Norepinephrine-induced calcium signaling and expression of adrenoceptors in avian tendon cells

Michelle E. Wall,1,2 James E. Faber,3 Xi Yang,2 Mari Tsuzaki,2 and Albert J. Banes1,2,4,5

1Department of Biomedical Engineering, 2Department of Orthopaedics, 3Department of Cell and Molecular Physiology, and 4Curriculum in Applied and Material Sciences, University of North Carolina at Chapel Hill, Chapel Hill 27599; and 5Flexcell International Corporation, Hillsborough, North Carolina 27278

Submitted 19 February 2004 ; accepted in final form 9 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sympathetic efferent nerves are present in tendons, but their function within tendon is unknown. {alpha}1-Adrenoceptors are expressed by a variety of cell types. In the presence of norepinephrine (NE), adrenoceptors activate Gq/11 signaling pathways that subsequently increase intracellular Ca2+ concentration ([Ca2+]ic). It was hypothesized that avian tendon cells express functional adrenoceptors that respond to NE by increasing [Ca2+]ic. Avian tendon cells were analyzed for mRNA expression of {alpha}1-adrenoceptors by RT-PCR. Avian tendons expressed the {alpha}1A- and {alpha}1B-adrenoceptor subtypes. Furthermore, both tendon surface epitenon cells and internal fibroblasts infused with a Ca2+-sensitive dye, fura 2, and stimulated with NE responded by increasing [Ca2+]ic. KMD-3213, an {alpha}1A-adrenoceptor antagonist, significantly reduced the Ca2+ response. Other adrenoceptor antagonists had no effect on the Ca2+ response. The absence of extracellular Ca2+ also significantly reduced the response to NE, indicating that Ca2+ influx contributed to the rise in [Ca2+]ic. This study provides the first evidence that tendon cells express adrenoceptors and that the NE-induced Ca2+ response is coupled to the {alpha}1A-adrenoceptor subtype.

{alpha}-adrenoceptors; fibroblasts; catecholamines; tenocytes


GROWTH FACTORS, HORMONES, the mechanical environment, and catecholamines help mediate tissue growth, development, and repair. Norepinephrine (NE) stimulation of vascular {alpha}-adrenoceptors induces mitosis (14, 35, 52), vascular wall growth, contraction, and an increase in blood pressure as well as actions in other cell types (see Refs. 17 and 43 for review). Despite the presence of sympathetic efferent nerves in tendon, ligament, and other connective tissues (2, 9, 19, 46), little is known about the expression and function of adrenoceptors in these tissues.

Adrenoceptors are G protein-linked cell membrane receptors that are divided into three main families: {alpha}1, {alpha}2, and {beta}. Stimulation of {alpha}1-adrenoceptors can liberate inositol trisphosphate, diaclyglycerol, arachidonic acid, and phosphatidic acid and produce changes in cAMP and intracellular Ca2+ concentration ([Ca2+]ic) (12, 17, 22, 43, 51). Stimulation of {alpha}2-adrenoceptors can inhibit (block or close) Ca2+ channels, open potassium channels, and inhibit adenyl cyclase (12, 17, 43). However, data also indicate that {alpha}2-adrenoceptors are linked to Ca2+ influx (43). Stimulation of {beta}-adrenoceptors activates adenyl cyclase and thus increases cAMP (12).

There are three {alpha}1-adrenoceptor subtypes: {alpha}1A, {alpha}1B, and {alpha}1D, the functions of which are widespread and the actions of which can depend on which cell type expresses them and the anatomical location (see Refs. 15, 17, 32, and 49 for reviews). For instance, stimulation of {alpha}1B-adrenoceptors on rat aorta adventitial fibroblasts induces proliferation, whereas {alpha}1A-adrenoceptors mediate this action on medial smooth muscle cells. In contrast, {alpha}1D-adrenoceptors mediate constriction of this vessel (52). With the vascular system, all {alpha}1-adrenoceptor subtypes are important for controlling contraction, vessel tone, and blood pressure.

