Neuroscience Vol. 66, No. 2, pp. 433~141, 1995
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M E C H A N I C A L STIMULATION INCREASES INTRACELLULAR~ CALCIUM CONCENTRATION IN NODOSE SENSORY NEURONS R. V. S H A R M A , M. W. C H A P L E A U , G. H A J D U C Z O K , * R. E. W A C H T E L , L. J. W A I T E , R. C. B H A L L A and F. M. A B B O U D t Departments of Anatomy, Anesthesia, Internal Medicine, Physiology and Biophysics, The Cardiovascular Center, The University of Iowa, College of Medicine and The Department of Veterans Affairs Medical Center, Iowa City, IA 52242, U.S.A. A~traet--The cellular mechanisms involved in activation of mechanosensitive visceral sensory nerves are poorly understood. The major goal of this study was to determine the effect of mechanical stimulation on intracellular calcium concentration ([Ca2+]i) using nodose sensory neurons grown in culture. Primary cultures of nodose sensory neurons were prepared by enzymatic dispersion from nodose ganglia of 4~8 week old Sprague-Dawley rats. Whole cell [Ca2+]i was measured by a microscopic digital image analysis system in fura-2 loaded single neurons. Brief mechanical stimulation of individual nodose sensory neurons was achieved by deformation of the cell surface with a glass micropipette. In 31 of 50 neurons (62%), mechanical stimulation increased [Ca2+]i from 125 + 8 to 763 + 89 nM measured approximately 10 s after stimulation. [Ca2÷]i then declined gradually, returning to near basal levels over a period of minutes. [Ca 2+]i failed to increase after mechanical stimulation in the remaining 19 neurons. The mechanically-induced rise in [Ca2+]i was essentially abolished after the neurons were incubated for 5-10 min in zero Ca 2÷ buffer (n = 7) or after addition of gadolinium (10 # M), a blocker of stretch-activated ion channels (n = 5). The effect of gadolinium was reversed after removal of gadolinium. The results indicate that: (1) mechanical stretch increases [Ca2+]i in a subpopulation of nodose sensory neurons in culture; and (2) the stretch-induced increase in [Ca2÷]i is dependent on influx of Ca 2÷ from extracellular fluid and is reversibly blocked by gadolinium. The findings suggest that opening of stretch-activated ion channels in response to mechanical deformation leads to an increase in Ca 2÷ concentration in visceral sensory neurons. Stretch-activated channel opening and increased [Ca2+]~may also occur at the sensory endings of these neurons in vivo. These fundamental cellular responses may enable sensing of mechanical deformation in various tissues innervated by nodose neurons, including the heart, aorta, lungs and gastrointestinal tract.
The viscera including the heart and major arteries, the lungs, and the gastrointestinal structures are innervated by mechanically-sensitive sensory neurons that are stimulated by distension. 5'4° The activity of these neurons is transmitted centrally to the nucleus tractus solitarius and triggers reflex adjustments that are of major importance in the regulation of autonomic function. The cellular mechanisms that participate in mechanoelectrical transduction of visceral mechanoreceptors are poorly understood. The small size and inaccessibility of the nerve endings precludes direct electrophysiological and biochemical measurements. To overcome this limitation we have initiated studies of the soma of sensory neurons isolated from nodose ganglia and maintained in culture.
tTo whom correspondence should be addressed. *Present address: Department of Physiology, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A. Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco's modified eagle's medium; EGTA, Ethyleneglycolbis(aminoethylether)tetra-acetate; FBS, Fetal Bovine Serum; HEPES, N-2-hydroxyethylpiperazineN'-2-ethanesulphonic acid.
