Published online before print December 9, 2002, doi: 10.1161/01.HYP.0000047513.75459.7E
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 2003;41:56.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
Modulation of Angiotensin II Responses in Sympathetic Neurons by Cytosolic Calcium
From the Departments of Pharmacology (S.F.F., J.L.I.), Medicine (J.L.I.), and Anesthesiology (B.A.D., P.R.K.), School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York; and University of Arizona Sarver Heart Center (M.-H.H.), Tuscon, Ariz.
Correspondence to Joseph L. Izzo, Jr, MD, Department of Medicine, Millard Fillmore Health System, 3 Gates Circle, Buffalo, NY 14209. E-mail JIzzo{at}KaleidaHealth.Org
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Both stimulatory and suppressive responses of the sympathetic nervous system to angiotensin II (AII) have been reported in intact animals. To elucidate possible cellular mechanisms, we studied AII-induced changes in cytosolic Ca2+ ([Ca2+]i) in primary cultures of rat stellate ganglion neurons. Two different patterns of [Ca2+]i responses to AII were observed: dose-dependent increases in [Ca2+]i in cells with intrinsically low baseline [Ca2+]i (n=64) and dose-dependent suppression of [Ca2+]i in neurons with intrinsically higher baseline [Ca2+]i (n=46). Individual neurons could express both response patterns to AII. In neurons with low basal [Ca2+]i, superfusion with Ca2+ ionophore (ionomycin) increased [Ca2+]i and reversed the initial AII-induced stimulatory pattern. L-type Ca2+ channel antagonism (nifedipine) in neurons with high baseline [Ca2+]i lowered [Ca2+]i and reversed the initial AII-induced suppressive response. Both stimulatory and suppressive responses were abolished by AT1 receptor antagonism (losartan). AII-induced stimulatory responses were blocked by IP3 receptor antagonism (2-APB) and by thapsigargin. AII-induced suppression of neuronal [Ca2+]i was blunted when Na-Ca exchange was impaired. We conclude that [Ca2+]i acts as a switch for AII-mediated stimulatory and suppressive responses in individual sympathetic neurons. AT1 receptor-mediated neuronal stimulation and suppression may allow local homeostatic adaptation to meet complex systemic needs.
Key Words: sympathetic nervous system • angiotensin II • calcium • calcium channel blockers • norepinephrine • inositol
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The renin-angiotensin and sympathetic nervous systems are homeostatically interconnected by a complex series of mutually reinforcing and mutually inhibiting interactions. In general, each of these systems tends to reinforce the pressor effects of the other as the two systems act to defend arterial pressure. Sympathetic stimulation is a major controller of juxtaglomerular cell renin release through the actions of catecholamines on juxtaglomerular cell ß1 receptors.1 The angiotensin II (AII) subsequently generated in the bloodstream can exert positive feedback on sympathetic nervous activity by several complex mechanisms, including direct stimulatory actions of AII on central and peripheral sympathetic neuronal activity and catecholamine release.1–3
In addition to its stimulatory effects, AII can also suppress sympathetic neuronal activity in vivo. In animals and humans, complex reflex inhibition occurs when increases in arterial pressure stimulate arterial baroreflexes to cause reflex suppression of sympathetic nervous outflow. A more confusing picture emerges in whole-animal experiments designed to isolate the contributions of the peripheral sympathetic nervous system: AII infusion can facilitate,4 suppress,5 or have no effect6 on norepinephrine release from postganglionic sympathetic neurons. In humans, a similar pattern of sympathetic neural responses has been reported, with some studies showing increased norepinephrine spillover3 and others showing no response7 or decreased norepinephrine release3,7,8 in response to AII. In other excitable cell types such as cardiomyocytes or vascular smooth muscle cells, a paradoxical response has also been reported. AII treatment was shown to increase cytosolic calcium ([Ca2+]i) by stimulation of IP3-dependent pathways9,10 and was also reported to decrease [Ca2+]i through enhanced calcium efflux.11,12
The current studies were undertaken to examine the effects of AII on neuronal [Ca2+]i and to investigate whether alterations in [Ca2+]i play a role in the apparent "bidirectional" excitatory and suppressive responses to AII seen in sympathetic neurons studied in vivo. Using isolated primary sympathetic neurons from rats, we first related neuronal responses to the dose of AII and to the baseline [Ca2+]i. To confirm that the same neuron is capable of both stimulatory and suppressive responses to AII, cells with spontaneously low [Ca2+]i were exposed to a Ca2+ ionophore and cells with spontaneously high [Ca2+]i were exposed to nifedipine. We used losartan to investigate the role of the AT1 receptors and confirmed IP3 pathway dependency with an IP3 receptor blocker and by prior calcium depletion. The role of Na-Ca exchange was studied by using Na+-free buffers.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Culture of Rat Stellate Ganglion Neurons
Details of this method have been described elsewhere.13 Briefly, adult and newborn (2 to 4 days old) Sprague-Dawley rats were anesthetized with halothane. Thoracotomy-exposed stellate ganglia were removed, rinsed, and minced in Tyrode solution containing (in mmol/L) NaCl 124, KCl 2.6, MgCl2 1.2, KH2PO4 1.2, glucose 5, HEPES 5, NaHCO3 3.6, and CaCl2 1.5, pH 7.4. Minced neuronal tissues were placed in an enzyme solution containing 0.25% trypsin and 0.1% collagenase and incubated in a 36°C shaker bath for 30 minutes. The cell suspension was centrifuged at 80g for 4 minutes. The supernatant was discarded and the dissociated cells were resuspended in growth media containing MEM (Sigma), nerve growth factor-ß 100 ng/mL (Sigma), and 10% FBS. Resuspended neurons were plated onto Thermanox TM tissue culture coverslips (Lux, Flow Laboratory Inc) precoated with laminin (20 µg/mL). Cells were maintained in an incubator at 37°C under a 95% air and 8% CO2 atmosphere. Culture media were changed twice per week. Typically, after 3 to 4 days in culture, neurons attached firmly on coverslips and developed extensive neurites. Neurons were selected for study after at least 3 days in culture.
