Chemical signaling from colonic smooth muscle cells to DRG neurons in culture

H. S. Ennes, S. H. Young, J. A. Goliger, and E. A. Mayer

Center for Ulcer Research and Education Digestive Diseases Research Center/Neuroenteric Disease Program, and Departments of Physiology and Medicine, School of Medicine, University of California, Los Angeles, California 90024


    ABSTRACT
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Transduction mechanisms between target cells within the intestinal wall and peripheral terminals of extrinsic primary afferent neurons are poorly understood. The purpose of this study was to characterize the interactions between smooth muscle cells from the rat distal colon and lumbar dorsal root ganglion (DRG) neurons in coculture. DRG neurons visually appeared to make contact with several myocytes. We show that brief mechanical stimulation of these myocytes resulted in intracellular Ca2+ concentration ([Ca2+]i) transients that propagated into 57% of the contacting neurites. Direct mechanical stimulation of DRG neurites cultured without smooth muscle had no effect. We also show that colonic smooth muscle cells express multiple connexin mRNAs and that these connexins formed functional gap junctions, as evidenced by the intercellular transfer of Lucifer yellow. Furthermore, thapsigargin pretreatment and neuronal heparin injection abolished the increase in neurite [Ca2+]i, indicating that the neuronal Ca2+ signal was triggered by inositol 1,4,5-trisphosphate-mediated Ca2+ release from intracellular stores. Our results provide evidence for intercellular chemical communication between DRG neurites and intestinal smooth muscle cells that mediates the exchange of second messenger molecules between different cell types.

dorsal root ganglion neuron; smooth muscle; intracellular calcium; calcium waves; gap junctions; inositol 1,4,5-trisphosphate signaling


    INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

THE PRIMARY FUNCTION OF extrinsic afferents innervating the gastrointestinal tract is thought to be reflex regulation and mediation of sensations such as fullness, urgency, and pain (23). It has been generally assumed that mechanical deformation of the intestinal wall during contraction or distension opens stretch-activated cation channels on mechanosensitive peripheral afferent nerve terminals, resulting in membrane depolarization, Ca2+ influx, and triggered action potentials that propagate to the soma and induce voltage-sensitive Ca2+ influx (4, 34). However, single-fiber recordings from pelvic nerve afferents show that, depending on the species, 40-90% of the afferent fibers do not generate action potentials in response to mechanical stimulation of the innervated organ (35). This suggests that the majority of peripheral nerve terminals are not mechanosensitive or that mechanical stimulation does not trigger propagated action potentials.

Several pieces of evidence suggest that local reciprocal interactions between smooth muscle and dorsal root ganglion (DRG) neurons play a role in slower signaling events related to adaptive growth (26) and local defense responses to tissue irritation (19). In a variety of cell types, including astrocytes (11) and colonic smooth muscle cells (39), intercellular signaling through gap junction-mediated exchange of second messenger molecules such as inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or cAMP has been demonstrated. In astrocytes, such gap junction-mediated intercellular signaling has been demonstrated in response to mechanical, chemical, and electrical stimulation (6). The mechanism by which mechanosensory information is transmitted from target cells to contacting afferent nerve terminals is poorly understood. Most published studies in vivo have been hindered by an inability to assess local changes in peripheral DRG terminals in response to mechanical stimulation. In addition, the responses of sensory neurons in vitro to chemical and mechanical stimulation have generally been obtained from the DRG somata and do not take into account possible differences in the activation of soma and neurites.

In a previous paper, we described a model system involving the coculture of rat DRG neurons and colonic myocytes (10). Mechanical stimulation of the myocytes caused increases in intracellular Ca2+ concentration ([Ca2+]i) that propagated as waves into contacting DRG neurites. Intercellular propagation of this Ca2+ wave did not depend on extracellular Ca2+ but could be inhibited by octanol. These results suggested that colonic myocytes and DRG neurites in coculture communicated via transfer of second messenger molecules through intercellular gap junctions. In this report, we describe experimental results confirming this hypothesis. We show that colonic smooth muscle cells express multiple connexin mRNAs and that these connexins form functional gap junctions, as evidenced by intercellular transfer of Lucifer yellow. We also show that DRG neurons couple less efficiently to intestinal epithelial cells than to colonic and vascular smooth muscle cells due to differences in connexin expression among the three target cell types. Last, we demonstrate that thapsigargin pretreatment and neuronal heparin injection abolish neurite [Ca2+]i increases, indicating that neuronal Ca2+ signals are triggered by Ins(1,4,5)P3-mediated Ca2+ release from intracellular stores.


    MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Cell cultures and cocultures. Colonic smooth muscle cells and lumbosacral DRG neurons were isolated from male Sprague-Dawley rats (200-300 g) as previously described (9). A7r5 embryonic rat thoracic aorta smooth muscle cells and IEC-6 rat intestinal epithelial cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained under 5% CO2 at 37°C in growth medium (DMEM, 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin). For cocultures, target cells were initially grown until ~50% confluent (7-9 days for primary colonic smooth muscle cells; 2-4 days for cell lines). Medium was then decanted, and freshly isolated DRG neurons in DMEM containing 10% fetal bovine serum, 10% horse serum (Sigma, St. Louis, MO), 5 ng/ml nerve growth factor (GIBCO, Grand Island, NY), and 0.5 mg/ml DNase I were seeded on top of target cells at a density of ~2,000 neurons/coverslip. These cocultures were left undisturbed for 10 min at room temperature before being moved to a 5% CO2 incubator at 36°C and grown for an additional 24-48 h. For coculture experiments, target cells and DRG neurons were distinguished by visual inspection of cell morphology.

Mechanical stimulation. Cells were mechanically stimulated by depressing the plasma membrane 2 µm for 0.5 s with a small (1-3 µm in diameter) fire-polished glass micropipette (39). At the end of each imaging experiment, tip deflections were observed under white light to verify that membrane depressions were no greater than 2 µm and that cells had not become visibly damaged. For some experiments, the cells were repetitively stimulated with four or five mechanical pulses at a frequency of 1.0 pulse/s. In other experiments, the area of cell contact was increased by using glass rods with larger diameters (4-6 µm).

Ca2+ fluorescence imaging. Cocultures growing on coverslips were incubated for 1 h at 37°C with 5 µM fura 2-AM (Molecular Probes, Eugene, OR) in external solution (Hanks' balanced salt solution containing 20 mM HEPES, pH 7.4). Cultures were subsequently placed in 1-ml coverslip chambers, and loading solution was replaced with external solution. Chambers were mounted on the stage of a Zeiss 100 TV inverted microscope, with a ×40 objective (Fluar, Zeiss, New York, NY). Mounted cells were perfused with external saline (~1 ml/min) using a two-channel peristaltic pump (Rainin, Woburn, MA). All experiments were performed at room temperature (20-23°C). Intracellular Ca2+ was measured with a commercially available fluorescence videomicroscopy system (RatioVision, Atto Instruments, Rockville, MD) using 334- and 380-nm excitation filters as described (13). Calibration of the fura 2 fluorescence ratios was accomplished in vitro using a series of buffered Ca2+ standards containing Mg2+ (Molecular Probes). [Ca2+]i was measured within regions of interest (ROI) consisting of pixel arrays (100 points, 8 µm × 6 µm) and calculated as the average value of all points. The propagation velocity of a Ca2+ wave was calculated as the distance between two ROI divided by the difference in arrival times of the start (or half maximal concentration) of the Ca2+ wave at the two ROI. Propagation delays between nerve and muscle were determined by measuring the difference in arrival times between ROI on neurite and muscle spaced no more than 4 µm apart. In general, full ratio images were obtained at sampling rates of 1.0-1.5 images/s.

Lucifer yellow dye transfer analyses. The gap junction-permeant dye Lucifer yellow (1% in buffer containing, in mM, 92 KCl, 40 KOH, 1 CaCl2, 1 MgCl2, 11 EGTA, and 20 HEPES, pH 7.4) was microinjected into myocytes using a pressure ejection system (Eppendorf, Madison, WI). Lucifer yellow was also introduced into DRG soma by either pressure or diffusion through a whole cell patch clamp (28, 40). Dye was allowed to transfer for 2-5 min while live cells were imaged using a fluorescence videomicroscopy system (RatioVision, Atto Instruments).

RT-PCR analyses of connexin mRNA. Total RNA was extracted from colonic smooth muscle cell cultures, DRG cultures, colonic smooth muscle cell-DRG cocultures, and freshly dissociated DRG neurons using RNAzol B as per manufacturer's instructions (TEL-TEST, Friendswood, TX). First-strand cDNAs were synthesized using the Advantage Rt-for-PCR kit (Clonetech, Palo Alto, CA) and then used as template for PCR, as per manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN). Nucleotide sequences of the sense and antisense primers for connexin 43 (Cx43), Cx32, and Cx26 were as described (27). The oligonucleotides 5'-CTTGGCAAGATGGGTGA-3' and 5'-GACTGGTCCTCCCTAAGAGG-3' were used as sense and antisense primers, respectively, to amplify a 1104-bp fragment of Cx40 DNA. PCR reactions were heated to 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1.5 min. Control PCR reactions containing equal amounts of corresponding total RNA as template were performed for each connexin primer set.

