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(Circulation Research. 1997;80:861-867.)
© 1997 American Heart Association, Inc.

Articles

Non–Voltage-Gated Ca²⁺ Influx Through Mechanosensitive Ion Channels in Aortic Baroreceptor Neurons

Margaret J. Sullivan, Ram V. Sharma, Ruth E. Wachtel, Mark W. Chapleau, Laurie J. Waite, Ramesh C. Bhalla, , Francois M. Abboud

From the Cardiovascular Center and Departments of Internal Medicine, Anatomy, Anesthesia, Physiology and Biophysics, University of Iowa, Iowa City, and Veterans Affairs Medical Center, Iowa City, Iowa.

Correspondence to Francois M. Abboud, Internal Medicine, SE 308 GH, University of Iowa, Iowa City, IA 52242.

	Abstract

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Abstract The mechanisms underlying mechanotransduction in baroreceptor neurons (BRNs) are undefined. In this study, we specifically identified aortic baroreceptor neurons in primary neuronal cell cultures from nodose ganglia of rats. Aortic baroreceptor neurons were identified by labeling their soma with the fluorescent dye 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) applied to the aortic arch. Using Ca²⁺ imaging with fura 2, we examined these BRNs for evidence of Ca²⁺ influx and determined its mechanosensitivity and voltage dependence. Mechanical stimuli were produced by ejecting buffer from a micropipette onto the cell surface with a pneumatic picopump, producing a shift in the center of mass of the cell that was related to intensity of stimulation. Ninety-three percent of DiI-labeled neurons responded to mechanical stimulation with an increase in [Ca²⁺]_i. The magnitude of the increases in [Ca²⁺]_i was directly related to the intensity of the stimulus and required the presence of external Ca²⁺. The trivalent cations Gd³⁺ or La³⁺ in equimolar concentrations (20 µmol/L) eliminated the K⁺-induced rises in [Ca²⁺]_i, demonstrating that both trivalent cations are equally effective at blocking voltage-gated Ca²⁺ channels in these baroreceptor neurons. In contrast, the mechanically induced increases in [Ca²⁺]_i were blocked by Gd³⁺ (20 µmol/L) only and not by La³⁺ (20 µmol/L). Stretch-activated channels (SACs) have been shown in other preparations to be blocked by Gd³⁺ specifically. Our data demonstrate that (1) BRNs, specifically identified as projecting to the aortic arch, have ion channels that are sensitive to mechanical stimuli; (2) mechanically induced Ca²⁺ influx in these cells is mediated by a Gd³⁺-sensitive ion channel and not by voltage-gated Ca²⁺ channels; (3) the magnitude of the Ca²⁺ influx is dependent on the intensity of the stimulus and the degree and duration of deformation; and (4) repeated stimuli of the same intensity result in comparable increases in [Ca²⁺]_i. We conclude that mechanical stimulation increases Ca²⁺ influx into aortic BRNs independent of voltage-gated Ca²⁺ channels. The results suggest that Gd³⁺-sensitive SACs are the mechanoelectrical transducers in baroreceptors.

Key Words: baroreceptor • mechanosensitive channel • stretch-activated ion channel • Ca²⁺ • Gd³⁺

	Introduction

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Baroreceptor neurons respond to increases in arterial pressure with an increase in activity, ultimately resulting in a reflex reduction in sympathetic tone and the buffering of rises in pressure. A great deal is known about the responses of the carotid sinus nerve and the aortic depressor nerve to various stimuli and chemical mediators in vivo,^{1 2 3} but the relative inaccessibility of the nerve endings has limited the exploration of the mechanism of mechanotransduction in baroreceptor neurons. Stretch causes the direct activation of ion channels in a variety of cells from vertebrates and invertebrates, including muscle,^{4 5 6} neuronal,^{7 8 9} endothelial, and epithelial^{10 11 12 13} cells. In several cell preparations, the trivalent lanthanide Gd³⁺ blocks these stretch-activated channels (SACs).^{6 13 14 15 16 17}

In the isolated carotid sinus of rabbit, we have shown that Gd³⁺ blocks carotid sinus nerve activity during increases in carotid sinus pressure.¹⁸ Andresen and Yang¹⁹ studied the isolated aortic arch preparation in rats and found that Gd³⁺ had no effect on the increase in nerve activity with pressure. These results raise a question about the fundamental role of Gd³⁺-sensitive SACs in mechanotransduction of baroreceptor activity. One could also question whether Gd³⁺ reaches the SACs on the nerve endings in the aortic arch preparation.