Fura-2 Loading of Sympathetic Neurons
Fura-2 am (Molecular Probes) was dissolved in DMSO to make a stock solution of 1 mmol/L. Loading solution (6 µmol/L Fura-2) was prepared by vigorously mixing the Fura-2 stock solution with Tyrode solution containing 1% BSA. Neurons in cultures were incubated in the loading solution for 30 minutes in an incubator. The Fura-2-loaded cells were perfused with Tyrode solution for 20 minutes, allowing de-esterification of Fura-2 in the cells and its removal from the extracellular space.
Measurement of [Ca2+]i Transients
A coverslip containing Fura-2-loaded cells was mounted in a cell chamber (Bioptechs FCS2, Bioptechs Inc) that was continuously perfused on the stage of an inverted microscope. A ratio-based microscopic fluorescent spectrophotometer (Photon Technology International Inc) was used to excite the dye at 340/380 nm, alternately at 2-Hz sampling rate. Measurements of 510-nm emissions were recorded as fluorescence ratios (R340/380) from a video screen window encompassing a single neuronal soma. All fluorescence signals were corrected for background by subtracting the average fluorescence intensity in the cell-attached configuration. [Ca2+]i was calculated by the equation [Ca2+]i=Kd · (R-Rmin)/(Rmax-R) · Sf2/Sb2. Kd was determined experimentally by using an in vitro calibration method, with calcium concentrations increased stepwise from 0 to 10 mmol/L.14 Rmax and Rmin were the fluorescence ratios obtained from the cells exposed to solution containing 1.5 mmol/L CaCl2 and 15 µmol/L ionomycin, which saturated [Ca2+]i, and then to a Ca2+-free solution containing 10 mmol/L EGTA and 15 µmol/L ionomycin, which would deplete intracellular Ca2+. Sf2 and Sb2 were fluorescence intensities at 380 nm with Ca2+ free and Ca2+ saturated solutions, respectively.
Perfusion System
Experimental solution (control or drug-containing) flowed continuously through the cell chamber by gravity; the temperature of the perfusate was kept near 36°C. The volume of fluid in the cell chamber was 
0.1 mL; the rate of perfusion was 0.3 to 0.4 mL/min. Perfusate containing drugs could be selected from several reservoirs with multichannel valve without interruption of flow. O2 was continuously bubbled through the perfusion reservoirs.
Pharmacological Interventions
Once the baseline [Ca2+]i became stabilized, AII was administered at different concentrations (1 pmol/L to 1 µmol/L) to neuronal cultures. The role of the AII receptor subtype was determined by administering AII to the neurons in the presence of the AII type I (AT1)-receptor blocker losartan and AII type II (AT2)-receptor blocker PD-123319. To assess whether the AII-induced increase in [Ca2+]i was due to intracellular Ca2+ release, neurons with a positive response to AII were rechallenged by AII in a Ca2+-free buffer and after pretreatment with thapsigargin. Furthermore, to determine whether an IP3-dependent mechanism was involved in intracellular Ca2+ release, the effect of AII was tested in the presence of 2-aminoethoxydiphenyl borate (2-APB, 200 µmol/L, Calbiochem), a specific IP3 receptor blocker.15 To assess the contribution of the Na-Ca exchange mechanism, AII experiments were performed under extracellular Na+-free conditions. Na+-free buffer was prepared by either substituting 124 mmol/L Na+ with equimolar N-methyl-D-glucamine (NMG) or with LiCl (Sigma-Aldrich). Finally, to determine whether altering [Ca2+]i could change neuronal response in the same cells, neurons were exposed to the calcium ionophore ionomycin (3 µmol/L) to raise baseline [Ca2+]i or to the L-type Ca2+ channel blocker nifedipine (1 µmol/L) to lower baseline [Ca2+]i. AII was then administered to these cells after a new baseline [Ca2+]i was obtained.