Intracellular calcium ([Ca2+]i) is an important second messenger involved in signal transduction and neurotransmitter release. 2'14'29'39'5° Mechanical stimulation increases [Ca2+]~ in a variety of nonneuronal cells including epithelia, 42'51 cardiac, skeletal and vascular muscles; ~3'44'46 vascular endothelium, 17'37'45 glia; 7 and mechanosensitive chick hair cells 3s suggesting that [Ca2+]i may function as an intracellular signal linking stretch-activated ion channels on the cell surface to dynamic changes in cell function. Stretch-activated channels have been identified in numerous cell types including the crayfish stretch receptor neuron ~5 and in isolated membrane patches. 19,41,44,5z53The probability of channel opening is increased by increased membrane tension. Most stretch-activated channels in mammalian cells are permeable to cations including sodium, calcium and potassium, ~9'4~'44 and can be blocked in a relatively selective manner by gadolinium (Gd3+). 44'52'53 However, a physiological role for stretch-activated channels, identified at the single-channel level, has been recently questioned based on an inability to measure a corresponding whole-cell response to stretch in certain types of cells. 36 433
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T h e m a j o r goals o f this study were to determine the effect o f mechanical d e f o r m a t i o n o n [Ca 2+]i in nodose sensory n e u r o n s a n d to evaluate the role of stretchactivated c h a n n e l s in m e d i a t i n g this response. A n additional aim was to determine the role of extracellular calcium in the whole-cell [Ca2+]~ response to stretch. The finding o f a stretch-induced increase in [Ca2+]i in n o d o s e sensory n e u r o n s t h a t is blocked by G d 3÷ would d e m o n s t r a t e a whole-cell response a n d provide additional s u p p o r t for a physiological role for stretch-activated channels. The nodose ganglia c o n t a i n n e u r o n s with b o t h mechanically-sensitive a n d chemically-sensitive nerve e n d i n g s f l '4° W e h y p o t h esized t h a t some, b u t not all, nodose n e u r o n s would r e s p o n d to mechanical stimulation.
Primary cultures of nodose sensory neurons were prepared from nodose ganglia of 4 8 week old Sprague-Dawley rats (Harlan, Indianapolis) using a modification of published procedures. 26 Rats were anesthetized with halothane (1%), decapitated and the nodose ganglia were located by following the vagus nerves towards the base of the skull. Each ganglion was excised, cleaned of connective tissue, desheathed and placed in ice cold, high glucose Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 4raM L-glutamine, 20 pg/ml gentamicin
and 20 mM HEPES. The tissue was minced into fine pieces using micro-scissors in the presence of 2 ml of trypsin enzyme mixture containing DMEM, trypsin (type III-1 mg/ml), collagenase (type I A - - I mg/ml) and DNase (type IV--0.1 mg/ml). The dish was placed on a stirring platform in a 5% CO2 incubator at 37°C for 1 h. After incubation with trypsin-collagenase, the tissue was triturated using sterile siliconized pipettes. The tissue digestion was stopped by adding 5 ml of inhibitor solution containing DMEM, soybean trypsin inhibitor (2 mg/ml), BSA (l mg/ml) and calcium (3 mM). The cell suspension was spun in a centrifuge for 5 min at 800 rpm. The supernatant was drawn off and the cells were resuspended in modified DMEM containing 15% FBS, 4 mM L-glutamine, 20/~ g/ml gentamicin and 20 mM HEPES. The suspended cells were plated on poly-L-lysine coated 25 mm cover slips and placed in a 5% CO 2 incubator until cells were attached (12 h). After cell attachment the dish was filled with DMEM supplemented with 15% FBS. 5-Fluorodeoxy-2'-uridine (80/~M) was added to the cultures at 24 h to decrease the number of dividing non-neuronal cells. All chemicals were from Sigma Chemical Co. (St. Louis, MO). Studies were performed on neurons maintained in culture for 5-12 days at which time well-developed neurites were present (Fig. 1). Measurement of [Ca2+]i [Ca2+]i was measured using a video microscope digital image analysis system (Photon Technology International Inc., South Brunswick, NJ). 3'43 Nodose sensory neurons grown on 25 mm coverslips were incubated at 37°C in DMEM containing 0.1% BSA and 2/~M fura-2/AM for 30-45 min. After washing once with DMEM/BSA, cells were again incubated in DMEM/BSA for 30-45 min at
Fig. 1. Rat nodose neuron after 7 days in culture. Photographs were taken using Hoffman modulation contrast optics. Neurons were isolated and cultured as described in the Methods section. The neurons in culture developed neurites. Smaller bipolar cell may be a Schwann cell. Magnification = 500 ×.