To address the question of mechanotransduction in neurons at the cellular level, we recently isolated nodose ganglion neurons from rats and demonstrated that 60% of the dissociated neurons responded to mechanical stimulation with a rise in [Ca²⁺]_i.²⁰ Since nodose ganglion neurons contain both chemosensitive and mechanosensitive neurons, we reasoned that a selective isolation of baroreceptor neurons from nodose ganglion would result in a much higher percentage of mechanosensitive cells and would allow us to test the hypothesis that stretch-activated Gd³⁺-sensitive ion channels mediate mechanoelectrical transduction in baroreceptor neurons. Thus, the goals of the present study were (1) to identify specifically aortic baroreceptor neurons in culture and determine their response to mechanical stimulation, (2) to determine whether a direct relationship exists between the intensity of graded mechanical stimulation and the resulting increase in [Ca²⁺]_i, and (3) to distinguish the rise in [Ca²⁺]_i resulting from activation of mechanosensitive ion channels from that attributable to voltage-gated channels in these baroreceptor neurons. The results suggest the presence of mechanosensitive ion channels in aortic baroreceptor neurons similar to SACs described in other preparations. These channels may be the important mediators of mechanoelectrical transduction in baroreceptor neurons and, hence, in the regulation of blood pressure and sympathetic tone.

	Materials and Methods

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Fluorescent Labeling of Baroreceptor Neurons
Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, Ind) weighing 250 to 300 g were housed with food and water available ad libitum and a 12 h/12 h light/dark cycle. Rats were anesthetized with ketamine (100 mg/kg IP) and acepromazine (10 mg/kg IP) and artificially ventilated. A thoracotomy was performed to permit access to the aortic arch, and 5 µL of the carbocyanine dye 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine (DiI, 50 mg/mL in dimethyl sulfoxide or ethanol, Sigma Chemical Co) was applied to the aortic arch using a fine-tipped glass pipette. The wound was closed, and the animal allowed to recover. A minimum of 1 week was allowed for transport of the dye to the neuron cell bodies in the nodose ganglion.

Experiments conformed to the guidelines for care and use of animals established by the University of Iowa, the National Institutes of Health, and the Society for Neuroscience.

Neuronal Cell Culture
The nodose ganglion neurons were dissociated according to the method of Ikeda et al,²¹ and culturing methods were adapted from De Koninck et al.²² Briefly, rats were anesthetized with halothane and decapitated, and the nodose ganglia were removed. The ganglia were incubated with trypsin (1 mg/mL), collagenase (1 mg/mL), and DNase (0.1 mg/mL) in modified L-15 medium²² for 1 hour at 37°C. Enzymatic activity was terminated by the addition of soybean trypsin inhibitor (2 mg/mL), bovine serum albumin (1 mg/mL), and CaCl₂ (3 mmol/L) in modified L-15 medium, and the ganglia were triturated using sterile siliconized Pasteur pipettes to dissociate individual cells. After centrifugation, the cells were resuspended in a modified L-15 medium with 5% rat serum and 2% chick embryo extract and plated on polylysine-coated glass coverslips. 5-Fluorodeoxy-2-uridine (80 µmol/L) was added to prevent the proliferation of nonneuronal cells. Neurons were studied 3 to 7 days after plating. No loss of DiI fluorescence was noted during this period of time in culture.