Norepinephrine Release Assay
Primary sympathetic neuronal cultures were prepared as described above. Dissociated stellate ganglion cells pooled from 3 to 5 rats in each preparation were distributed to 3 to 5 collagen-coated culture dishes (Fisher Scientific) to maximize cell density. Culture medium was removed from culture dishes and replaced with a buffer solution containing NaCl 124 mmol/L, HEPES 30 mmol/L, NaHCO3 3.6 mmol/L, KCl 4 mmol/L, MgCl2 1 mmol/L, CaCl2 1.5 mmol/L, glucose 5.6 mmol/L, ascorbic acid 0.2 mmol/L, and 50 nmol/L [3H]NE (39 Ci/mmol, Amersham International). After incubation for 50 minutes at 37°C, the isotope-supplemented medium was removed. The excess [3H]NE was washed from the monolayer by 6 consecutive 0.5-mL aliquots of the buffer solution without [3H]NE at 37°C. Release of [3H]NE was followed by exposing the monolayer to 0.4 mL buffer for 10 minutes at 37°C in the absence (basal release) and presence of AII. After each AII treatment, medium was collected and the monolayers washed with 5 mL buffer solution for 10 minutes. Basal [3H]NE release was reestablished before applying new concentrations of AII. The effect of AII on [3H]NE release was also tested in the presence of losartan (10 µmol/L). Unreleased [3H]NE in the monolayer was extracted with perchloric acid (0.4 mol/L) at the end of experiment. The amount of [3H]NE released into the medium was expressed as a percentage of the total amount of radioactivity in the cell monolayer at the beginning of the experiment.
Data Analysis
The amplitude of Ca2+ transients was measured with Felix software (PTI). Neuronal responses after drug treatment were expressed as mean of [Ca2+]i (mean±SD). The paired Student t test, ANOVA, and Tukey posttest analysis were used for statistical studies. A probability value of <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Patterns of Neuronal Response to AII
Primary cultures of neonatal rat sympathetic neurons when exposed to brief pulses (3 minutes) of AII results in acute changes in intracellular calcium concentration ([Ca2+]i). Based on morphology of observed Ca2+ tracings, responses were grouped into two categories. In 45% (n=64 of 142 cells) of the neurons tested, AII exposure resulted in an increase in [Ca2+]i (stimulatory pattern, Figure 1A). This pattern showed an initial phase of rapid transient increase in [Ca2+]i followed by a sustained plateau phase. In 32% (n=46 of 142 cells), AII caused a paradoxical decrease in [Ca2+]i (suppressive pattern, Figure 1B). In 22% (n=32 of 142 cells), AII did not elicit any response. Microscopic examination of neurons from both groups did not exhibit any morphological differences. Similar results were seen when using adult rat sympathetic neurons.
|
Role of AT1 and AT2 Receptor Subtypes in AII-Induced Responses
To identify AII receptor subtypes involved in mediating changes in [Ca2+]i, specific AT1 and AT2 receptor blockers were used. Neurons that showed stimulatory responses to 10 pmol/L AII were incubated with 10 µmol/L losartan, an AT1 receptor blocker, for 5 minutes. Losartan pretreatment completely abolished stimulatory responses when rechallenged with AII (Figure 2A). In neurons that initially showed suppressive responses to 10 pmol/L AII, pretreatment with 10 µmol/L losartan likewise inhibited these suppressive responses when rechallenged (Figure 2B). Treatment with 1 µmol/L PD-123319, an AT2 receptor blocker, for 5 minutes did not modify the stimulatory nor suppressive responses to 10 pmol/L AII (data not shown). These results suggested that both stimulatory and suppressive responses caused by AII were primarily mediated by the activation of AT1 receptors. Involvement of AT2 receptors was not apparent.
|
Role of Baseline [Ca2+]i in AII-Induced Change in [Ca2+]i
Mean baseline [Ca2+]i for neurons that responded with stimulatory patterns was 78±39 nmol/L (n=64). In neurons that showed suppressive responses, mean baseline [Ca2+]i was 230±48 nmol/L. The difference in mean baseline [Ca2+]i was statistically significant (P<0.01). When data points were arbitrarily segregated into two groups, based on neuronal baseline [Ca2+]i, <200 nmol/L or >200 nmol/L, cells with low baseline [Ca2+]i (<200 nmol/L) predominantly showed increases in [Ca2+]i with AII treatment (n=76, Figure 3). In neurons with high baseline [Ca2+]i (>200 nmol/L), AII largely promoted decreases in [Ca2+]i (n=34, Figure 3). This relation between AII response and baseline [Ca2+]i exists for all AII doses, as shown in the scatterplot and 3-D model (Figure 4). The 3D model derived from 110 aggregated data points correlated baseline [Ca2+]i, AII dose applied, and the [Ca2+]i response to AII (
[Ca2+]i). At lower baseline [Ca2+]i, incremental increase in AII dose (1 pmol/L to 1 µmol/L) showed a dose-dependent increase in [Ca2+]i response. On the other hand, at higher baseline [Ca2+]i, increase in AII concentration showed a dose-dependent decrease in [Ca2+]i. These findings suggested that baseline [Ca2+]i in part determined specific patterns of AII-induced responses.