Adrenergic nerves (i.e., containing tyrosine hydroxylase and neuropeptide Y immunoreactivity) are associated with the microvasculature within and around tendons and in the paratenon (2). Sympathetic, hemodynamic, and biomechanical factors modulate blood flow in tendon and ligament as in most other tissues (39, 48). In several cell types, NE or epinephrine induces an increase in [Ca2+]ic through both Ca2+ influx and/or release from intracellular stores (11, 29, 31, 33, 38, 41, 44).

Evidence of adrenoceptors in connective tissues is found in UMR-106 cells, a rat osteosarcoma cell line, wherein cAMP and intracellular Ca2+ increased in response to NE (10, 33). The Ca2+ response was linked to both {alpha}1- and {beta}-adrenoceptors. Ca2+ release from bone is also modulated by {alpha}- and {beta}-adrenoceptors (47). Furthermore, adrenoceptors were unexpectedly found in relatively high abundance in vascular adventitial fibroblasts (20) that migrated and proliferated in response to {alpha}1-adrenoceptor stimulation (20, 52, 53). Mouse embryonic fibroblasts (3) as well as rat cardiac fibroblasts (35) also proliferated in response to {beta}-adrenoceptor stimulation. However, no published data implicate a particular adrenoceptor subtype expressed in tendon cells.

Within tendons, an endotenon is continuous with the epitenon that encases the whole tendon. The endotenon, a sheet of loose connective tissue, binds together the blood vessels, nerves, and collagen fascicles. Thus the surface cells of the epitenon are more likely to be exposed to NE under normal physiological conditions than the internal fibroblasts that reside amidst the collagen fibrils. NE may have a greater effect on these epitenon cells than the internal fibroblasts. However, both cell groups would be exposed to NE during traumatic injury. Therefore, it was hypothesized that avian tendon surface epitenon cells (ATSC) and internal fibroblasts (ATIF) express adrenoceptors and respond to NE with an increase in [Ca2+]ic.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation and culture. Surface epitenon cells and internal fibroblasts were isolated from 6–8 pooled digital flexor digitorum profundus (FDP) tendons of 42-day-old Cornish chickens (Perdue Farms, Robbins, NC) by sequential collagenase and trypsin digestions modified from Banes et al. (5). The tendon was severed proximal to the oseotendinous junction and at the level of the third metatarsal bone. Cells were cultured in DMEM with 5% fetal bovine serum, 100 µg streptomycin, and 100 units/ml penicillin. To evaluate the in vitro Ca2+ response, both ATSC and ATIF between passages 1 and 6 were plated in micromass, spot cultures at 2,000 cells/10-µl spot (2 spots/well) in six-well plates, grown to confluence, and made quiescent by halving the serum content of the medium on days 3 and 5 after plating. All cell culture media and chemicals were purchased from Sigma (St. Louis, MO), GIBCO BRL (Grand Island, NY), or HyClone (Logan, UT). Avian feet were processed according to the approved protocol for specimens from the abattoir and to regulations of the Institutional Animal Care and Use Committee.

Tendon explant isolation. To evaluate the Ca2+ response in tendon explants, the FDPs were removed from the middle toe of both feet of 5- to 6-wk-old White Leghorn chickens (Cackle Hatchery, Lebanon, MO). Each FDP was cut into three sections, labeled with fura 2-acetoxy methylester (fura 2-AM; Molecular Probes, Eugene, OR), and then potted in 1% agarose gel (to stabilize the tendon during addition of the agents) in Earle's balanced salt solution (EBSS) with 20 mM HEPES, pH 7.2, 1.80 mM calcium chloride, and 0.8 mM magnesium sulfate (Tissue Culture Facility, Chapel Hill, NC).