Ca²⁺ Imaging With Fura 2
Cells were loaded with 5 µmol/L fura 2-AM in serum-free HEPES-buffered (10 mmol/L) DMEM containing 0.1% BSA and 0.02% pluronic 127 for 45 to 60 minutes. Cells were then incubated 30 minutes in serum-free DMEM with BSA to metabolize the fura 2-AM to fura 2 free acid.

[Ca²⁺]_i levels were measured using a video microscopic digital analysis system (Photon International Technology, Inc) as described by Bhalla et al.²³ Dual-wavelength imaging with fura 2 was used to detect changes in [Ca²⁺]_i in DiI-labeled neurons. Image pairs were acquired at 5-second intervals using excitation wavelengths of 340 and 380 nm and emissions at 510 nm. Absolute Ca²⁺ levels were determined from the 340/380 ratio of the emissions as described previously.²⁰ Any cell exhibiting a decrease in intensity of emission at the 340- and 380-nm wavelengths simultaneously was discarded, because this is an indication of a decrease in the concentration of the dye, such as might occur with a leaky cell membrane or a break in the membrane.

Mechanical Stimulation of Baroreceptor Neuronal Cell Bodies
The coverslip containing the neurons formed the bottom of the imaging chamber, which was filled with a buffered saline solution containing (mmol/L) NaCl 120, KCl 3, CaCl₂ 2, MgCl₂ 1, glucose 10, mannitol 35, and HEPES 10 at pH 7.4. In some experiments, CaCl₂ was excluded from the buffer, MgCl₂ was increased to 3 mmol/L, and 5 mmol/L EGTA was added to the solution. The chamber was placed on the stage of an inverted microscope, and a buffer-filled micropipette (6- to 8-µm tip) was positioned 50 µm above the cell surface. The micropipette was connected to a pneumatic picopump, which delivered a 30-millisecond pulse of 5-, 10-, and 15-psi air pressure to eject fluid onto the cell surface. Each cell received all three stimuli in order of increasing intensity with an interval of 25 seconds between application of each stimulus.

For some cells, the silicone-intensified camera of the imaging system was replaced with a video camera, and the responses of the cells to mechanical stimulation were videotaped. When trivalent cations were used, Gd³⁺ or La³⁺ was added to the buffer at a concentration of 20 µmol/L. The pipette was always filled with the same solution as the bath. Quantification of mechanical deformation of neurons produced by mechanical stimulation was performed using the computer program Vtrace developed by the Image Analysis Core Facility, University of Iowa (Vaytek, Fairfield, Iowa). Images were captured from videotapes 1 second before the first stimulus (control) and at maximum deformation after 5-, 10-, and 15-psi pulses, and then after the final 15-psi stimulus at 50 milliseconds, 100 milliseconds, 2 seconds, 10 seconds, and 40 seconds. Displacement of the center of mass of the cell from control values was determined. The center of mass was defined as the point at which the major axis and the minor axis intersect.

K⁺ Depolarization
Cells were depolarized in buffer containing 50 mmol/L KCl (83 mmol/L NaCl). Basal Ca²⁺ values were obtained before the replacement of normal buffer with high-K⁺ buffer. When 20 µmol/L Gd³⁺ or 20 µmol/L La³⁺ was used, both the normal and high-K⁺ buffers were used.

Analysis
A response was defined as an increase of >30 nmol/L [Ca²⁺]_i. Peak values of the responses were analyzed using repeated measures ANOVA. Significant responses were further examined using Fisher's least significant difference test. The level of significance was P<.05. Results are presented as mean±SEM.

	Results

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Putative baroreceptor neurons labeled from the aortic arch with DiI produced a red fluorescence detected with the imaging system with an excitation wavelength of 540 nm and a dichroic mirror and barrier filter suitable for rhodamine. In culture, the nodose ganglion neurons ranged between 30 and 50 µm in diameter and exhibited pseudounipolar or bipolar morphologies as previously described in the literature.²²

Fig 1 shows that the increases in [Ca²⁺]_i were progressively higher than basal values after 5-, 10-, and 15-psi stimuli and returned slowly toward control levels during the 25-second period following each stimulation. When cells were allowed to recover fully after the 15-psi stimulation, it took from 3 to 13 minutes for the [Ca²⁺]_i to return to basal values. After recovery, the responsiveness of the neuron was reproducible (Fig 1).