|
|
Reversibility of AII Responses Through Control of Baseline [Ca2+]i
To experimentally mimic observational data correlating baseline [Ca2+]i with specific AII responses, baseline [Ca2+]i was manipulated to a desired level. Baseline [Ca2+]i was either raised to a higher level by using a Ca2+ ionophore (ionomycin) or lowered by using an L-type Ca2+ channel blocker (nifedipine). In neurons that initially responded to 100 nmol/L AII with stimulatory patterns ([Ca2+]i=+133±42 nmol/L), baseline [Ca2+]i levels were raised by using 3 µmol/L ionomycin. Ionomycin treatment elevated [Ca2+]i levels from 78±53 nmol/L to 667±150 nmol/L. Rechallenge with the same AII dose (100 nmol/L) after elevation of baseline [Ca2+]i resulted in reversal of responses from stimulatory to suppressive patterns, [Ca2+]i=-438±107 nmol/L (Figure 5A). In neurons that initially responded to 100 nmol/L AII with suppressive patterns, [Ca2+]= -36±10 nmol/L, baseline [Ca2+]i levels were lowered by using 1 µmol/L nifedipine. Nifedipine treatment modestly lowered [Ca2+]i from 301±12 nmol/L to 244±16 nmol/L. Rechallenge with 100 nmol/L AII after lowering of baseline [Ca2+]i resulted in reversal of responses from suppressive to robust stimulatory patterns, [Ca2+]i=+246±50 nmol/L (Figure 5B). Induction of both stimulatory and suppressive responses in the same neuron through modification of baseline [Ca2+]i provided strong evidence that baseline [Ca2+]i determined the type of neuronal response to AII.
|
P<0.05.
Involvement of Inositol Trisphosphate Pathway in AII-Induced Stimulatory Responses
To characterize the secondary signals involved in the stimulatory response, the IP3 pathway was investigated. To directly block IP3 receptors, neurons were incubated with 200 µmol/L 2-aminoethoxydiphenyl borate (2-APB) for 5 minutes. 2-APB, by binding noncompetitively to IP3 receptors, effectively blocks any IP3-mediated response.15 Five minutes’ preincubation with 2-APB reduced stimulatory responses by 80% (Figure 6A). 2-APB treatment did not alter suppressive responses to AII (data not shown). To identify the source of Ca stores that resulted in the increase in [Ca2+]i, neurons that initially showed stimulatory patterns to 100 nmol/L AII ([Ca2+]i=+155±31 nmol/L) were incubated with 1 µmol/L thapsigargin for 10 minutes (Figure 6B). Restimulation showed minimal increases in [Ca2+]i ([Ca2+]i=+12±7 nmol/L). Dependence on intracellular Ca2+ stores and inhibition by 2-APB of stimulatory responses indicated that formation of IP3 was necessary for generating stimulatory responses.
|
Involvement of Na-Ca Exchange in AII-Induced Suppressive Responses
To explore the role of Na-Ca exchange in mediating suppression of [Ca2+]i, AII exposures were performed under extracellular Na+-free conditions. Na+-free buffers were prepared by equimolar replacement (124 mmol/L) of Na+ with N-methyl D-glucamine (+NMG) or with lithium chloride (+LiCl). Neurons with low baseline [Ca2+]i were treated with 3 µmol/L ionomycin to raise [Ca2+]i. Suppressive responses, [Ca2+]i=-467±29 nmol/L, were elicited by AII in these ionomycin-treated cells. Responses were completely inhibited when AII stimulations were performed with NMG buffer (Figure 7A) or LiCl buffer (Figure 7B). Recovery of suppressive responses were obtained when cells were restimulated under regular Na+-containing buffer conditions. Dependence of AII-induced decrease in [Ca2+]i on extracellular Na+ suggested that the Na-Ca exchange plays a role in generation of suppressive responses.
|
Norepinephrine Release Profiles
To assess the role of AII in modulating norepinephrine (NE) release, we compared NE release rates between baseline and post-AII treatment. AII exposure resulted in both stimulatory and inhibitory effects on NE release (Figure 8). In cultures with low baseline NE release (1.7±0.2%/10 minutes), AII induced a significant increase in NE release (n=18). In cultures with an initially high baseline NE release (3.5±0.5%/10 minutes), AII promoted a significant inhibition of NE release (n=10). This bidirectional effect of AII on NE release paralleled the observation seen with AII-induced changes in cytosolic Ca2+.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of this study identify complex postreceptor effects of AII on individual sympathetic neurons and identify the role of cytosolic calcium ([Ca2+]i) as an intrinsic "calcium switch mechanism" that modulates responses to AT1-receptor stimulation. In cells with intrinsically low cytosolic calcium ([Ca2+]i), which are presumably quiescent, AII activates IP3-mediated release of calcium from the endoplasmic reticulum, thereby activating neurons through acute increases in [Ca2+]i. When intraneuronal [Ca2+]i is elevated, AII causes AT1 receptor-mediated Na-Ca exchange, which tends to reduce [Ca2+]i, presumably passivating the activated neuron. Thus, the neuronal effects of AII (ie, stimulation or suppression) are at least partially dependent on baseline [Ca2+]i and in turn on the cellular mechanisms that affect Ca2+ influx, release, and extrusion. It would appear that each neuron can use its "Ca2+ switch" to modulate its own activity level according to complex needs. After periods of neuronal quiescence, activation of the renin-angiotensin system may enhance and sustain sympathetic activation. During periods of chronic sympathetic activation, however, AII may exert a passivating effect.
