem catecholaminergic (CAergic) activity, sympathetic output, and blood pressure is documented very extensively (reviewed in Reference 5) but as yet not well understood. Based on previous studies, 3 main differences between CAergic transmission in the normotensive rat and SHR may be hypothesized.

First, noradrenaline (NA) or adrenaline (ADR) in SHRs may be stored and released differently. This hypothesis is based on evidence of morphological changes in storage vesicles in SHRs and their increased ability to take up 3H-NA,6 higher expression of norepinephrine transporter in SHR brain stem cultures,7 and increased tyrosine hydroxylase expression in the SHR ventrolateral medulla.8 Structural differences in NA-storing vesicles and/or NA synthesis and transport could translate into enhanced packaging and release of catecholamine (CA) in SHRs. Thus, it could be that, in SHRs, CAergic volume transmission is amplified, increasing the effect of CA on various cellular targets in areas critical for control of sympathetic outflow.

Second, central CAergic neurons in the SHR could be electrically "hyperactive" and generate action potentials at higher rates than in normotensive animals. Higher discharge rates were detected in neurons (some of which could be CA containing) of the rostral ventrolateral medulla of neonatal SHRs.9

The third hypothetical difference is that the excitatory effect of angiotensin II (Ang II) on these neurons or a characteristic Ang II–induced Ca2+ mobilization10 may be enhanced in SHRs. Central Ang II has been implicated in pathogenesis of excessive sympathetic activity and hypertension by numerous studies11–13 (reviewed in Reference 14). In rat hypothalamus/brain stem primary cultures, Ang II excited a significant proportion of neurons,15 and Ang II effects were enhanced in primary cultures of neonatal SHRs.16,17 It is, therefore, conceivable that stronger activation of CAergic neurons by central Ang II could lead to a higher transmitter release in SHRs.

Finally, CAergic neurons are located within several discrete clusters within the brain stem (referred to as "A" groups, which release norepinephrine, and "C" groups, which have the biochemical machinery for synthesis of epinephrine), and it appears from the literature that the roles of distinct CAergic cell groups in control of blood pressure are different or even opposite. For example, C1 neurons are widely believed to play an important role in maintaining sympathetic outflow,18–22 whereas A2 neurons are likely to be a part of the central homeostatic "antihypertensive" mechanism.23–26 It follows that if the hypertensive phenotype is associated with altered signaling in central CAergic neurons, the changes must be cell group specific rather than universal.

Although the first clear links between hypertension and central CA transmission were made >30 years ago, to date it has not been possible to pinpoint any differences in the signaling in these neurons between hypertensive and normotensive phenotypes, because suitable methods were lacking. We have recently developed transgene-based approaches that allow us, for the first time, to study intracellular signaling and quantal characteristics of CA release from identified clusters of CA neurons in organotypic slices of rat brain stem.27,28 Using these strategies, we have been able to directly test the hypotheses listed above. The present study compared the quantal transmitter release characteristics, electrophysiological properties, and Ca2+ handling in C1 and A2 neurons in response to Ang II from SHRs and the normotensive Wistar rat (WR).


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Methods Summary
For a detailed account of the methods please see the data supplement (available online at http://hyper.ahajournals.org). In brief, all of the experiments were performed in organotypic cultured slices of rat brain stem from P7–9 rat pups, where NA/ADR-producing neurons were induced to express enhanced green fluorescent protein (EGFP) by an adenoviral vector with a PRSx8 promoter.28–30 For each part of the study, pups from ≥3 different litters have been used. It has been demonstrated by numerous earlier studies that changes in the activity and biochemistry of central neurons are detectable in tissues from postnatal SHRs in vitro31–34 and that there are alterations in sympathetic activity in juvenile SHRs before the development of hypertension,3 consistent with recent unpublished observations from this laboratory. We have focused on 3 main characteristics of either C1/A1 or A2 neurons. The transverse brain stem slices for C1 were taken >1 mm more rostrally than for A2, but it is acknowledged that the distributions of phenylethanolamine–N-methyltransferase–positive, ie, putative adrenergic, and of phenylethanolamine–N-methyltransferase–negative but dopamine-β-hydroxylase (DBH)–positive, ie, noradrenergic, neurons partially overlap in the ventrolateral medulla. Transmitter release and packaging characteristics were assessed using microamperometry (see Figure 1) as described previously.28 Membrane potential, ongoing firing activity, and electric and intracellular Ca2+ concentration ([Ca2+]i) responses to Ang II were assessed using patch clamp and confocal fluorescence imaging of Rhod-2, essentially as described previously35 (Figure 2).