Total RNA isolation from tissue. Total RNA was isolated from tissue samples (1–5 g wet wt each of brain, pectoralis major muscle, spleen, kidney, lung, heart, FDP tendon, metatarsal bone, liver, and cartilage from the tip of the metatarsal bone) from six 52-day-old White Leghorn chickens (Goldkist, Sanford, NC) as previously described by Banes et al. (7). Briefly, samples were weighed (1 g wet wt each), frozen in liquid nitrogen, pulverized, and solubilized with RNA Stat-60 (Tel-Test B, Friendwood, TX). Samples were homogenized with a Polytron (Fisher Scientific, Pittsburgh, PA), treated with chloroform, and vortexed. After sedimentation, the supernatant fluid was extracted with RNA Stat-60 and chloroform, mixed, and sedimented. Then, 0.7 vol of isopropanol was added to the supernatant fluid and sedimented. Pellets were reconstituted in 1 ml of diethylpyrocarbonate-treated deionized water.

Total RNA was also isolated from the following tissue samples (0.2–1.2 g wet wt) from six 122-day-old White Leghorn chickens (Cackle Hatchery): proximal and distal FDP, proximal and distal extensor digitorum longus, anterior cruciate ligament, and the medial and lateral menisci (distal denotes the toe and proximal the ankle regions of the chicken foot). After being weighed, samples were pulverized in liquid nitrogen and solubilized in TRIzol (1 ml/100 mg of tissue; GIBCO). Samples were homogenized with a Polytron, incubated at room temperature for 5 min, and then treated with 1-bromo-3-chloropropane (0.1 ml/ml TRIzol; Molecular Research Center, Cincinnati, OH). Samples were mixed, incubated at room temperature for 3 min, and sedimented at 7,200 g at 4°C for 15 min. The aqueous phase was collected, treated with isopropanol (0.5 ml/ml TRIzol), and precipitated at 14,000 g at 4°C for 10 min. Pellets were washed with an equal volume of 70% ethanol and sedimented at 14,000 g at 4°C for 5 min. Pellets were reconstituted in 100 µl of RNase-free water.

Quantitative RT-PCR. Total RNA was used in quantitative RT-PCR experiments to determine the presence of mRNA for {alpha}1-adrenoceptor subtypes in the avian tissues. In the RT reaction, 10 µg total RNA were mixed with the following in final concentrations: 1 mM each of 2-deoxynucleoside 5-triphosphates, 1 ng/µl random hexamers, 0.8 units/µl RNaseOUT recombinant ribonuclease inhibitor, 0.01 M dithiothreitol, 4 units/µl Superscript II RNase RT, and PCR buffer containing 50 mM Tris·HCl, 75 mM KCl, and 3 mM MgCl2 (Invitrogen, Carlsbad, CA; GIBCO). The RT mix was incubated at 25°C for 10 min and then heated at 42°C for 120 min, at 99°C for 5 min, and at 5°C for 5 min. The following sequences were used as sense and antisense adrenoceptor primers, respectively (20): {alpha}1A-adrenoceptor (204 bp), 5'-CGAGTCTACGTAGTAGCC-3' and 5'-GGCTTGGCAGCTTTCTTC-3' [there is 1 base pair difference (underlined) in the cloned avian sequence and the sequence for rat published by Faber et al. (20)]; {alpha}1B-adrenoceptors (201 bp), 5'-ATCGTGGCCAAGAGGACC-3' and 5'-TTTGGCTGCTTTCTTTTC-3'; and {alpha}1D-adrenoceptors (218 bp), 5'-CGCGTGTACGTGGTCGCAC-3' and 5'-CTTGGCAGCCTTTTTC-3'. In the PCR reaction, the cDNAs from the avian tissues were mixed with the following agents at final concentrations: 0.05 units/µl Platinum Taq DNA polymerase (Invitrogen), 0.5 µM internal standard, 0.5 µM of sense and antisense primers, PCR buffer containing 16 mM (NH4)2SO4, 67 mM Tris, pH 8.8, 0.1 mM each of 2-deoxynucleoside 5-triphosphates, 2.25 mM MgCl2, and 0.2 nl Tween 20. The cDNAs from the tissue of the 52-day-old White Leghorn chickens (including the spleen, muscle, liver, kidney, heart, lung, cartilage, bone, tendon, and brain) and the 112-day-old White Leghorn chickens (including proximal and distal FDP, proximal and distal extensor, anterior cruciate ligament, and meniscus) were amplified with QuantumRNA 18s rRNA (Ambion, Austin, TX) as the internal standard. The Competimer technology controls for the amplification of 18s cDNA, thus keeping 18s rRNA within the same linear range as the mRNA of interest. Samples were initially denatured at 94°C for 5 min followed by 35 cycles of denaturing, annealing, and extension at 94°C, 62°C, and 72°C, respectively, for 30 s each and a final extension at 72°C for 5 min in a Perkin-Elmer 2400 thermocycler (Wellesley, MA). Amplification products were separated in 1.8% agarose gels for 22 min at 93 V (20 mA). Gels were stained with ethidium bromide (Fisher Scientific) and photographed with a Polaroid camera (Waltham, MA). The relative band intensity was quantified by image analysis.