The current findings are completely consistent with the well-known tachyphylaxis to infused AII that arises in humans within hours to days, a phenomenon that led to the abandonment of AII as a reliable pressor hormone in humans.16 Our results also verify the apparent divergence in the scientific literature that includes credible descriptions of both stimulatory1,3,17–19 and suppressive effects5,8,20 of AII on the sympathetic nervous system. Finally, the enhancement of AII-dependent neuronal stimulation in the presence of the L-channel blocker nifedipine is consistent with the sympathoexcitation seen during long-term use of this agent in humans21 and may help explain the potential adverse effects observed with calcium antagonists in high-risk patients.22 The finding that both stimulatory and suppressive responses can be elicited in the same neuron argues strongly that the observed physiological effects are not mediated entirely by systemic reflexes. Furthermore, the "bidirectional" effect of AII on sympathetic neurons is clearly not due to activation of different (ie, stimulatory and inhibitory) subclasses of sympathetic neurons within sympathetic ganglia or to the stimulation of different subclasses of AII receptors, because both stimulatory and suppressive effects are blocked by the AT1-receptor blocker losartan (Figure 2). Thus, the modulation of AII effects occurs at the postreceptor level and is clearly affected by the cytosolic calcium concentration (Figure 5).
Modulation of neuronal function by AII in vivo could occur as a result of the effects of either locally generated tissue AII or circulating AII. Plasma AII levels are 5 pmol/L in normal individuals,3 so that the responses to picomolar dosages of AII in the current study are well within the range observed routinely in humans. Quantification of tissue concentrations of AII is considerably more difficult at present, but it has been assumed that locally generated AII modulates neuronal function as well.23 Small changes in plasma AII would be expected to exert significant effects on the sympathetic nervous system because critical AII-dependent sympathetic modulatory effects have been attributed to the actions of AII on circumventricular control centers such as the area postrema of the medulla oblongata, which does not have a blood-brain barrier.24 Generation of AII within the central nervous system is also well documented, and the brain synthesizes all components of the renin-angiotensin system within neurons or perineurocytes.23 Whether the observations from this study pertain more to plasma or tissue AII cannot be determined. The current data are also consistent with histochemical findings of high concentrations of AT1 receptors in rat sympathetic ganglion neurons,25,26 including stellate ganglion neurons that provide the primary sympathetic supply to the heart.
At the level of individual neurons, responses to 2-APB (an IP3 receptor blocker) and thapsigargin (which depletes endoplasmic reticular Ca2+ stores) demonstrate that AT1-receptor-mediated increases in [Ca2+]i are due to activation of the phospholipase C-IP3 pathway, with AII-dependent Ca2+ release from the endoplasmic reticulum (ER)(Figure 6). Further support for the idea that AII causes increased [Ca2+]i by releasing ER Ca2+ stores comes from other confirmatory studies in our laboratory demonstrating that removal of Ca2+ from the extracellular buffer only slightly reduces the acute [Ca2+]i responses to AII (data not included). Activation of the phospholipase C-IP3 signaling pathway is a well-known mechanism for AII-mediated cytosolic Ca2+ release from the ER of many types of mammalian cells27–30; the current results extend these findings to include sympathetic neurons.
Suppression of neuronal [Ca2+]i responses to AII appears to be tightly linked to altered Na-Ca exchange. This finding may serve to reconcile existing differences of opinion as to the overall effect of AII on the sympathetic nervous system. Teleologically, AII-induced sympathetic suppression may represent a conservation response that prevents exhaustion of a chronically stimulated sympathetic nervous system. Dependence of this suppressive response to AII on Na-Ca exchange is clearly demonstrated by the current experiments that altered the transmembrane sodium gradient in neurons with high baseline [Ca2+]I, using LiCl or NMG, which reduced the Na+ gradient-dependent extrusion of Ca2+ through Na-Ca exchange (Figure 7).