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		Figure 1. Microamperometric recording of transmitter release from varicosities within the C1 area of the rat brain stem. A, EGFP-expressing fluorescent varicosities can be identified. Under visual control, a microamperometric electrode (fine carbon fiber embedded into a glass sleeve) is placed on a varicosity. i, Low-power image of the ventral edge of a slice culture (a superimposition of differential interference contrast microscopy and fluorescent images). Many EGFP-expressing neurons are visible toward the lower right edge of the slice, corresponding with the expected location of the C1 area. ii, High-power image of an axon with fluorescent varicosities. B, Oxidation of the electroactive transmitter at the charged surface of the microamperometric electrode results in currents that are directly proportional to the quantity of CA released. Upper trace, A representative recording from a WR C1 area varicosity. No large quanta are evident. Many small events are not easily detectable at this scale, but some of them are indicated with blue arrows. Lower trace, C1 varicosity of SHR. In contrast to WR, 2 very large release events are evident (red arrows). C, Examples of release events registered using microamperometry, including single spike events characterized by a well-defined single peak and exponential decay and complex events with >1 peak. Note that, whereas the majority of the release events had charges <0.5 pC (defined as "small events" in the text), more rare but very large events could also be detected (defined as "large events"). Some of the large events delivered transmitter loads equivalent to hundreds of small events.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Figure 2. Simultaneous patch clamp recording and confocal Ca2+ imaging of an identified A2 neuron. A, To assess the electric membrane properties of A2 and C1 neurons and their responsiveness to Ang II, the cells were identified based on EGFP fluorescence and patched with a pipette containing a red-shifted Ca2+ indicator Rhod-2 (red arrow). Electric characteristics of the cells and Rhod-2 fluorescence were assessed before and during application of Ang II (200 nmol/L) by bath (3 to 5 minutes after the delivery of the drug into the chamber). The changes in intracellular Ca2+ were assessed based on the relative change (%F) in Rhod2-emitted fluorescence (590 to 650 nm). B and C, A typical recording from a C1 area cell of WR. Upper trace, A plot of fluorescence intensity over time during Ang II application (images of the cell at time points indicated by arrows 1 and 2 are on the right). Lower trace, Membrane potential recording of the same cell. Note that in some cells, as in this one, a clear increase in [Ca2+]i could be detected in spite of the lack of any visible depolarization. This suggests that Ang II–induced [Ca2+]i increases are largely because of the release from intracellular stores, consistent with data published previously.10 Note also a series of membrane responses to hyperpolarizing and depolarizing current injections at the end of Ang II application; this was routinely performed to assess membrane resistance in quiescent cells. The last depolarizing current pulse evoked an action potential (black arrow).

Drugs and Their Application
In amperometric and electrophysiolgical/Ca2+ imaging studies, Ang II (Sigma) and other drugs were introduced into the perfusion media, as described previously.35 For the experiments presented here we used a 200 nmol/L concentration, which is similar to or lower than those used in many previous in vitro studies.16,17,36–40 In a few preliminary experiments we applied 50 and 100 nmol/L Ang II and observed qualitatively similar effects, albeit smaller (data not shown).

Statistical Analysis
All of the values in this text are expressed as means±SEMs unless indicated otherwise. Statistical evaluation was carried out using Microsoft Excel and GraphPad Prism 4.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Quantal Characteristics of Transmitter Release
Microamperometric currents appeared as either separate single-peaked events with monophasic rising and falling phases (Figure 1B and 1C, top) or complex events (Figure 1B and 1C, bottom). Single events (Figure 1C, top) could be subdivided into relatively frequent small-to-medium-sized quanta (referred to as "small" events below; median quanta: {approx}0.02 pC; main populations on Figure 3A and 3B; examples shown in Figure 1C) and more rare "large" events >0.5 pC (median quanta: {approx}1 pC; average: {approx}2.1 pC; events within the right part of the distributions in Figure 3A and 3B; examples shown in Figure 1C). Event frequencies varied widely between individual axonal varicosities (0.01 to 2.50 Hz) and were not significantly different between CAergic areas or rat strains (n=6 varicosities for each area in each strain). Because the size of a release event determines the spread of the CA in the extracellular space, we analyzed the characteristics and relative contributions of various types of events to the overall amount of transmitter released (Table 1).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Figure 3. Amperometric analysis of transmitter release from varicosities of C1 and A2 neurons. A, Distribution of quantal sizes (pC1/3) including single and complex events at C1 varicosities in WR and SHR. In SHR, as compared with WR, the population of small events is decreased but that of large events is increased. B, Quantal size distribution of single and complex events at A2 varicosities in WR and SHR. Frequencies of small and large fractions are slightly increased in SHR. C, Relative contributions of small (top half) and large (lower half) single (plain) and complex (dotted) release events to total CA release in WR and SHR. Note that the majority of the CA release in C1 varicosities of SHR occurs in large quanta, whereas the contribution of small events in C1 varicosities is reduced. Release from large quanta in SHR was increased and small quanta decreased, not only in relative, but also in absolute terms (see text). These differences are area specific and not evident in A2 varicosities.