View larger version (40K): [in this window] [in a new window]   Figure 1. Sequential pseudocolor images of a baroreceptor neuron demonstrating [Ca2+]i levels before (basal) and 5, 10, and 25 seconds after stimulation at 5 psi (top row), 10 psi (second row), and 15 psi (third row). The intensity of fluorescence was clearly related to the intensity of mechanical stimulation. Within 25 seconds after stimulation, the intensity had declined significantly but remained higher than basal. Full recovery time to basal values after the 15-psi stimulus was 7 minutes. After recovery, repeated stimulation at 10 psi (fourth row) caused increases in [Ca2+]i comparable to those seen with the earlier 10-psi stimulus (second row). Several neurons demonstrated similar patterns of response, with full recovery times ranging from 3 to 13 minutes.

Ninety-three percent (27 of 29) of putative baroreceptor neurons responded to mechanical stimulation with an increase in [Ca²⁺]_i (Figs 1 through 3); 66% (10 of 15) of nonlabeled nodose ganglion neurons increased [Ca²⁺]_i in response to mechanical stimulation. In the 27 baroreceptor neurons that responded, basal levels of [Ca²⁺]_i were 153±38 nmol/L. After stimulation intensities of 5, 10, and 15 psi, the [Ca²⁺]_i increased to peak values of 173±33, 261±37, and 373±41 nmol/L, respectively (Fig 4B).

View larger version (25K): [in this window] [in a new window]   Figure 2. Baroreceptor neurons responded to graded mechanical stimulation with incremental increases in [Ca2+]i (, n=29). No responses occurred in the presence of zero Ca2+ buffer (, n=8). Symbols and error bars represent mean±SEM.

View larger version (31K): [in this window] [in a new window]   Figure 3. Representative examples of the responses of baroreceptor neurons in the presence of normal buffer (top row), zero Ca2+ (second row), 20 µmol/L Gd3+ (third row), and 20 µmol/L La3+ (bottom row). Shown (left to right) are basal levels of [Ca2+]i and peak values in response to 5-, 10-, and 15-psi stimuli. Conc. indicates concentration.

View larger version (15K): [in this window] [in a new window]   Figure 4. A, Distance the center of mass of the neuron moved after a 5-, 10-, and 15-psi pulse of fluid onto the surface of the neuron (n=7). Bars and error bars represent mean±SEM. A straight linear relationship is apparent between the displacement and the intensity of the stimulus. B, Direct correlation between movement of center of mass and peak increases in [Ca2+]i at 5, 10, and 15 psi. Symbols and error bars indicate mean±SEM.

Repeated stimulation of the same intensity caused comparable increases in [Ca²⁺]_i (Fig 1). In eight DiI-labeled cells, three successive 10-psi stimuli resulted in [Ca²⁺]_i levels averaging 421±133, 315±127, and 296±122 nmol/L. The peaks differed significantly from the average basal level of 88±10 nmol/L Ca²⁺ but did not differ from each other.

Analysis of videotapes of seven neurons during mechanical stimulation demonstrated that the center of mass of the neurons shifted with each stimulus. The magnitude of the shift was related to the intensity of the stimulus and correlated with the peak increases in [Ca²⁺]_i at 5, 10, and 15 psi (Fig 4). On average, the center of mass moved 6.8±2.7 µm with the maximum stimulus of 15 psi; relative to the size of the soma (30 to 50 µm), this represented a distance of 14% to 23% of the cell diameter. The center of mass returned to within 2 µm of the starting point by 2 seconds but had not returned to control values by 40 seconds after the stimulus.