Mechanosensitive whole cell calcium response in nodose neurons 37°C to allow complete hydrolysis of fura-2/AM to fura-2 inside the cell. Complete hydrolysis of fura-2/AM to fura-2 was verified by the absence of a shoulder or red-shifted peak at 390-400 nm in the presence of saturating calcium and 5/~M ionomycin. Excitation wavelengths of 34Q nm and 380 nm were obtained with a 75 W xenon arc lamp, two separate monochromators and a chopper. The 340 and 380 nm excitation light was delivered from the monochromators to the Nikon Diaphot microscope through a bifurcated fiber optics bundle. A 405nm dichroic mirror separated the excitation and emitted light. Emitted fura-2 fluorescence was observed through a Nikon Diaphot microscope after passing through a 510rim broad-band filter. Photo bleaching of fura-2 loaded cells was minimized by using a low-intensity excitation light and by blocking off the light source between acquisition of image pairs. 340 and 380 nm excitation image pairs were acquired every 10 s with a silicon intensified target camera (Hamamatsu) and a 512 × 480 frame grabber and recorded onto a 500 MB computer hard drive. Eight to sixteen frames (acquired at video frame rate) were averaged to reduce the noise level. Pseudo-color maps of absolute [Ca2+]i distribution in the cell were constructed by substituting the 340/380 nm ratio (R) on a pixel by pixel basis into the equation: [Ca2+]i = K d ( S f / S b ) ( R - Rmin)/(Rma x - R )
where Ka is the dissociation constant of fnra-2 that equals 224 nM; 1~ Rmax is the 340/380 nm ratio at saturating levels of calcium and R,~o is the ratio at zero calcium; Sf is the 380 nm excitation fluorescence in the presence of EGTA and Sb is the 380 nm excitation fluorescence in the presence of saturating levels of calcium. Values of R~n, Rma~ and S f / S b (0.45, 8.5 and 8.1, respectively) were obtained by exposing the cells to 5/tM ionomycin, I0 #M nigericin and 2 #M gramicidin in 100 mM K + salt solution buffered to pH 7.36 with 25 mM HEPES containing either 0 or 10 mM calcium. [Ca2÷]~ values over the entire cell were averaged from the masked images to obtain the whole cell calcium concentration in each neuron and to construct line graphs of the. changes in [Ca2+]i over time. E x p e r i m e n t a l solutions
Fura-2 loaded cells were bathed in bicarbonate-free Eagle's salt solution buffered to pH7.36 with 25mM HEPES. For experiments in calcium-free solution neurons were incubated in a modified Eagle's salt solution containing 2 mM EGTA for 5 10 mix before stretching. For experiments with Gd 3+, bicarbonate- and phosphate-free Eagle's salt solution buffered with 25 mM HEPES was used to avoid problems of Gd 3÷ precipitation. Neurons were incubated in the presence of Gd 3÷ for 10-15 mix before mechanical stimulation was performed.
Fig. 2. Time course of stretch-induced increase in [Ca2+]~. The neuron was stimulated by probing with a micropipette at the site indicated by the arrow. Basal refers to [Ca2÷]~ before stretch. Each image of the cell shows the [Ca2+]i at sequential times indicated below each image in seconds. Mechanical stimulation increased [Ca2+]i in the entire cell. Pseudo-color scale is displayed on the right. Numbers on scale indicate both 340/380 ratio as well as [Ca2÷]i. The inset shows the levels of [Ca2+]i averaged from the entire cell before and after mechanical stimulation.
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Fig. 3. Reproducibility of stretch-induced increase in [Ca2+]i. The neuron was stimulated by probing with a micropipette at the site indicated by the arrow. Basal refers to [Ca2+]i before stretch. In this example, the same neuron was probed with a micropipette three times with 10-20min intervals between each stimulation. The [Ca2÷]~ increased repeatedly with repeated stretch.