Intracellular injection of heparin-chondroitin sulfate. Heparin (20 mg/ml in 75 mM KOH with 280 mM HEPES, pH 7.2) and chondroitin sulfate (10 mg/ml in 75 mM KOH with 280 mM HEPES, pH 7.2) were introduced into DRG neurons by a pressurized microinjection system (Eppendorf). Injection pressures ranged from 300 to 600 hPa, and injection times ranged from 0.3 to 0.9 s. Texas red-coated dextran (0.5 mM; mol wt 10,000; Molecular Probes) was included in the heparin injection solution to assay injection efficacy; within 10 s of injection, Texas red-dextran could be seen along the entire length of all visible neuritic projections. Only injections that produced no detectable effect on size and shape of the soma or any of the neuritic processes were used for experiments.

Statistical analyses. Resting [Ca2+]i levels and the amplitude of the transient response were measured. Data are reported as means ± SE. Statistical analysis was accomplished using Student's t-test performed after logarithmic transformation of data, and significance was expressed at the P < 0.05 level throughout. To take into account variability in [Ca2+]i levels that occurs between cultures and between cells, paired experiments were performed for most conditions. Control values from a single cell in normal saline were compared with values from the same cell obtained after exposure to an experimental solution.


    RESULTS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

We have previously described a model system involving the coculture of primary colonic myocytes and DRG neurons (10). We have now used this model system to examine an extensive number of neurite-myocyte contacts (n = 33), as well as neurite contacts with other cell types. As expected, brief mechanical stimulation of colonic myocytes in contact with DRG neurites, either directly at the site of neurite innervation (<2 µm from the neurite, n = 7), in close proximity to the neurite-myocyte contact (2-8 µm from the neurite; n = 20), or at a site distal from the point of contact (>8 µm from neurite; n = 6), caused >90% of the muscle cells to respond with an increase in [Ca2+]i (average resting level = 91 ± 9 nM; average peak transient = 306 ± 41 nM; n = 28). This [Ca2+]i transient propagated within the stimulated myocyte at a mean velocity of 14 ± 2 µm/s (n = 22). Myocyte stimulation also caused an increase in [Ca2+]i in 57% of the contacting neurites (19 of 33 apparent contacts; average neurite resting level = 60 ± 9 nM; average neurite peak transient = 252 ± 33 nM; also see Figs. 1 and 2). For reasons that are not yet understood, the time required to detect an increase in neurite [Ca2+]i varied greatly, from <1 s to several minutes after myocyte stimulation. A representative example of a slowly propagating wave is shown in Fig. 1. The mean propagation distance of the [Ca2+]i wave within the neurites was 55 ± 7 µm (n = 29), and propagation to the soma was observed in 5 of 33 contacts. The frequency and rate of intercellular wave propagation from myocyte to neuron, the average distance of propagation, and the peak transient levels of [Ca2+]i did not vary with the site of myocyte stimulation. In addition, the intercellular wave propagation into a contacting neurite was not detectably dependent on the rate or direction of solution perfusion (S. H. Young, unpublished observations), suggesting that extracellular mediators do not play a significant role in this process.


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Fig. 1.   Propagation of intracellular Ca2+ concentration ([Ca2+]i) transient from mechanically stimulated myocyte into dorsal root ganglion (DRG) neuron shows complex temporal characteristics. A single mechanical stimulation of neurite-contacted muscle cell produced an intercellular [Ca2+]i wave that then propagated intracellularly into adjoining muscle cells, as well as into neurites of connecting neuron. a: White light illumination of experimental preparation. * Stimulation point of muscle cell (m1). Muscle m1 is in direct contact with a DRG neuron (neurite segments labeled n1 and n2; neuronal soma is "off screen") and another muscle cell (m2). A 3rd muscle cell (m3) contacts muscle cell m2 as well as DRG neurite. Scale bar, 30 µm. b: Image of preparation taken seconds after mechanostimulation and pseudocolored for [Ca2+]i shows a large increase in [Ca2+]i spreading from point of stimulation throughout cell. Color scale is labeled for [Ca2+]i in nM. c: At 454 s after single mechanostimulation of muscle cell m1, [Ca2+]i increase has now spread into muscle cell m2 and from muscle cell m2 into muscle cell m3 but not into neurite. d: Image taken 40 s after c. [Ca2+]i wave has now started in cell neurite and is spreading away from point of contact.