In the absence of extracellular Ca²⁺, mechanical stimulation did not increase [Ca²⁺]_i in any of the eight DiI-labeled neurons studied (Figs 2 and 3). In the presence of Gd³⁺, only 1 of 11 baroreceptor neurons responded to mechanical stimulation with an increase in [Ca²⁺]_i (Figs 2 and 4). Responses were preserved in the presence of La³⁺ with 8 of 10 baroreceptor neurons responding. In the presence of La³⁺, peak values of [Ca²⁺]_i were significantly higher than basal levels after stimulation by 10- and 15-psi pulses of fluid (Figs 3 and 5).

View larger version (24K): [in this window] [in a new window]   Figure 5. In the presence of 20 µmol/L Gd3+ (, n=10) mechanically induced increases in [Ca2+]i were blocked. At the same concentration, La3+ (, n=11) did not block increases in [Ca2+]i. Symbols and error bars represent mean±SEM.

Depolarization of baroreceptor neurons with 50 mmol/L K⁺ produced significant increases in [Ca²⁺]_i (Fig 6). Although Gd³⁺ and La³⁺ did not change basal levels of [Ca²⁺]_i, K⁺-induced increases in [Ca²⁺]_i were completely obliterated in the presence of either trivalent cation Gd³⁺ or La³⁺.

View larger version (31K): [in this window] [in a new window]   Figure 6. K+ (50 mmol/L) induced increases in [Ca2+]i in baroreceptor neurons (n=13), which were blocked by both 20 µmol/L Gd3+ (n=7) and 20 µmol/L La3+ (n=8). Bars and error bars represent mean±SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we specifically examined baroreceptor neurons, particularly the somata of neurons with baroreceptor nerve endings projecting to the aortic arch, to determine their response to mechanical deformation. The results indirectly support the fundamental hypothesis that Gd³⁺-sensitive SACs mediate mechanoelectrical transduction in isolated baroreceptor neurons. Mechanical stimulation of these neurons triggered a significant rise in [Ca²⁺]_i, reflecting Ca²⁺ entry through mechanosensitive channels and not through voltage-gated Ca²⁺ channels. The magnitude of the rise in [Ca²⁺]_i was dependent on the intensity of the stimulus. The video analysis of the motion of the neurons during stimulation indicated that a shift in the center of mass of the cell was also related to the intensity of the stimulation. There was a positive correlation between the displacement and the rise in [Ca²⁺]_i. This was important in order to link the responses to mechanical deformation. Furthermore, repeated stimuli of the same intensity caused comparable increases in [Ca²⁺]_i.

Our discussion will deal with four issues: (1) the evidence that mechanosensitive ion channels rather than voltage-gated Ca²⁺ channels mediate the response, (2) the advantages and limitations of the neuronal culture preparation, (3) the magnitude and duration of the rise in [Ca²⁺]_i, and (4) the relation of this work to recent work from our laboratory and from other investigators.

Evidence That Mechanosensitive Ion Channels Rather Than Voltage-Gated Ca²⁺ Channels Mediate the Response to Mechanical Stimulation
SACs have been described in several preparations and are blocked by the trivalent cation Gd³⁺.^{6 13 14 15 16 17} The channels are less sensitive to other trivalent cations of this class, such as La³⁺.¹⁷ In our preparation, 20 µmol/L Gd³⁺ blocked the mechanically induced increases in [Ca²⁺]_i. La³⁺ at the same concentration did not block the increase in [Ca²⁺]_i produced by mechanical stimulation. In the presence of 20 µmol/L La³⁺, the mechanically induced increases in [Ca²⁺]_i in response to the 10- and 15-psi stimuli were similar to those obtained under control conditions. This selective blockade of the responses to mechanical stimulation by Gd³⁺ is consistent with the known pharmacology of SACs.^{15 16 17} These results also suggest that a nonspecific leak of Ca²⁺ into the cell is highly unlikely, since one trivalent cation, namely Gd³⁺, blocked the Ca²⁺ entry, whereas another, La³⁺, was ineffective.