Mechanical stimulation Mechanical stimulation of single nodose sensory neurons was achieved with a brief (<0.5 s) deformation of the cell surface with a rounded and sealed glass micropipette (tip diameter ~ 1 0 # m ) . The micropipette was attached to a micromanipulator and positioned near the cell. Neurons were manually stimulated by a lateral movement of the micropipette parallel to the bottom of the culture dish. Stretching of the cell membrane was directly visualized through the microscope. Some of the cells were apparently damaged by the stretch and rapidly lost fura-2 fluorescence; these cells were excluded from the data analysis. Protocols Three measurements o f [Ca2+]i were made before mechanically stimulating the neurons to assure a stable basal level of [Ca2+]i. [Ca2+]i was then measured repeatedly at approximately 10 s intervals after brief mechanical deformation of the individual neurons. The first measurement was obtained 7-12 s after the mechanical stimulation. The influence of mechanical stimulation o n [Ca2+]i was determined in 50 nodose neurons. In 5 neurons, the reproducibility of the response to stretch was evaluated by repeatedly stimulating the neurons 2 3 times with 10 30 min intervals between stimuli.
A stretch-induced increase in [Ca2+]i may result from influx of calcium from extracellular fluid and/or release of calcium from intracellular stores. 7'13'37'38'42'14'45Therefore, in 7 neurons, the influence of mechanical stimulation on [Ca2+]i was determined in the presence (1.8mM) and absence of extracellular calcium. Stretch-activated channels have been implicated as playing an important role in the response to stretch in many types of cells. 8'15'21'28'32'41'49 Therefore, in five neurons that were responsive to stretch, the influence of mechanical stimulation o n [Ca2+]i was determined before, during and after washout of Gd 3+ (10 gM).
Statistical analyses The results are expressed as the mean whole cell [Ca2+]i __+SEM. The comparisons between different treatment groups were carried out by Student's t-test analysis. Differences were considered significant when P < 0.05.
Basal levels o f [Ca2+]i w e r e stable in n o d o s e n e u r o n s a n d r a n g e d f r o m 50 to 2 3 0 n M , a v e r a g i n g 124 + 7 n M (n = 41). F i g u r e 2 s h o w s a whole-cell
Mechanosensitive whole cell calcium response in nodose neurons 2800J 27004 16004 1500D
14001300.m + eq
800" 700. 600. 500 400 300 2001000 0
To investigate the mechanism of the stretchinduced rise in [Ca2+]i, mechanical stimulation was performed first in the presence of 1.8 mM calcium and then in the absence of extracellular calcium (Fig. 5). Mechanical stimulation increased [Ca2+]~ significantly in the presence of extracellular calcium (136 _+ 19 to 812 + 145 nM, P < 0.002, n = 7) but the response was not significant in the absence of calcium (79___9 to 151 + 4 9 n M , P =0.09, n = 7). Addition of 10#M Gd 3+ to the bath essentially abolished the stretch-induced increase in [Ca2+]~ (n =5). In the neuron shown in Fig. 6, [Ca2+Ji increased from 203 to 842 nM in response to stretch. In the presence of Gd 3÷, stretch did not increase [Ca2+]i (191 vs 190nM). After washout of Gd 3., stretch increased [Ca2+]~ from 200 to 560 nM. The effect of Gd 3+ on the response to stretch was examined in five neurons that responded to stretch. Stretch increased [Ca2+]i before Gd 3+, but not in the presence of Gd 3÷. The response to stretch was restored in all neurons after washout of Gd 3+.
Basal [Ca2+] i Fig. 4. Shown are the data from individual experiments with the [Ca2+]i measured 10s after stretch plotted as a function of the basal [Ca2+]i. In 31 cells (responders, closed circles), stretch induced a significant increase in [Ca2+]i. In 19 cells (non-responders, open squares), stretch did not produce a significant increase in [Ca2+]~.The line represents the line of identity indicating absence of a response to stretch (basal = stimulated [Ca~+]i) in these 19 neurons.