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Fig. 2.   [Ca2+]i within regions of interest (ROI) plotted vs. time for experiment shown in Fig. 1. ROI are followed for muscle cells m1, m2, and m3 and from 2 regions along neurite of DRG cell (n1 and n2). Regions are identified in Fig. 1, a and c. Muscle cell m1: after a single stimulation, [Ca2+]i in muscle cell m1 rose rapidly to ~500 nM above resting level (50 nM). After initial rise, 3 subsequent spontaneous transient increases occurred, the last ~210 s after initial stimulation. Muscle cell m2: this cell did not respond immediately after initial increase of muscle cell m1. The 3rd spontaneous increase of [Ca2+]i in muscle cell m1 is linked with a small increase in [Ca2+]i in muscle cell m2. Muscle cell m2 then showed a small increase ~100 s later, followed by a larger and longer lasting transient after another 100 s. Muscle cell m3: this cell, which is connected to cell m2, showed a [Ca2+]i increase that begins after 3rd and largest increase of [Ca2+]i in cell m2. DRG neurite locations n1 and n2: [Ca2+]i increases in this neurite started in area that overlies muscle cell m2 and then spread along neurite in both directions at a speed of 8 µm/s at distances up to 90 µm. However, start of response is not clearly linked to start of any of [Ca2+]i transients experienced by cell m2 but does appear after start of largest [Ca2+]i transient appearing in cell m2. Small transient on declining phase of cell m3 is linked to increases in n1 and n2.

We also performed a series of direct mechanical stimulations of the neurites in DRG cultures without cocultured muscle. [Ca2+]i was monitored along the neurite as well as at the soma of stimulated neurons. No changes in [Ca2+]i were observed following deformation of 20 neurites from 18 neurons. One neuron, however, showed an increase in [Ca2+]i in the soma following mechanical stimulation of the neurite. In contrast, 20% of the somata showed [Ca2+]i transients following mechanical stimulation of the soma, a response similar to our previous observations (29). These data argue against the likelihood that neuritic [Ca2+]i transients observed in response to myocyte stimulation arise from indirect mechanical stimulation of the nerve terminal.

We sought to determine whether tetrodotoxin (TTX)-sensitive Na+ channels or Gd3+-sensitive stretch-activated channels contributed to the formation and/or intercellular propagation of [Ca2+]i transients between colonic myocytes and contacting DRG neurites. In paired experiments, addition of 1 µM TTX to the external saline did not significantly change resting neurite [Ca2+]i, the amplitude of the neurite [Ca2+]i transient, or its propagation velocity (Fig. 3 and Table 1). Cells were also superfused with 100 µM Gd3+, a concentration previously shown to block L-type voltage-sensitive Ca2+ channels and to greatly reduce mechanically induced [Ca2+]i transients in DRG somata (29). However, Gd3+ did not significantly change the resting [Ca2+]i of the colonic smooth muscle cell or the DRG neurite (Table 1). Gd3+ also had no effect on the peak amplitude of the [Ca2+]i transient in the stimulated muscle or the neurite, nor did it affect the propagation velocity of the [Ca2+]i transient (Table 1). Thus TTX-sensitive Na+ channels, Gd3+-sensitive stretch-activated channels, and L-type voltage-sensitive Ca2+ channels do not contribute to the mechanically induced increase in myocyte and neurite [Ca2+]i or to the propagation of the Ca2+ wave into the neurite. Instead, our results are more consistent with the hypothesis that Ca2+ waves propagate from colonic smooth muscle cells to DRG neurons via intercellular gap junctions.


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Fig. 3.   Tetrodotoxin (TTX) does not affect [Ca2+]i transient in neurite. A: [Ca2+]i vs. time for a neurite-contacted muscle cell. Mechanical stimulus was applied to muscle cell in close proximity to neurite in normal saline, resulting in [Ca2+]i transients in both myocyte and neuron. Although addition of 1 µM TTX to bath slightly reduced amplitude of [Ca2+]i transient in depicted cell, there was no significant effect of TTX on average [Ca2+]i transient amplitude calculated from several independent experiments (see Table 1). B: [Ca2+]i vs. time for soma of connecting neurite, with same time scale as top. The 1st stimulation of muscle cell produced a [Ca2+]i transient that propagated from contacting neurite into soma. In presence of 1 µM TTX, [Ca2+]i wave continued to propagate into soma.