Voltage-gated Ca²⁺ channels have been described in neurons from the nodose ganglion. Mendelowitz and Kunze²⁴ describe low-threshold and -conotoxin–sensitive high-threshold voltage-gated Ca²⁺ channels in nodose ganglion neurons. Because Gd³⁺ and La³⁺ are known to block voltage-gated Ca²⁺ channels,^{25 26 27 28} we tested the effects of these cations on increases in [Ca²⁺]_i in response to depolarization of the cells with a high-K⁺ solution. Depolarization increases [Ca²⁺]_i secondary to opening of voltage-dependent Ca²⁺ channels. Exposure of the neurons to K⁺ increased [Ca²⁺]_i. The K⁺-induced increases in [Ca²⁺]_i were abolished by both Gd³⁺ and La³⁺, presumably through blockade of voltage-gated Ca²⁺ channels.

There is, therefore, a dichotomy between the effects of trivalent cations on K⁺-induced and stretch-induced increases in [Ca²⁺]_i. Whereas Gd³⁺ blocks both responses, La³⁺ blocks only the K⁺-induced increases in [Ca²⁺]_i. These findings support the existence in baroreceptor neurons of distinct mechanosensitive ion channels that are selectively sensitive to Gd³⁺. Mechanically induced increases in [Ca²⁺]_i are dependent on the entry of extracellular Ca²⁺. Since the voltage-gated Ca²⁺ channels are blocked in the presence of La³⁺, the increase in [Ca²⁺]_i during mechanical stimulation in the presence of La³⁺ suggests that Ca²⁺ enters the neuron via the mechanosensitive ion channel and not through voltage-activated Ca²⁺ channels. Therefore, these data are consistent with the hypothesis that mechanical stimuli gate either a nonspecific cation-conducting SAC similar to that described by other investigators^{16 17} or a Ca²⁺-conducting mechanosensitive channel. The methods used in the present study, however, allow only an indirect assessment of the presence of these channels.

Advantages and Limitations of the Cultured Neuron Preparation
The study of the cellular mechanisms of mechanotransduction of baroreceptors has been hampered by the inaccessibility and the small size of the endings. Any attempt to infer what is happening at the nerve ending from measurements of afferent nerve activity has limitations. First, the afferent nerve activity represents action potentials generated at the spike-initiating zone, which only indirectly represents the events occurring at the nerve ending, where generator potentials are produced. Second, the sensory nerve ending is embedded in the adventitia of the vessel wall and is exposed to paracrine factors, which can modulate the process of mechanoelectrical transduction and its effect on afferent nerve activity.

Neuronal cell culture allows direct access to the neuronal membrane. The cultured cell model assumes that the membrane elements in the neuronal perikaryon are representative of those found in the neuron terminals. Indeed, receptors and ion channels have been demonstrated to be common to both cell bodies and sensory or central endings of nodose ganglion neurons. For instance, receptors for cholecystokinin octapeptide (CCK-8), which acts on vagal afferents in the gut, are found on the cell bodies of a subpopulation of nodose ganglion neurons. Application of CCK-8 to the cell body results in depolarization.²⁹ 5-Hydroxytryptamine has been shown to act at the sensory terminals of vagal afferent fibers² and to depolarize the neuron cell bodies in the nodose ganglion as well.³⁰ A fast transient K⁺ channel was characterized in the cell bodies of nodose ganglion neurons by Cooper and Shrier,³¹ and we have demonstrated that this channel is involved in the adaptation of the baroreceptor nerve activity recorded from the afferent fibers.³² Therefore, although the existence and action of membrane proteins in the cell body are not proof of their existence and function in the sensory endings, the cell body has served as a useful model in many instances.^{24 29 30 32 33 34 35} In the present study, we have found indirect evidence that mechanosensitive ion channels are present on the cell body of baroreceptor neurons. We speculate that the same mechanosensitive ion channels are present in the sensory terminals of the neuron and mediate the mechanotransduction of arterial pressure.