[Ca2+]i response to stretch. Stretch produced an increase in [CaZ+]i that reached a maximum value within the first 10 s after mechanical stimulation and then declined gradually returning to near basal levels within 2-3 min. Repeated mechanical stimulation at 10-30 min intervals again produced an increase in [Ca2+]i (Fig. 3). [Ca2+]i increased to 798-t-155 and 472 + 87 nM after the first and second probes, respectively (n = 4). Figure 4 shows individual data obtained from 50 neurons. Mechanical stimulation of single neurons in primary culture increased [Ca2+]i in 31 of 50 neurons. Neurons that showed a stretch-induced increase in [Ca2+]i were classified as responders. In responders, [Ca2+]i averaged 126+__8nM before stretch and 763 + 89 nM approximately 10 s after stretch. The magnitude of stretch-induced increase in [Ca2+]i did not correlate with resting levels of [Ca2+]i (Fig. 4). The neurons which did not show a distinct increase in [Ca2+]i above basal values in response to mechanical stimulation (<25 nM increase in [Ca2+]0 were classified as non-responders. The [Ca2+]~ averaged - 1 1 3 + 9 and 1 2 0 + 9 n M (n = 19) before and after stretch, respectively in non-responsive neurons. Six of these neurons were probed more than once and no response was observed.
Role of stretch-activated channels in mediating increase in [Ca2+]/ The blockade of the stretch-induced increase in [CaZ+]i by Gd 3+ suggests that stretch-activated ion channels are present on nodose sensory neurons and that opening of these channels leads to the increase in [Ca2+]i. Experiments in our laboratory have provided electrophysiological evidence of stretch-activated channels in nodose neurons} 2 Whole-cell voltage-clamp recordings demonstrated that exposure of the neurons to a hypo-osmotic extracellular solution to swell or stretch the cells increases an inward current that is blocked by Gd 3+.~2 Furthermore, the stretch-activated current is not blocked by antagonists of voltage-gated channels, including tetrodotoxin, tetraethylammonium, 4aminopyridine or lanthanum./2 In addition, we have identified stretch-activated channels at the singlechannel level in nodose neurons as those activated by suction applied through the recording electrode. These results taken together are consistant with the hypothesis that stretch-activated channels are involved in the stretch-induced increase in [Ca2+]i in nodose neurons. One should also consider the possibility that opening of voltage-gated Ca 2+ channels contributes to the stretch-induced influx of Ca z+. In addition to blocking stretch-activated channels, Gd 3+ also blocks voltage-gated Ca z+ channels in several cell types? °m'35 Mendeiowitz and Kunze34 have demonstrated voltage-gated Ca z+ currents in nodose sensory neurons; the neurons contain omega conotoxinsensitive and insensitive currents (N and P type) but do not contain nifedipine-sensitive L-type Ca 2+
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current. 34 Although influx of Ca 2+ through voltagegated Ca 2÷ channels may contribute to the s~retchinduced increase in [Ca2+]i, several lines of evidence suggest that stretch-activated channels may play a more important role in the nodose neurons mechanosensitivity. First, there is increasing evidence that stretch-activated channels are physiologically important mechanotransducers in a variety of cells. 15,28,37,38,44,49 For example, opening of stretchactivated channels and a stretch-induced increase in [Ca2+]i have been demonstrated in the same cell type 17'32'37'44'45with both responses being blocked by G d 3+ but not blocked by inhibitors of voltage-dependent calcium channels. 37'44 Second, cells lacking stretch-activated channels fail to demonstrate a stretch-induced increase in [Ca2+]~.44 Third, preliminary results from a separate study in our laboratory show that exposure of nodose neurons to l a n t h a n u m (20/~M) blocks depolarization-induced increase in [Ca2+]~ without blocking the stretch-induced increase
in [Ca2+]i.48 Finally, it has been recently reported that mechanical stimulation of anterior pituitary cells inhibits voltage-activated whole cell calcium currents. 4 Such an effect, if present in nodose neurons, would favor stretch-activated channels over voltage-gated calcium channels in mediating the stretch-induced increase in [Ca2+]i. Relevance to mechanoelectricaI transduction at sensory endings
An important consideration is whether the ionic mechanisms studied at the level of the cell soma reflect the mechanisms operating at the sensory nerve endings. There is considerable evidence that the ion channels and receptors responsible for fundamental sensory transduction processes are present not only on the nerve endings but also on the cell soma. Chemical factors, including bradykinin, prostaglandins and serotonin, that are known to activate visceral sensory nerve endings, also depolarize and
Fig. 5. Effect of extracellular calcium on stretch-induced increase in [Ca2+]i. The neuron was stimulated by probing with a micropipette at the site indicated by the arrow. Basal refers to [Ca2+]~before stretch. In this example, the same neuron was probed with a micropipette, first in salt solution containing 1.8 mM calcium and a second time after a 10 min period of equilibration in zero calcium salt solution. The increase in [Ca2+]~ with stretch was abolished in calcium-free solution.