                              
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Table 1.   Effects of TTX and Gd3+ on mechanically induced increases in myocyte and neurite [Ca2+]i

Evidence for gap junction-mediated intercellular communication between myocytes and DRG neurites. To demonstrate direct intercellular communication between colonic myocytes and DRG neurons, we performed dye transfer assays with the gap junction-permeant tracer Lucifer yellow (521 mol wt). A 1% solution of Lucifer yellow was microinjected into nine myocytes in contact with neuritic processes. We observed prompt transfer of the dye into the contacting neurites in three of the cell pairs. A representative experiment is shown in Fig. 4. Thus functional gap junctions are present between myocytes and DRG neurons. We also injected Lucifer yellow into DRG somata to assess communication in the opposite direction, i.e., from neuron to myocyte, but failed to detect any dye transfer (data not shown).


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Fig. 4.   Dye coupling between myocytes and DRG neurites. a: Phase photomicrograph of a muscle cell-neuron coculture in which two muscle cells (muscle 1, muscle 2) are in contact with each other as well as in contact with several neurites from 5 different somata. Scale bar, 25 µm. b: Fluorescence image obtained 1 min after muscle cell 1 was injected with internal solution containing 1% Lucifer yellow. Dye can be seen in muscle cell 2 and in connecting neurites from cluster. c: Image obtained 5 min after injection. Dye signal has increased in muscle cell 2 and can be measured in 3 of neuronal somata.

Differences in communication efficiency between DRG neurons and various target cell types. We examined the specificity of intercellular Ca2+ wave propagation between DRG neurons and other putative target cell types. Neural cocultures were established with A7r5 vascular smooth muscle cells and with IEC-6 intestinal epithelial cells. After 48 h of coculture, innervated target cells were mechanically stimulated as described above. Both A7r5 and IEC-6 cells displayed mechanically inducible increases in [Ca2+]i that propagated as intracellular waves. We observed intercellular propagation of [Ca2+]i transients from A7r5 cells into 50% of the contacting DRG neurites (n = 6 contacts). In contrast, cocultures with IEC-6 cells showed intercellular propagation of [Ca2+]i transients into only 13% of the contacting neurites (n = 15 contacts). These results suggest that cultured DRG neurons communicate through gap junctions more efficiently with colonic and vascular smooth muscle target cells than with intestinal epithelial cells.

Connexin expression in DRG neurons and target cells. RT-PCR was used to characterize the connexin mRNAs expressed by DRG neurons, primary colonic myocytes, A7r5 vascular smooth muscle cells, and IEC-6 intestinal epithelial cells. We detected expression of Cx43 and Cx40 mRNA (Fig. 5) but not Cx32 or Cx26 mRNA (data not shown) in the cultured DRG neurons. Little or no amplification was observed in control reactions containing total RNA as template, indicating that the DNA fragments amplified in the PCR reactions were derived from first-strand cDNAs and not from contaminating genomic DNA. We also detected expression of Cx43 and Cx40 mRNA in freshly dissociated DRG neurons, suggesting that these connexin genes are expressed in vivo as well as in culture (Fig. 5). Primary colonic smooth muscle cells and the A7r5 vascular smooth muscle cells similarly expressed Cx43 and Cx40 mRNA (Fig. 5) but not Cx32 or Cx26 mRNA (data not shown). This agrees with electrophysiological data demonstrating expression of both Cx43 and Cx40 in A7r5 cells (24). In contrast, IEC-6 intestinal epithelial cells expressed Cx43 mRNA but not Cx40 (Fig. 5), Cx32, or Cx26 (data not shown). These results are consistent with results showing greater efficiency of intercellular Ca2+ wave propagation between DRG neurons and smooth muscle cells than between DRG neurons and IEC-6 epithelial cells, as further discussed below (see Interactions between DRG neurons and target cells in coculture).


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Fig. 5.   Connexin expression in DRG neurons and target cells. RT-PCR was used to examine expression of connexin 40 (Cx40) and Cx43 mRNA in freshly isolated DRG neurons (F-DRG), cultured DRG neurons (C-DRG), primary colonic smooth muscle cells (CSMC), A7r5 vascular smooth muscle cells, and IEC-6 intestinal epithelial cells. Each panel shows 4 lanes displaying ethidium bromide-stained products of PCR reactions containing primers for Cx43 (lanes 1 and 2) or Cx40 (lanes 3 and 4) and either total RNA (lanes 1 and 3) or first-strand cDNA (lanes 2 and 4) as template. Results show expression of Cx43 (1275-bp fragment) and Cx40 (1105-bp fragment) in F-DRG, C-DRG, CSMC, and A7r5 cells but show expression of only Cx43 in IEC-6 cells.