View this table:
[in this window]
[in a new window]

  		Table 1. Characteristics of Single Small and Large (<0.5 pC) Events in Areas C1 and A2 of WRs and SHRs

C1 Varicosities
Total CA release (in pC/min) from C1 SHR varicosities was larger than in WRs (+55%). Median quantal content or t1/2 of single small events did not significantly differ between WRs and SHRs (Table 1). Although the kinetics of large events were similar between WRs and SHRs, their frequency increased {approx}1.5 times in SHRs (Table 1 and Figure 3A). More importantly, the relative contribution of large and small events to total CA release was different between SHRs and WRs ({chi}2 test values <0.05 for all fractions; Figure 3C). Although in WRs single and complex large events delivered 1.6x106 CA molecules per minute, this parameter more than doubled in SHRs (3.7x106 molecules per minute). This was because of a combination of both higher incidence of large events in C1 varicosities of SHRs (Figure 3A) and a much higher transmitter load of such events ({approx}270%; P<0.01; Table 2). Thus, whereas in WRs large events were only responsible for 57% of the total release, in SHRs they delivered 86% of the total CA because of a dramatic increase in both single and complex fractions. In contrast, release from small events in SHRs decreased from 1.2x106 to 5.9x105 molecules per minute, corresponding with a fall from 43% to 14% of the total release. As a result, the bulk of the transmitter released from C1 varicosities in SHRs arrives in large packages (Figure 1C and 3UpC) in excess of 0.5 pC, or 1.6x106 molecules.


View this table:
[in this window]
[in a new window]

  		Table 2. Charges (in fC) of Large Events (single and complex) in C1 and A2 Varicosities of WR and SHR

A2 Varicosities
There were no significant differences in median quantal content between the populations of single small events at A2 release sites of WRs versus SHRs, but small events in SHR had a significantly longer t1/2 (P<0.05; Table 1). Kinetics of large quanta were not different between WRs and SHRs. The total quantity of NA released from the pool of 6 A2 varicosities was 35% greater in SHRs because of slightly higher frequencies of all of the fractions of events. However, the relative contribution of various types of quanta to the total transmitter output was similar between the strains (Figure 3C). Moreover, the transmitter load of the large release events (Figure 1C) in SHR A2 varicosities tended to be smaller than in WRs (Table 2).

Electrophysiological Characteristics
C1 Area Neurons
More C1 cells were active (ie, showed ongoing action potential firing) in WRs (47% of 43 neurons) than in SHRs (9% of 11 neurons; Figure 4A). "Quiescent" neurons in WRs and SHRs had identical resting membrane potential (–54.5±0.9 mV, n=23, versus –54.6±2.0 mV, n=10). Actively firing C1 WR neurons had a resting membrane potential of –47.9±1 mV (P<0.01 compared with silent C1 WRs). Because only 1 C1 SHR cell had ongoing discharge, such a comparison is not possible for that strain.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Figure 4. Ongoing activity and responses to Ang II of C1 and A2 neurons. A, Distribution of silent vs active neurons in C1 and A2 areas. In both areas fewer cells in SHR exhibited ongoing action potential activity. B, Effect of Ang II (200 nmol/L) on membrane potential of C1 and A2 neurons. Because we have only encountered 1 C1 cell with ongoing activity in SHR slices, the effect of Ang II is not shown here. C, Effect of Ang II (200 nmol/L) on [Ca2+]i estimated using Rhod-2. In A2 cells of SHR, Ang II evoked much weaker Ca2+ response. Note that, in C1 cells, the trend was opposite.