Although we have referred to these neurons as baroreceptor neurons, we recognize that their membrane properties may reflect not only the receptive components at the sensory terminals but also the central components. The latter may reflect properties of synaptic terminals in the nucleus tractus solitarius. Since our experiments relate to the effects of mechanical deformation, we have accepted the notion that the responses represent the receptive component of the sensory terminals.

The results also indicate that the majority (93%) of putative aortic baroreceptor neurons cultured from nodose ganglia responded to mechanical stimulation by increasing [Ca²⁺]_i, whereas only 66% of nonlabeled neurons responded. This difference in the number of DiI-labeled neurons responding to mechanical stimulation compared with unlabeled nodose ganglion neurons was anticipated. Nearly all the neurons labeled from the aortic arch of rats would be expected to be baroreceptor neurons responsive to stretch. A significant number of the nonlabeled nodose neurons presumably have chemosensitive endings and would not be responsive to mechanical stimulation, whereas some of the neurons innervating the viscera may also be mechanosensitive and responsive to mechanical events such as gastric distention, intestinal motility, cardiac volume, and lung expansion. Furthermore, the thoroughness with which all aortic baroreceptor neuronal endings have been labeled with injection of DiI can be challenged; there may indeed be aortic baroreceptor neurons in the nonlabeled population.

Magnitude and Duration of the Increase in [Ca²⁺]_i
The baroreceptor neurons showed an incremental rise in [Ca²⁺]_i in response to increasing intensities of mechanical stimulation. A stimulus-response relationship was established. Repeated stimuli of the same intensity caused comparable rather than incremental increases in [Ca²⁺]_i. The increase in [Ca²⁺]_i declined at a variable rate during the 25-second period between stimuli but remained significantly elevated above basal values. When full recovery was allowed, it took 3 to 13 minutes for the complete return to basal values. Clearly, some rise in [Ca²⁺]_i far outlasted the brief stimulus.

Video analysis of the mechanical response indicates that the increases in [Ca²⁺]_i correlated with the shift in the center of mass of the cell and the intensity of the stimulus. Relative to the size of the soma, this shift ranged from 2% to 6% of the cell diameter at 5 psi to 14% to 23% at 15 psi. As with the increase in [Ca²⁺]_i, the duration of the shift outlasted the brief pulse of buffer as the center of mass of the neurons only approached the starting position 40 seconds after the stimulus. If, as others in the field have suggested, gating of SACs is dependent on tension transmitted via the cytoskeleton, the sustained shift in the center of mass may represent continued tension on the cytoskeletal elements of the cell, which may account for the prolonged increase in [Ca²⁺]_i.

We do not believe that the sustained increase in [Ca²⁺]_i following the stimulus is a reflection of cell damage or membrane tearing for several reasons. First, as mentioned in "Materials and Methods," a break or a tear in the membrane would result in a decrease in emission intensity at both 340 and 380 nm simultaneously, and such cells were discarded. Second, there was a strong correlation between the magnitude of the shift in the center of mass of the cell, the increase in [Ca²⁺]_i, and the intensity of the sequentially applied stimuli at 5, 10, and 15 psi. Third, both the increase in [Ca²⁺]_i and the mechanical displacement of the cell outlasted the stimulus by at least 40 seconds, and when the cells were allowed to recover after a stimulus of 15 psi, it took 3 to 13 minutes for [Ca²⁺]_i to return completely to basal level. Fourth, after recovery, a repeated stimulus caused an increase in [Ca²⁺]_i comparable to the earlier stimulus of similar intensity.

An additional factor in the sustained increase in [Ca²⁺]_i may be a Ca²⁺-dependent Ca²⁺ release in response to the initial entry of extracellular Ca²⁺. Naruse and Sokabe¹³ have shown that this occurs with mechanical stimulation of endothelial cells. Furthermore, the experiments were run at room temperature, and the kinetic properties of Ca²⁺ handling by isolated soma at 25°C may be slower than the kinetics in vivo.