Ca2+ is not detectably transferred from neurons to myocytes. The mechanism by which mechanical stimulation of target cells leads to increased [Ca2+]i in DRG neurites is not known. To examine whether Ca2+ can directly move through the gap junction, we selectively increased [Ca2+]i in DRG neurons by dropwise application of ~1 µM capsaicin to the external bath solution. Capsaicin has been shown to activate a Ca2+-permeable cation channel in small DRG neurons (41), but it has no effect on smooth muscle cells cultured alone (Young, unpublished observations). In coculture, capsaicin treatments caused an average increase in [Ca2+]i in DRG neurons, from a baseline value of 136 ± 28 nM to a stimulated value of 549 ± 121 nM (n = 5). However, this dramatic increase in neurite [Ca2+]i did not cause any detectable increases in [Ca2+]i within smooth muscle cells. Although these experiments do not rule out the possibility that Ca2+ can traverse gap junctions only in one direction from myocyte to DRG, we do not favor a model of asymmetrical intercellular communication. Instead, these results suggest either that too little Ca2+ diffused into the myocyte to be detected or, more likely, that Ca2+ does not traverse the gap junction.

Mechanically induced [Ca2+]i increase results from release of Ca2+ from thapsigargin-sensitive intracellular Ca2+ stores within the neurite. We next examined whether Ins(1,4,5)P3 mediates communication between DRG neurons and target cells, since this signaling molecule is thought to regulate Ca2+ wave propagation between other cell types (6, 31, 39). Colonic myocytes were mechanically stimulated in the presence of thapsigargin, which depletes intracellular stores of Ca2+. In four paired experiments, muscle-neurite communication was initially demonstrated in the absence of inhibitor, and then thapsigargin (1 µM) was added to the bath. Thapsigargin produced a transient increase in [Ca2+]i in the neurites and soma of the DRG cells and in myocytes, which returned to near (98 ± 10%) resting levels after an average of 145 ± 14 s, at which time the myocytes were stimulated again. Muscle cell responses to mechanical stimulation in the presence of thapsigargin were moderately reduced to 81 ± 4% of their initial (before thapsigargin) responses, which is similar to the effects of thapsigargin on mechanically induced [Ca2+]i transients within rabbit colonic cells (39). In contrast, thapsigargin completely blocked the propagation of [Ca2+]i transients into DRG neurites in all cell pairs examined (n = 5). This is consistent with our previous report showing thapsigargin-mediated inhibition of [Ca2+]i wave propagation between rabbit colonic myocytes (39).

We also evaluated the effect of Ins(1,4,5)P3 receptor antagonism by heparin on neuronal [Ca2+]i. Introduction of heparin into cultured rabbit tracheal epithelial cells has been shown to block intracellular [Ca2+]i wave propagation (2). Heparin injections were performed in paired trials in which we first identified myocyte-neurite contacts capable of intracellular [Ca2+]i wave propagation to the neuron soma and then injected the soma with heparin (8,000 mol wt; 20 mg/ml; Fig. 6). In all cell pairs (n = 6), heparin injection prevented the [Ca2+]i wave from traveling into the neuron without affecting the mechanically induced [Ca2+]i response in the muscle. Control experiments with intracellular injection of chondroitin sulfate had no effect on [Ca2+]i wave propagation into the neurites (n = 4). These experiments demonstrate that transient mechanical stimulation of colonic smooth muscle cells in contact with DRG neurites results in the release of Ca2+ from thapsigargin-sensitive stores within the neurites.


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Fig. 6.   Injection of heparin into DRG soma prevents propagation of [Ca2+]i wave into neuron. At time 0, muscle cell was stimulated and a [Ca2+]i transient was produced (A), which propagated into soma of a connecting neuron (B). Soma was then microinjected with a saline containing heparin; 600 s after first stimulus, muscle cell was stimulated again. Muscle cell (A) responded with 2nd [Ca2+]i transient, but transient did not propagate into soma (B).


    DISCUSSION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

The current study demonstrates that transient mechanical stimulation of colonic smooth muscle cells in contact with DRG neurites results in the release of Ca2+ from thapsigargin-sensitive stores within the neurites. The signal for Ca2+ release from neurite Ca2+ stores is not due to direct mechanical stimulation of the DRG nerve terminal and does not involve Gd3+-sensitive or TTX-sensitive Ca2+ influx through the neurite plasma membrane. Instead, these increases in neurite [Ca2+]i require a chemical messenger molecule, most likely Ins(1,4,5)P3, which is generated in response to mechanical stimulation of the myocytes.