Mechanosensitive whole cell calcium response in nodose neurons
Fig. 6. Gadolinium (10 pM) reversibly blocks the stretch-induced increase in [Ca2+]i. The neuron was stimulated by probing with a micropipette at the site indicated by the arrow. Basal refers to [Ca:+], before stretch. The same neuron was probed first in HEPES (control), then in HEPES containing gadolinium, and then again after washout of gadolinium (recovery). The inhibitory effect of gadolinium was reversed after removal of gadolinium; stretch again increased [Ca2+]~.
increase the action potential frequency of nodose neurons when applied to the cell soma. 9'~6'24Furthermore, K + channels susceptible to blockade by 4aminopyridine are present on mechanosensitive baroreceptor endings in the carotid sinus6 as well as on the cell soma of nodose neurons. 1°'47 Mechanosensitive somatic sensory neurons also show similarities between the properties of the cell soma and the sensory endings]2 Recordings of spike frequency from the cell soma of mechanosensitive neurons innervating the skin demonstrate that the rate of spike frequency adaptation during sustained mechanical stimulation of the endings correlates well with the rate of adaptation during sustained depolarization of the cell soma? 2 Of particular relevance to the present study is our finding that Gd 3÷, placed in the isolated carotid sinus of anesthetized rabbits, reversibly blocks activation of baroreceptor endings during ramp increases in carotid sinus pressure; suggesting that SA channels are the mechanotransducers in the sensory nerve endings. 2° Gd 3+ has also been shown to block stretch-
induced depolarization of crayfish stretch receptors. ~9 However, Andresen and Yang ~ failed to observe an effect of Gd 3+ on baroreceptor activity measured in the isolated rat aortic arch. The reasons for the different results are not clear.
Possible role of [Ca2+]i in mechanotransduction The physiological significance of a stretch-induced increase in [Ca2+]i in nodose sensory endings has not been established. Calcium influx into mechanosensory terminals of somatic nerves can contribute to the local, graded depolarization that occurs during stretch but the contribution is, in general, small compared with that of sodium influx. ~4 In addition, the stretch-induced rise in [Ca2+]i may lead to activation of potassium channels and a subsequent hyperpolarization in many types of cells including mechanoreceptors ~4,27'39 and nodose neuronsY The hyperpolarization may contribute to adaptation of mechanoreceptors during sustained stretch. In fact, experimentally-induced increases in [Ca2+]i enhance adaptation and decreases in [Ca2+]~ attenuate adap-
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suggesting a critical role of calcium influx. Furthermore, the stretch-induced increase in [Ca2+]~ was reversibly blocked by G d 3+ . These results suggest that the stretch-induced increase in [Ca2+]i in nodose neurons is triggered, at least in part, by opening of stretch-activated channels. We suggest that the stretch-insensitive nodose neurons may represent a class of cells that serve functions other than mechanoreception. The nodose ganglia contain numerous types of neurons, some of which have chemosensitive endings that do not respond to mechanical d e f o r m a t i o n ) 4,4°
Mechanical stretch increases [Ca2+]i in a subpopulation of nodose neurons in culture. The stretchinduced rise in [Ca2+]i was greatly attenuated by removal of calcium from the extracellular fluid,
Acknowledgements--This work was supported by USPHS grants HL-14388 and HL-44546. We wish to thank Drs Margaret Sullivan and Thomas Cunningham for helpful comments and suggestions concerning the manuscript.
tation during sustained stretch of mechanosensitive chick hair cells H,29 and lobster stretch receptor neurons. 14,39 Increased [Ca2+]i may also lead to release of neuropeptides from sensory neurons 23'33'5°and to changes in gene expression. 2 Therefore, we speculate that the stretch-induced increase in [Ca2+]i may cause longerterm changes in neuronal excitability or exert trophic influences on nodose sensory neurons.
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(Accepted 19 October 1994)