Although the [Ca²⁺]_i recovery in the soma is prolonged and reflects the duration of the mechanical stimulus, we still should not extrapolate these findings to events at the baroreceptor nerve endings during a pulsatile or a sustained rise in pressure. We believe the rise in [Ca²⁺]_i to be a signal of activation of "mechanosensitive channels" similar to the "stretch-activated channels" demonstrated in other preparations.

In the absence of Ca²⁺ in the bathing medium, mechanical stimulation did not increase [Ca²⁺]_i. Thus, the increased [Ca²⁺]_i observed in the baroreceptor neuron is dependent on an influx of extracellular Ca²⁺; however, Ca²⁺ may not be the only, or even the predominant, ion entering the neuron through these mechanosensitive channels. Our approach does not allow us to speculate whether or not the mechanical stimulus that we used in the present study actually brings the membrane potential of these cells into the range necessary to depolarize the cell sufficiently to activate voltage-gated Ca²⁺ channels or to produce action potentials. There may have been some contribution from voltage-gated Ca²⁺ channels to the signal, as evidenced by the suppression of the response to the 5-psi stimulus by La³⁺; however, the major rise in [Ca²⁺]_i with 10 and 15 psi was not altered by La³⁺ and is, therefore, not dependent on voltage-gated Ca²⁺ channels.

Relation of the Present Study to Other Work
Several other investigators have explored the possibility that SACs are involved in transducing mechanical stimuli in other preparations by examining the effects of Gd³⁺. Naruse and Sokabe,¹³ for instance, report that in endothelial cells stretch-induced increases in [Ca²⁺]_i are nearly abolished by Gd³⁺. Gd³⁺ inhibits arrhythmias induced by stretching canine ventricles.³⁶ This dose-dependent inhibition was attributed to blockade of SACs in the sarcolemma. Mechanotransduction in the crayfish stretch receptor neuron, which is believed to be mediated by SACs, is attenuated by the application of Gd³⁺.¹⁴

In our previous studies of mechanotransduction by baroreceptor neurons, we have reported that Gd³⁺ in the isolated carotid sinus of the rabbit blocks the baroreceptor afferent nerve activity during ramp increases in carotid sinus pressure.¹⁸ The suppression of afferent activity was dose dependent and reversible. Andresen and Yang,¹⁹ however, did not find an effect of Gd³⁺ on aortic depressor nerve activity in the isolated aortic arch of the rat. The cause of the disparity in these results is not apparent, especially in view of the present study of aortic baroreceptor neurons in the rat. It is possible that the differences may be due to differences in the time course of the exposure to Gd³⁺ used in the protocols and/or greater penetration or access of Gd³⁺ to the SACs on the nerve terminals in the carotid sinus of rabbit than in the aortic arch of rat. Recent work from our laboratory³⁷ provides electrophysiological evidence of SACs in cultured nodose ganglion neurons. Using a hypo-osmotic solution to induce swelling of the baroreceptor neuron as a mechanical stimulus, we have demonstrated an ionic current that is Gd³⁺ sensitive and is not blocked by La³⁺, the Ca²⁺ channel blocker conotoxin, or the Na⁺ channel blocker tetrodotoxin. Although hypo-osmotic stretch represents a slow onset stimulus, those data were also suggestive of the presence of SACs in the cell membranes of baroreceptor neurons.

To summarize, the data from these studies indicate that deformation of the nodose baroreceptor neuronal membrane results in influx of Ca²⁺ into the neuron and a rise in [Ca²⁺]_i. The increase in [Ca²⁺]_i is related to the magnitude of the deformation, is blocked by Gd³⁺, and is distinct from that produced by K⁺ depolarization in that the mechanically induced response is La³⁺ insensitive. These data suggest that in addition to voltage-gated Ca²⁺ channels, mechanosensitive ion channels are present in the baroreceptor cell membrane. The sensory endings of the neuron may use such Gd³⁺-sensitive SACs as the mechanoelectrical transducers and as regulators of arterial pressure and sympathetic activity.

Received February 3, 1995; accepted February 24, 1997.

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