Mechanically induced elevation of DRG neurite [Ca2+]i is not associated with influx of Ca2+ through plasma membrane. Transient mechanical stimulation of DRG somata is associated with robust and reproducible [Ca2+]i transients in approximately one-third of stimulated cells (29). These [Ca2+]i transients are dependent on influx of extracellular Ca2+ via Gd3+-sensitive pathways, presumably stretch-activated cation channels (29). However, in this study, the failure of Gd3+ and TTX to affect the amplitude and propagation of the neurite [Ca2+]i transient rules out a significant role for stretch-activated cation channels, L-type voltage-sensitive Ca2+ channels, or mechanically induced Na+-dependent action potentials in the generation and spatial propagation of neurite [Ca2+]i changes. This is consistent with previous studies showing myocyte-to-neurite wave propagation in the absence of extracellular Ca2+ (10). The inability of a mechanical stimulus to induce Ca2+ influx in the neurite could be related to constitutive differences in expression of stretch-activated cation channels between soma and neurite or to a selective target inhibition of the peripheral expression of these channels in certain DRG neurites (38). A specific inhibitory effect of the cocultured smooth muscle on the expression of mechanosensitive channels is unlikely in view of our findings showing a similar lack of neurite mechanosensitivity when DRG neurons were cultured alone. However, by analogy with other neuron-target cell interactions (14, 15), it is possible that the expression of mechanosensitive channels in DRG neurites requires stimulatory signals from other specific target cells within the bowel wall, such as the interstitial cells of Cajal or intrinsic enteric neurons.

Interactions between DRG neurons and target cells in coculture. Our previous studies (10) suggested that Ca2+ wave propagation between primary colonic myocytes and DRG neurites was mediated by gap junctions, since propagation could be inhibited by octanol, a nonspecific inhibitor of gap junction permeability (8). We now provide multiple lines of evidence for gap junction-mediated intercellular communication between DRG neurons and target cells. 1) When the low-molecular-weight dye Lucifer yellow was injected into colonic myocytes, it promptly diffused into a subset of contacting DRG neurites, indicating the presence of functional gap junctions. The fact that not all contacting neurites received detectable amounts of Lucifer yellow suggests that DRG neurons and myocytes are not highly coupled or perhaps are transiently coupled. Alternatively, gap junctions between neurons and myocytes may have preferred permeabilities that do not support the facile transfer of Lucifer yellow (12). We failed to detect dye transfer in the opposite direction, i.e., from neurite into myocyte. It is possible that Lucifer yellow dye transfer between myocytes and DRG neurons is asymmetrical, as has been suggested for dye transfer between vascular smooth muscle cells and endothelial cells (21). However, it seems more likely that too little Lucifer yellow diffused through the small neuritic processes during the short experimental duration (2-5 min) to be detected in the larger smooth muscle cells. 2) Intracellular Ca2+ transients induced in smooth muscle cells by mechanical stimulation showed propagation into DRG neurites with a characteristic delay at the intercellular junction. The delay observed in the current study was similar in magnitude to that reported in intercellular [Ca2+]i wave propagation in rabbit tracheal cells (33) and bovine endothelial cells (7). It has been postulated that the delay is due to low-level junctional coupling between the cells, slowing the diffusion of Ins(1,4,5)P3 from one cell into its neighbor (31). Alternatively, it may reflect the time required for the generation of Ins(1,4,5)P3 in response to the mechanically induced Ca2+ increase in the stimulated cell. 3) The intercellular propagation of [Ca2+]i waves occurs with varying efficacy between DRG neurons and other target cell types. Such differences in coupling efficiencies may reflect the specific patterns of connexins expressed by these cell types (see below). 4) DRG neurites and target cells express multiple connexin mRNAs. We screened for 4 of the 12 known connexins (12) and detected expression of Cx43 and Cx40 mRNA in DRG neurons as well as colonic and vascular smooth muscle cells but detected only expression of Cx43 in intestinal epithelial cells. These two connexins are incompatible, i.e., connexons (gap junction hemichannels) composed of Cx43 do not interact and form functional intercellular channels with connexons composed of Cx40 (3). If Cx43 and Cx40 are the predominant connexins present in these cell types, then we would expect homotypic channels composed of either Cx43 or Cx40 to form between DRG neurons and smooth muscle cells, whereas only channels composed of Cx43 would form between DRG neurons and IEC-6 cells. This may explain why DRG cells showed decreased coupling efficiencies with IEC-6 cells. Channels composed of Cx43 and Cx40 also display differences in voltage sensitivity (3), suggesting possible differences in the properties of intercellular communication between DRG neurons and their various target cell types.