An additional mechanistic link relating cytosolic Ca2+ to modulation of AII-mediated neuronal activation probably occurs at the level of ER IP3 receptors. The degree of binding of IP3 to its ER receptors is tightly regulated by cytosolic Ca2+.31,32 This link may potentially explain the nifedipine responses observed in cells with spontaneously high [Ca2+]i. By decreasing Ca2+ influx, nifedipine may indirectly increase IP3 receptor binding, thereby promoting augmented ER Ca2+ release in response to AT1 receptor stimulation (Figure 5B). There may also be clinical relevance to these findings. L-Type calcium channel antagonists such as nifedipine cause acute and chronic sympathetic activation,21 a phenomenon that may be especially problematic in patients with hypertension, congestive heart failure, and myocardial infarction.22,33–35 Altered baroreflex inhibition could be one mechanism of sympathoinhibition in response to chronic vasodilation, but enhancement of AII-mediated neuronal activation in chronically stimulated sympathetic neurons may be another mechanism underlying chronic sympathoexcitation in high-risk patients with congestive heart failure and acute coronary syndromes.36,37
Whether or not these findings can be fully extrapolated to the intact nervous system remains unclear. The primary advantage of using an in vitro system is the absence of confounding physiological or reflex effects. However, this advantage is also an important drawback because the relative role of cellular and physiological modulating effects cannot be directly compared. Also, potential artifacts of culture preparation cannot be easily eliminated. Cultured sympathetic neurons go through a rigid dissection and isolation procedure that may affect cell membranes and will certainly disrupt cell-cell interactions. The use of nerve growth factor and other constituents of culture media may also influence the results. The fluorescence microscopic technique that was used measures integrated changes in [Ca2+]i over the cell body of individual neurons. Whether these integrated changes represent similar changes in axons or dendrites is also not known. In other cell types, Ca2+ is not uniformly distributed throughout the cytoplasm. Acute Ca2+ transients in many somatic cells occur as dense "sparks" or waves of sparks38,39 rather than uniform increases in [Ca2+]i. Whether comparable [Ca2+]i sparks or waves also occur in sympathetic neurons is not yet known.
These findings of an apparent "calcium switch" mechanism and the associated bidirectional neuronal response to AII open up multiple future research directions. On a cellular level, experiments can be designed to characterize the physiological significance of 
200 nmol/L baseline [Ca2+]i, the critical deflection point for the calcium switch and the divergent neuronal responses to AII (Figures 3 and 4). The interaction and regulation of IP3 receptors and Na-Ca exchangers should also be examined. Initial reports have suggested that IP3 receptors and Na-Ca exchangers colocalize in the same cellular subdomain.40 This physical proximity may provide further evidence for the counterregulatory function of these two signaling systems. Another area of study that will be of interest is the differential expression of protein isoforms. Several IP3 receptor and Na-Ca exchanger isoforms have been reported; an important question that can be addressed is how changes in isoform expression may affect overall regulation of cytosolic Ca2+.
Perspectives
Mutually reinforcing stimulatory responses of the sympathetic nervous and renin-angiotensin systems would tend to favor marked vasoconstriction and runaway increases in blood pressure unless a parallel system of negative interactions between the two systems also exists. The current results suggest that both positive and negative interactions between the two systems are controlled at the cellular level and are closely related to the calcium content of the cell. Teleologically, if the sympathetic nervous system (SNS) is chronically activated (neurons with high baseline calcium concentrations), additional stimulation by AII is unnecessary and may even be deleterious. On the other hand, if the SNS has been largely quiescent (cells with low baseline calcium), AII may be useful to extend or amplify the initial stress response. By integrating these whole-animal responses around the calcium metabolism of efferent SNS neurons, a "fail-safe" system may exist that integrates metabolic and neurophysiologic signals without requiring interposed central nervous system inputs or baroreflex modulation. The functional coupling between IP3-mediated Ca release and Ca efflux mediated by the Na-Ca exchanger is evidence of the ability of an individual neuron to integrate different stimuli. Future studies can be directed toward understanding how other cell signals (metabolic, toxic, and so forth) may influence neuronal Ca balance and whether imbalances exist in conditions such as hypertension and heart failure.
|
Acknowledgments |
|---|
This work was supported by the FDA Pilot Clinical Pharmacology Training Program Grant FDT-00889 and by the Departments of Medicine and Anesthesiology, State University of New York at Buffalo. We are grateful to Jadwiga Helinski for technical assistance.
Received June 18, 2002; first decision July 25, 2002; accepted November 6, 2002.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Reid IA. Interactions between ANG II, sympathetic nervous system, and baroreceptor reflexes in regulation of blood pressure. Am J Physiol. 1992; 262: E763–E778.[Medline] [Order article via Infotrieve]
2. Aiken JW, Reit E. Stimulation of the cat stellate ganglion by angiotensin. J Pharmacol Exp Ther. 1968; 159: 107–114.
3. Clemson B, Gaul L, Gubin SS, Campsey DM, McConville J, Nussberger J, Zelis R. Prejunctional angiotensin II receptors: facilitation of norepinephrine release in the human forearm. J Clin Invest. 1994; 93: 684–691.[Medline] [Order article via Infotrieve]
4. Kaufman LJ, Vollmer RR. Endogenous angiotensin II facilitates sympathetically mediated hemodynamic responses in pithed rats. J Pharmacol Exp Ther. 1985; 125: 128–134.