A2 Neurons
A2 cells (silent or quiet cells) of WRs and SHRs had similar resting membrane potential (–56±2 versus –58±3 mV, n=8 and 9, correspondingly; P>0.05). A total of 38% of WR and 22% of SHR A2 neurons were active (Figure 4A).

Responses to Ang II
C1 Area Neurons
Ang II (200 nmol/L) evoked a larger depolarization in the active rather than in silent C1 WR cells (+4.8±0.6 mV, n=20 and 2.8±0.7 mV, n=23, correspondingly; P<0.05; Figures 4B and 5Down). The average amplitude of depolarization in silent SHR C1 cells (2.2±0.6) was not different from the silent WR cells (P>0.1). Only silent WR neurons were used for comparison because we only found 1 active cell in slices from SHRs. Ang II evoked distinct [Ca2+]i increases in 61% silent C1 neurons from WRs and 55% silent cells from SHRs. There was a trend of a higher [Ca2+]i response in SHR neurons (Figure 4C), but this was not significant.


Figure 5
View larger version (40K):
[in this window]
[in a new window]

  		Figure 5. Losartan (1 µmol/L) blocks the excitatory effect of Ang II on an A2 neuron of WR. Upper Trace, first application of Ang II. Lower trace, Second application of Ang II after 25 minutes of wash in the presence of losartan (1 µmol/L).

A2 Neurons
Ang II depolarized the majority of WR and SHR A2 neurons (+5.6±1.3 versus +4±0.8 mV), but there was no difference between rat strains (P>0.05; Figure 4B). Ang II triggered a clear increase in [Ca2+]i in 75% of WR A2 neurons (average increase: +39±9% in 6 responding neurons), but in SHR A2 cells we only found a response in 2 of 9 cells (ie, 22%). Thus, the average increase in [Ca2+]i in WRs was greater than in SHRs (129±9% versus 106±3%; P<0.05; Figure 4C). All of the effects of Ang II were reversible and/or could be abolished or prevented by 1 µmol/L of losartan (Figures 5 and 6Down).


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Figure 6. Losartan blocks the elevation in [Ca2+]i induced by Ang II in C1 cells with ongoing activity in WR (n=5). To confirm that Ca2+ mobilization is also an Ang II type 1 receptor–mediated event in some cells, Ang II was applied for the second time after 25 minutes of wash. As expected, losartan completely prevented all of the effects of Ang II.

To assure that our conclusions were not dependent on the way the data have been analyzed, we also evaluated them by grouping cells in a variety of different ways, such as "Ang II" responsive and "nonresponsive" categories. However, this did not affect our conclusions (data not shown). In addition, we did not find significant differences in passive input resistance (measured by hyperpolarizing current pulses) of comparable populations of WR and SHR neurons (data not shown).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study is the first to directly compare various properties of identified CAergic neurons from C1 and A2 areas of SHRs with a normotensive WR strain. Although a number of differences have been documented, we believe that the most important finding of this study is the confirmation of the first hypothesis posed in the Introduction. It appears that C1 varicosities in SHRs release almost double the amount of CA via large quanta that have transmitter loads comparable to those found in peripheral chromaffin granules. We have also revealed that, whereas CAergic neurons of A2 and C1 cell groups in SHRs are not electrically "hyperactive," they are differentially sensitive to Ang II. Unexpectedly, SHR A2 neurons were less sensitive to this critical neuromodulator, whereas an opposite trend was evident in C1 cells. Taken together, these observations confirm that the changes associated with the hypertensive phenotype are not universal across all of the central neurons equipped to synthesize CA. Instead, the differences revealed in C1 are in some respects the opposite of those that we found in the A2 area.

Looking for the differences in central CA neurons in a hypertensive and a normotensive rat strain has been made possible by the introduction of new approaches, such as virally mediated genetic targeting of these neurons, which allows their direct identification and microamperometric analysis of CA release from their fluorescently labeled varicosities in organotypic slices of rat brain stem.28,29,41 Consistent with our previous study,28 release of CA from axonal varicosities of A2 and C1 neurons occurred in 2 types of quanta. The most frequent release events have a median of 0.28 pC1/3, (0.022 pC), corresponding with {approx}70 000 CA molecules. This is compatible with vesicles with an average size of 75 nm, assuming a 0.5-mol/L intravesicular CA concentration.28,42 In addition, there are rare but very large quanta28 for which the neuroanatomical substrate in the brain has not yet been identified, but they may be related to vesicular organelles described in sympathetic neurons.43 Complex events, small and large, make a considerable contribution to total release. Overall in A2 neurons, large events composed a similar fraction of total CA release in WRs and SHRs, whereas release from C1 varicosities of SHRs was altered: the contribution of large quanta rose from 57% in WRs to 86% in SHRs, whereas the fraction of small events decreased. Higher output from large events was a combination of their much higher transmitter load ({approx}270%) and higher frequency relative to WRs. Increase in the size of these events may be because of a higher norepinephrine transporter activity in the SHR brain stem7; an increased expression of tyrosine hydroxylase, the key enzyme of NA/ADR biosynthesis, in the SHR ventral medulla,44; and/or alterations in the vesicular storage apparatus.6 These changes, when combined, possibly alter the way the vesicles are formed or filled. Interestingly this was area specific and not evident in A2 varicosities.