Mechanically induced elevation of DRG neurite [Ca2+]i is associated with Ca2+ release from thapsigargin-sensitive stores. We performed several experiments examining mechanisms underlying propagation of the mechanically induced Ca2+ transient into DRG neurites. Capsaicin treatment of cocultures caused dramatic increases in neural [Ca2+]i but no resulting change in myocyte [Ca2+]i. As with the Lucifer yellow dye transfer experiments, it is possible that not enough Ca2+ diffused through the neuritic process to be detected in the myocyte or that the transfer of Ca2+ through myoneural gap junctions is asymmetrical. Alternatively, Ca2+ may simply not traverse gap junctions. We thus examined the effects of thapsigargin on intercellular Ca2+ wave propagation and found that thapsigargin abolished increases in neurite [Ca2+]i while only slightly inhibiting [Ca2+]i transients within myocytes. These findings suggest that a chemical messenger other than Ca2+ stimulates release of Ca2+ from thapsigargin-sensitive stores within the neurite. Plausible candidates include Ins(1,4,5)P3 and cyclic adenosine diphosphoribose, both of which have been proposed as long-range chemical messengers in neurons (17). The fact that blockade of neurite Ins(1,4,5)P3 receptors with heparin completely abolished intercellular propagation of the Ca2+ transient into the neurite strongly implicates Ins(1,4,5)P3 as the likely intercellular transfer signal. These findings are analogous to recent reports on Ins(1,4,5)P3-mediated intercellular Ca2+ waves between astrocytes (6) and between myocytes (39) in primary culture. Furthermore, direct transfer of caged Ins(1,4,5)P3 through gap junctions has recently been demonstrated (5).

Possible physiological implications of DRG myocyte interactions. Gap junctions mediate homologous intercellular communication in a great variety of cell types, including smooth muscle cells (39), glial cells (11), and neural cells (6, 11, 18, 25, 30, 36, 37). Coupling between neural and glial cells has also been reported (11, 18, 30, 36, 37), and one study even demonstrated unidirectional propagation of a Ca2+ wave from electrically stimulated astrocytes into cocultured neurons (25). However, we believe this is the first description of gap junction-mediated intercellular communication between target cells and neurons.

We detected expression of connexin mRNAs from freshly isolated DRG, suggesting that these neurons make gap junction proteins in vivo. However, we do not have or know of any direct evidence demonstrating intercellular communication between target cells and DRG neurons in vivo. Thus we can only speculate about the possible physiological implications of our data. The magnitudes of the neurite [Ca2+]i transients that we observed in vitro after mechanical stimulation of target cells were significantly below those thought to be required for secretion of peptides from DRG cell bodies (20). Thus, unless coupling between target cells and neurons in vivo is significantly more efficient, this form of intercellular communication may not regulate neurosecretion. However, it may play a modulatory role. For example, intercellular propagation of Ca2+ waves from myocyte to neurite could activate Ca2+-sensitive ion channels in the neurite membrane, thereby modulating the excitability of the terminals. Finally, gap junction-mediated communication between myocytes and a class of mechanically insensitive DRG neurons may provide a mechanism by which chemical, electrical, or mechanical stimuli acting on the muscle are signaled to peripheral networks of DRG terminals and perhaps by which neuronal events are signaled to the muscle. On the basis of findings in other cell types (6), the nature of the intercellular messenger may differ depending on the type of stimulus (i.e., chemical, mechanical, or electrical activation). The exchange of molecules such as Ins(1,4,5)P3, cAMP, or cyclic adenosine diphosphoribose may have important effects on phosphorylation and expression of plasma membrane proteins such as ion channels and receptors that may modulate the response properties of DRG neurons and/or the muscle itself (22, 16).


    ACKNOWLEDGEMENTS

We thank Drs. J. A. McRoberts and M. Gold for critical review of the manuscript.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-40919 and DK-48351 (to E. A. Mayer).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: H. S. Ennes, Dept. of Physiology, UCLA School of Medicine, Los Angeles, CA 90024.

Received 30 July 1998; accepted in final form 11 November 1998.


    REFERENCES
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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Am J Physiol Cell Physiol 276(3):C602-C610
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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