5. Kuo Y-JJ, Keeton TK. Captopril increases norepinephrine spillover rate in conscious spontaneously hypertensive rats. J Pharmacol Exp Ther. 1991; 258: 223–231.
6. Fujii AM, Vatner SF. Direct versus indirect effects pressor effects and vasoconstrictor actions of angiotensin in conscious dogs. Hypertension. 1985; 7: 253–261.
7. Goldsmith SR, Hasking GJ, Miller E. Angiotensin II and sympathetic activity in patients with congestive heart failure. J Am Coll Cardiol. 1993; 21: 1107–1113.[Abstract]
8. Chang PC, Grossman E, Kopin IJ, Goldstein DS, Folio CJ, Holmes C. On the existence of functional angiotensin II receptors on vascular sympathetic nerve terminals in the human forearm. J Hypertens. 1995; 13: 1275–1284.[CrossRef][Medline] [Order article via Infotrieve]
9. Sadoshima J-I, Izumo S. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro: roles of phospholipid-derived second messengers. Circ Res. 1993; 73: 424–438.
10. Haller H, Lindschau C, Quass P, Luft FC. Intracellular actions of angiotensin II in vascular smooth muscle cells. J Am Soc Nephrol. 1999; 10 (suppl 11): S75–S83.[CrossRef][Medline] [Order article via Infotrieve]
11. Monteith GR, Kable EPW, Chen S, Roufogalis BD. Plasma membrane calcium pump-mediated calcium efflux and bulk cytosolic free calcium in cultured aortic smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto normotensives rats. J Hypertens. 1996; 14: 435–442.[Medline] [Order article via Infotrieve]
12. Fukuta Y, Yoshizumi M, Kitagawa T, Hori T, Katoh I, Houchi H, Tamaki T. Effect of angiotensin II on Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchanger. Biochem Pharmacol. 1998; 55: 481–487.[CrossRef][Medline] [Order article via Infotrieve]
13. Horackova M, Huang MH, Armour JA, Hopkins DA, Mapplebeck C. Cocultures of adult ventricular myocytes with stellate ganglia or intrinsic cardiac neurons from guinea pig: spontaneous activity and pharmacological properties. Cardiovasc Res. 1993; 27: 1101–1108.
14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985; 260: 3400–3450.
15. Asher-Landsberg J, Saunders T, Elovitz M. The effects of 2-aminoethoxydiphenyl borate, a novel inositol 1,4,5-triphosphate receptor modulator on myometrial contractions. Biochem Biophys Res Commun. 1999; 264: 979–982.[CrossRef][Medline] [Order article via Infotrieve]
16. Lumbers ER, Whelan RF. Tachyphylaxis to angiotensin in man. Cardiovasc Res. 1970; 4: 312–318.
17. Taddei S, Favilla S, Duranti P, Simonini N, Salvetti A. Vascular renin-angiotensin system and neurotransmission in hypertensive persons. Hypertension. 1991; 18: 266–277.
18. Brown DA, Constanti A, Marsh S. Angiotensin mimics the action of muscarinic agonists on rat sympathetic neurones. Brain Res. 1980; 193: 614–619.[CrossRef][Medline] [Order article via Infotrieve]
19. Malik KU, Nasjletti RR. Facilitation of adrenergic transmission by locally generated angiotensin II in rat. Circ Res. 1976; 38: 26–30.
20. Goldsmith SR, Hasking GJ. Angiotensin II inhibits the forearm vascular response to increased arterial pressure in humans. J Am Coll Cardiol. 1995; 25: 246–250.[Abstract]
21. Kiowski W, Erne P, Bertel O, Bolli P, Buhler F. Acute and chronic sympathetic reflex activation and antihypertensive response to nifedipine. J Am Coll Cardiol. 1986; 7: 344–348.[Abstract]
22. Furberg CD, Psaty BM, Meyer JV. Nifedipine. Dose-related increase in mortality in patients with coronary heart disease. Circulation. 1995; 92: 1326–1331.
23. Bader M, Peters J, Baltatu O, Muller DN, Luft FC, Ganten D. Tissue renin-angiotensin systems: new insights from experimental animal models in hypertension research. J Mol Med. 2001; 79: 76–102.[CrossRef][Medline] [Order article via Infotrieve]
24. Hasser EM, Cunningham JT, Sullivan MJ, Curtis KS, Blaine EH, Hay M. Area postrema and sympathetic nervous system effects of vasopressin and angiotensin II. J Clin Exp Pharmacol Physiol. 2000; 27: 432–436.