We believe that such reorganization of the CA vesicular pools may have a major impact on downstream signaling by C1 varicosities. Because large release events are very fast in the brain,28 they deliver copious quantities of CA into the extracellular space essentially instantaneously. Consistent with the "volume" mode of transmission, varicosities of C1 neurons in the spinal cord seldom form tight oppositions with preganglionic sympathetic neurons.45 Thus, CA released in the volume mode of transmission may spread further from the release site than when released in small quanta. When released within synaptic appositions,45,46 a high quantal load may result in synaptic overspill and add to volume transmission.

The RVLM provides tonic stimulatory input to many central nervous system sites of autonomic control, including the sympathetic preganglionic neurons.18–20 Hence, by releasing bulk loads of the transmitter, C1 varicosities could signal to a wider range of cellular targets in SHRs. In addition, by analogy with granules in chromaffin cell, large vesicles are likely to release copious amounts of cotransmitters (such as ATP, chromogranin, and neuropeptide Y), which could play an additional important signaling role.

The total CA release/time was higher in SHRs than WRs for both C1 and A2 varicosities. This observation is at odds with the lower levels of ongoing action potential activity in SHRs and may suggest a prevalence of action potential–independent release in SHRs. However, at present, we have insufficient evidence to support this hypothesis, because microamperometry does not take into account the balance between the numbers of release-active and "dormant" varicosities.

Regarding the second objective of this study, we found no evidence that either C1 or A2 neurons of SHRs are "hyperexcitable" or "hyperactive," at least under our in vitro conditions. In fact, fewer C1 cells of SHRs had an ongoing discharge compared with WRs (10% versus 53%). The techniques used here required genetic labeling of the CA neurons with EGFP and their optical identification. Therefore, this study could only be conducted in vitro, and this limitation needs to be acknowledged. Obviously, within the context of the intact brain, CA neurons receive various synaptic and paracrine signals, which may well affect their firing frequencies. Therefore, differences in neuronal activity in slice cultures predominantly reflect limited synaptic inputs and the intrinsic features of these cells, which suggests that neither A2 nor C1 neurons are electrically hyperactive.

The third hypothesis tested was that the effects of Ang II on electric activity or Ca2+ of CA neurons mobilization are enhanced in SHRs. Here, we only analyzed the fast, nongenomic effects of Ang II, which occur within minutes and involve effects on ion channels, Ca2+ stores, and transmitter release, similar to those effects described previously in chromaffin cells.10 Consistent with the results obtained in peripheral models, Ang II depolarized many A2 and C1 neurons and triggered [Ca2+]i elevations. Based on previous evidence10,47 and the present experiments, such [Ca2+]i elevations are largely attributable to Ca2+ release from the intracellular stores. Interestingly, WR A2 neurons responded to Ang II more vigorously than SHRs, both in terms of depolarization and Ca2+ release. A2 neurons are most likely a part of an antihypertensive homeostatic mechanism, as several groups including us have shown.23,25,26 This may involve NA release from A2 neurons within the nucleus tractus solitarius and/or from A2 projections to the hypothalamus,48,49 as reviewed previously.5 Thus, reduced sensitivity of the A2 cells to Ang II might further compromise their homeostatic role in the SHR.

Perspectives
This study has uncovered previously unsuspected changes in central CA transmission in SHRs that are area specific. A2 cells of SHRs have a reduced sensitivity to Ang II, which could indicate their weaker restraining influence on blood pressure. C1 varicosities of the SHR, but not WR, preferentially release CA in large quanta. This is likely to translate into enhanced impact on the cellular recipients of C1 signals throughout the central nervous system. Both changes may potentially lead to elevated sympathetic activity and hypertension in SHRs. The molecular approaches of this study, in particular, the cell-specific viral vector–mediated gene expression, will be instrumental for future investigations into how differences at the single cell level in vitro translate into a genetic predisposition to hypertension in vivo.


*    Acknowledgments
 
Sources of Funding

A.G.T. is a recipient of a fellowship from the British Heart Foundation (FS/04/003). S.K. and J.F.R.P. are in receipt of British Heart Foundation Programme (RG/07/006/23634) and Project (PG/06/085) grants. S.W. and M.K.R. were supported by National Institutes of Health grant 5R37 HL033610.

Disclosures

None.

Received April 7, 2008; first decision April 24, 2008; accepted June 11, 2008.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Grassi G. Counteracting the sympathetic nervous system in essential hypertension. Curr Opin Nephrol Hypertension. 2004; 13: 513–519.[CrossRef][Medline] [Order article via Infotrieve]
  2. Schlaich MP, Lambert E, Kaye DM, Krozowski Z, Campbell DJ, Lambert G, Hastings J, Aggarwal A, Esler MD. Sympathetic augmentation in hypertension: role of nerve firing, norepinephrine reuptake, and angiotensin neuromodulation. Hypertension. 2004; 43: 169–175.[Abstract/Free Full Text]
  3. Judy WV, Farrell SK. Arterial baroreceptor reflex control of sympathetic nerve activity in the spontaneously hypertensive rat. Hypertension. 1979; 1: 605–614.[Abstract/Free Full Text]
  4. Iriuchijima J, Numao Y, Suga H. Effect of increasing age on hemodynamics of spontaneously hypertensive rats. Jpn Heart J. 1975; 16: 257–264.[Medline] [Order article via Infotrieve]
  5. Kasparov S, Teschemacher AG. Altered central catecholaminergic transmission and cardio-vascular disease. Exp Physiol. 2008; 93: 725–740.[Abstract/Free Full Text]
  6. Rho JH, Newman BL, Alexander N, Hough KL. Enhanced NE uptake by isolated hypothalamic storage vesicles of hypertensive rats. Hypertension. 1983; 5: 3–7.[Abstract/Free Full Text]
  7. 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.[Abstract/Free Full Text]
  8. Reja V, Goodchild AK, Phillips JK, Pilowsky PM. Tyrosine hydroxylase gene expression in ventrolateral medulla oblongata of WKY and SHR: a quantitative real-time polymerase chain reaction study. Autonomic Neurosci. 2002; 98: 79–84.[CrossRef][Medline] [Order article via Infotrieve]
  9. Matsuura T, Kumagai H, Kawai A, Onimaru H, Imai M, Oshima N, Sakata K, Saruta T. Rostral ventrolateral medulla neurons of neonatal Wistar-Kyoto and spontaneously hypertensive rats. Hypertension. 2002; 40: 560–565.[Abstract/Free Full Text]
  10. Teschemacher AG, Seward EP. Bidirectional modulation of exocytosis by angiotensin II involves multiple G-protein-regulated transduction pathways in chromaffin cells. J Neurosci. 2000; 20: 4776–4785.[Abstract/Free Full Text]
  11. Allen AM. Blockade of angiotensin AT1 receptors in the rostral ventrolateral medulla of spontaneously hypertensive rats reduces blood pressure and sympathetic nerve discharge. J Renin Angiotensin Aldosterone Syst. 2001; 2: S120–S124.[Abstract/Free Full Text]
  12. Berecek KH, Kirk KA, Nagahama S, Oparil S. Sympathetic function in spontaneously hypertensive rats after chronic administration of captopril. Am J Physiol. 1987; 252: H796–H806.[Medline] [Order article via Infotrieve]
  13. Mayorov DN, Head GA. AT1 receptors in the RVLM mediate pressor responses to emotional stress in rabbits. Hypertension. 2003; 41: 1168–1173.[Abstract/Free Full Text]
  14. Veerasingham SJ, Raizada MK. Brain renin-angiotensin system dysfunction in hypertension: recent advances and perspectives. Br J Pharmacol. 2003; 139: 191–202.[CrossRef][Medline] [Order article via Infotrieve]
  15. Pan SJ, Zhu M, Raizada MK, Sumners C, Gelband CH. ANG II-mediated inhibition of neuronal delayed rectifier K+ current: role of protein kinase C-alpha. Am J Physiol. 2001; 281: C17–C23.
  16. Sun C, Du J, Sumners C, Raizada MK. PI3-kinase inhibitors abolish the enhanced chronotropic effects of angiotensin II in spontaneously hypertensive rat brain neurons. J Neurophysiol. 2003; 90: 3155–3160.[Abstract/Free Full Text]