25. Castren E, Kurihara M, Gutkind NS, Saavedra JM. Specific angiotensin II binding sites in the rat stellate and superior cervical ganglia. Brain Res. 1987; 422: 347–351.[CrossRef][Medline] [Order article via Infotrieve]
26. Stromberg C, Tsutsumi K, Viswanathan M, Saavedra JM. Angiotensin II AT1 receptor in rat superior cervical ganglia: characterization and stimulation of phosphoinositide hydrolysis. Eur J Pharmacol. 1991; 208: 331–336.[CrossRef][Medline] [Order article via Infotrieve]
27. Dostal DE, Booz GW, Baker KM. Angiotensin II signalling pathways in cardiac fibroblasts: conventional versus novel mechanisms in mediating cardiac growth and function. Mol Cell Biochem. 1996; 157: 15–21.[Medline] [Order article via Infotrieve]
28. Touyz RM, Wu XH, He G, Park JB, Chen X, Vacher J, Rajapurohitam V, Schiffrin EL. Role of c-Src in the regulation of vascular contraction and Ca2+ signaling by angiotensin II in human vascular smooth muscle cells. J Hypertens. 2001; 19: 441–449.[CrossRef][Medline] [Order article via Infotrieve]
29. Ochsner M, Huwiler A, Fleck T, Pfeilschifter J. Protein kinase C inhibitors potentiate angiotensin II-induced phosphoinositide hydrolysis and intracellular Ca2+ mobilization in renal mesangial cells. Eur J Pharmacol. 1993; 245: 15–21.[CrossRef][Medline] [Order article via Infotrieve]
30. Sandnes D, Dajani O, Bjorneby A, Christoffersen T. The relationship between activation of phosphoinositide-specific phospholipase C and growth stimulation by Ca2+-mobilizing hormones in hepatocytes. Pharmacol Toxicol. 1999; 84: 234–240.[Medline] [Order article via Infotrieve]
31. Sipma H, DeSmet P, Sienaert I, Vanlingen S, Missiaen L, Parys J, B, and DeSmedt H. Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4,5-trisphosphate receptor by Ca2+ and calmodulin. J Biol Chem. 1999; 274: 12157–12162.
32. Cardy TJ, Traynor D, Taylor CW. Differential regulation of types-1 and 3 inositol trisphosphate receptors by cytolic Ca2+. Biochem J. 1997; 328: 785–793.[Medline] [Order article via Infotrieve]
33. Lindqvist M, Kahan T, Melcher A, Hjemdahl P. Acute and chronic calcium antagonist treatment elevates sympathetic activity in primary hypertension. Hypertension. 1994; 24: 287–296.
34. Muiesan G, Agabiti-Rosei E, Romanelli G, Muiesan ML, Castellano M, Beschi M. Adrenergic activity and left ventricular function during treatment of essential hypertension with calcium antagonists. Am J Cardiol. 1986; 57: 44D–49D.[CrossRef][Medline] [Order article via Infotrieve]
35. Lee DD, Rigonan K, DeQuattro V. Increased blood pressure and neural tone in the silent ischemia of hypertension: disparate effects of immediate release nifedipine. J Am Coll Cardiol. 1993; 22: 1438–1445.[Abstract]
36. McCance AJ, Thompson PA, Forfar JC. Increased cardiac sympathetic nervous activity in patients with unstable coronary heart disease. Eur Heart J. 1993; 14: 751–757.
37. Meredith IT, Eisenhofer G, Lambert GW, Dewar EM, Jennings GL, Esler MD. Cardiac sympathetic nervous activity in congestive heart failure. Evidence for increased neuronal norepinephrine release and preserved neuronal uptake. Circulation. 1993; 88: 136–145.
38. Pabelick CM, Sieck GC, Prakash YS. Invited review: significance of spatial and temporal heterogeneity of calcium transients in smooth muscle. J Appl Physiol. 2001; 91: 488–496.
39. Miura M, Boyden PA, ter Keurs HE. Ca2+ waves during triggered propagated contractions in intact trabeculae: determinants of the velocity of propagation. Circ Res. 1999; 84: 1459–1468.
40. Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev. 1999; 79: 763–854.
This article has been cited by other articles:
 |
|
 |
|
  A. G. Teschemacher, S. Wang, M. K. Raizada, J. F.R. Paton, and S. Kasparov Area-Specific Differences in Transmitter Release in Central Catecholaminergic Neurons of Spontaneously Hypertensive Rats Hypertension, August 1, 2008; 52(2): 351 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Y. Shim, J.-W. Ha, S. Park, E.-Y. Choi, D. Choi, S.-J. Rim, and N. Chung Exaggerated Blood Pressure Response to Exercise Is Associated With Augmented Rise of Angiotensin II During Exercise J. Am. Coll. Cardiol., July 22, 2008; 52(4): 287 - 292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wang, A. G. Teschemacher, J. F. R. Paton, and S. Kasparov Mechanism of nitric oxide action on inhibitory GABAergic signaling within the nucleus tractus solitarii FASEB J, July 1, 2006; 20(9): 1537 - 1539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F. Fernandez, M.-H. Huang, B. A. Davidson, P. R Knight III, and J. L. Izzo Jr Mechanisms of Angiotensin II-Mediated Decreases in Intraneuronal Ca2+ in Calcium-Loaded Stellate Ganglion Neurons Hypertension, February 1, 2005; 45(2): 276 - 282. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||