  17. Sun C, Li H, Gao Y, Matsuura T, Upchurch PA, Raizada MK, Sumners C. Lack of macrophage migration inhibitory factor regulation is linked to the increased chronotropic action of angiotensin II in SHR neurons. Hypertension. 2007; 49: 528–534.[Abstract/Free Full Text]
  18. Chalmers JP, Blessing WW, West MJ, Howe PR, Costa M, Furness JB. Importance of new catecholamine pathways in control of blood pressure. Clin Exp Hypertension. 1981; 3: 393–416.[CrossRef]
  19. Reis DJ, Ross CA, Ruggiero DA, Granata AR, Joh TH. Role of adrenaline neurons of ventrolateral medulla (the C1 group) in the tonic and phasic control of arterial pressure. Clin Exp Hypertension. 1984; 6: 221–241.[CrossRef]
  20. Dampney RA, Goodchild AK, Robertson LG, Montgomery W. Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study. Brain Res. 1982; 249: 223–235.[CrossRef][Medline] [Order article via Infotrieve]
  21. Smith JK, Barron KW. The rostral and caudal ventrolateral medulla in young spontaneously hypertensive rats. Brain Res. 1990; 506: 153–158.[CrossRef][Medline] [Order article via Infotrieve]
  22. Schreihofer AM, Guyenet PG. Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo. J Comp Neurol. 1997; 387: 524–536.[CrossRef][Medline] [Order article via Infotrieve]
  23. Snyder DW, Nathan MA, Reis DJ. Chronic lability of arterial pressure produced by selective destruction of the catecholamine innervation of the nucleus tractus solitarii in the rat. Circ Res. 1978; 43: 662–671.[Free Full Text]
  24. 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.[Free Full Text]
  25. Talman WT, Perrone MH, Reis DJ. Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers. Science. 1980; 209: 813–815.[Abstract/Free Full Text]
  26. Duale H, Waki H, Howorth P, Kasparov S, Teschemacher AG, Paton JFR. Restraining infulence of A2 neurones in chronic control of blood pressure in SHR. Cardiovasc Res. 2007; 76: 184–193.[Abstract/Free Full Text]
  27. Kasparov S, Teschemacher AG, Paton JFR. Application of viral gene transfer in studies of neurogenic hypertension. In: Raizada MK, Paton JFR, Katovich MJ, Kasparov S, eds. Cardiovascular Genomics. Totowa, NJ: Humana Press; 2005: 243–264.
  28. Chiti Z, Teschemacher AG. Exocytosis of norepinephrine at axon varicosities and neuronal cell bodies in the rat brain. FASEB J. 2007; 21: 2540–2550.[Abstract/Free Full Text]
  29. Teschemacher AG, Paton JFR, Kasparov S. Imaging living central neurones using viral gene transfer. Adv Drug Deliv Rev. 2005; 57: 79–93.[CrossRef][Medline] [Order article via Infotrieve]
  30. Teschemacher AG, Wang S, Lonergan T, Duale H, Waki H, Paton JFR, Kasparov S. Targeting specific neuronal populations in the brainstem using adeno- and lentiviral vectors: applications for imaging and studies of cell function. Exp Physiol. 2005; 90: 61–69.[Abstract/Free Full Text]
  31. Berecek KH, Swords BH, Lo S, Kirk KA. Effect of angiotensin converting enzyme inhibitors upon brain angiotensin II binding. J Hypertens. 1992; 10: 545–552.[CrossRef][Medline] [Order article via Infotrieve]
  32. Hermann K, Phillips MI, Raizada MK. Metabolism of angiotensin peptides by neuronal and glial cultures from rat brain. J Neurochem. 1989; 52: 863–868.[CrossRef][Medline] [Order article via Infotrieve]
  33. Raizada MK, Lu D, Yang H, Yu K. AT1-receptors and cellular actions of angiotensin II in neuronal cultures of stroke prone-spontaneously hypertensive rat brain. Adv Exp Med and Biol. 1996; 396: 71–78.
  34. 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.[Abstract/Free Full Text]
  35. Wang S, Teschemacher AG, Paton JFR, Kasparov S. Mechanism of nitric oxide action on inhibitory GABAergic signaling within the nucleus tractus solitarii. FASEB J. 2006; 20: 1537–1539.[Abstract/Free Full Text]
  36. Barnes KL, McQueeney AJ, Ferrario CM. Receptor subtype that mediates the neuronal effects of angiotensin II in the rat dorsal medulla. Brain Res Bull. 1993; 31: 195–200.[CrossRef][Medline] [Order article via Infotrieve]
  37. Ferguson AV, Li Z. Whole cell patch recordings from forebrain slices demonstrate angiotensin II inhibits potassium currents in subfornical organ neurons. Reg Peptides. 1996; 66: 55–58.[CrossRef][Medline] [Order article via Infotrieve]
  38. Li YW, Guyenet PG. Neuronal excitation by angiotensin II in the rostral ventrolateral medulla of the rat in vitro. Am J Physiol. 1995; 268: R272–R277.[Medline] [Order article via Infotrieve]
  39. Li YW, Guyenet PG. Angiotensin II decreases a resting K+ conductance in rat bulbospinal neurons of the C1 area. Circ Res. 1996; 78: 274–282.[Abstract/Free Full Text]
  40. Wang G, Anrather J, Glass MJ, Tarsitano MJ, Zhou P, Frys KA, Pickel VM, Iadecola C. Nox2, Ca2+, and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius. Hypertension. 2006; 48: 482–489.[Abstract/Free Full Text]
  41. Lonergan T, Teschemacher AG, Hwang DY, Kim KS, Kasparov S. Targeting brainstem centres of cardiovascular control using adenoviral vectors: impact of promoters on transgene expression. Physiol Genom. 2005; 20: 165–172.[Abstract/Free Full Text]
  42. Wightman RM, Jankowski JA, Kennedy RT, Kawagoe KT, Schroeder TJ, Leszczyszyn DJ, Near JA, Diliberto EJ Jr, Viveros OH. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc Natl Acad Sci U SÅ. 1991; 88: 10754–10758.[Abstract/Free Full Text]
  43. Schwab ME, Thoenen H. Mechanism of uptake and retrograde axonal transport of noradrenaline in sympathetic neurons in culture: reserpine-resistant large dense-core vesicles as transport vehicles. J Cell Biol. 1983; 96: 1538–1547.[Abstract/Free Full Text]
  44. Reja V, Goodchild AK, Pilowsky PM. Catecholamine-related gene expression correlates with blood pressures in SHR. Hypertension. 2002; 40: 342–347.[Abstract/Free Full Text]
  45. Anderson CR, McLachlan EM, Srb-Christie O. Distribution of sympathetic preganglionic neurons and monoaminergic nerve terminals in the spinal cord of the rat. J Com Neurol. 1989; 283: 269–284.[CrossRef][Medline] [Order article via Infotrieve]
  46. Morrison SF, Milner TA, Reis DJ. Reticulospinal vasomotor neurons of the rat rostral ventrolateral medulla: relationship to sympathetic nerve activity and the C1 adrenergic cell group. J Neurosci. 1988; 8: 1286–1301.[Abstract]
  47. Fernandez SF, Huang MH, Davidson BA, Knight PR III, Izzo JL Jr. Modulation of angiotensin II responses in sympathetic neurons by cytosolic calcium. Hypertension. 2003; 41: 56–63.[Abstract/Free Full Text]
  48. Simon OR, Basuray BN, West WL, Copeland R. Interaction between the baroreflex and anterior hypothalamic stimulation. Demonstration of a noradrenergic involvement. Neuropharmacol. 1985; 24: 665–675.[CrossRef][Medline] [Order article via Infotrieve]
  49. Kobilansky C, Lanzinger I, Philippu A. Release of endogenous catecholamines in the nucleus tractus solitarii during experimentally induced blood pressure changes. Naunyn-Schmiedebergs Arch Pharmacol. 1988; 337: 125–130.[Medline] [Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
52/2/351    most recent
HYPERTENSIONAHA.108.114371v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Teschemacher, A. G.
Right arrow Articles by Kasparov, S.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Teschemacher, A. G.
Right arrow Articles by Kasparov, S.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*2-PHENYLETHYLAMINE
*LOSARTAN POTASSIUM
Medline Plus Health Information
*High Blood Pressure
Related Collections
Right arrow ACE/Angiotension receptors
Right arrow Animal models of human disease
Right arrow Other hypertension
Right arrow Hypertension - basic studies
Right arrow Autonomic, reflex, and neurohumoral control of circulation

Hypertension Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2008 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.