Cloning. Primers for rat {alpha}1-adrenoceptors were used to amplify mRNA in the bone tissue from 52-day-old White Leghorn chickens. The cDNAs were extracted from the gel using a QIAquick gel extraction kit and purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA). The cDNAs were ligated using an Original TA cloning kit and transformed into TOP10F Escherichia coli cells using a One Shot kit (Invitrogen). Plasmid DNA was isolated from bacteria using a QIAwell 8 Ultra Plasmid kit (Qiagen) and then sequenced. Sequences were submitted to GenBank.

Intracellular Ca2+ measurements. On day 6 after plating, ATSC and ATIF were rinsed twice with EBSS. The cells were then incubated in the dark at room temperature in 2.5 or 5 µM fura 2-AM with 0.1% Pluronic-127 (Molecular Probes) for 45–60 min. After incubation, the cells were rinsed twice in EBSS to remove any unincorporated fura 2-AM. The six-well dish was mounted on the stage of an Olympus BX-51 upright fluorescence microscope (Melville, NY) equipped with a 40x water immersion ultraviolet objective lens, a Sutter Lambda DG4 wavelength switcher and light guide (Novato, CA), and a CoolSnap digital camera (Roper Scientific, Trenton, NJ). Image analysis software (ISee Imaging Systems, Raleigh, NC) was used to quantitate the changes in [Ca2+]ic. The ratio method, with 340 (bound)- to 380 (free)-nm excitation and 510-nm and above emission, was used to convert the fluorescence emission intensity of the labeled tendon cells to Ca2+ ion concentration by comparison to known Ca2+ standards (23). All cells in a field of view [9–77 cells in 2-dimensional (2-D) cultures, 12–151 cells in explants] were selected and outlined, and the pixel intensity relating to Ca2+ concentration was quantified for each cell. Background images were obtained from an area without cells at the start of each experiment. Background pixel values were subtracted from each image to correct for nonuniformity due to variation in the fluorescence excitation beam across the field.

Treatment with NE and differentiation of adrenoceptor subtypes. Avian tendon cells were exposed to 0.01, 0.03, 0.1, 0.3, 1, or 10 µM NE (n = 2–8 isolations with 2–12 replicates). Cells in any one specimen were exposed to only one NE concentration. Basal Ca2+ levels were determined by averaging the [Ca2+]ic over 30–60 s before NE treatment. For 2-D cultures, a cellular response was mathematically represented as an increase in [Ca2+]ic that averaged three standard deviations above the basal level of the cell. Avian FDP explant sections were treated with 1 or 10 µM NE, and an FDP section from the opposing leg was treated with 1, 10, or 100 µM ATP as a positive control. Each section received only one drug concentration. For explants, a cellular response was mathematically represented as an increase in [Ca2+]ic that averaged two standard deviations above the basal level, since there was a large change in the background across the data set from the autofluorescence of collagen. The response was averaged from the initial increase in [Ca2+]ic after treatment until the Ca2+ signal output returned to a steady level.

ATSC and ATIF were also exposed to 1 µM NE in EBSS that contained no exogenous Ca2+ to differentiate between extracellular Ca2+ influx vs. release of Ca2+ from intracellular stores (n = 4 isolations with 2–6 replicates). Cells were incubated in EBSS without Ca2+ for at least 30 min before NE treatment. To differentiate among the possible adrenoceptors activated by NE, ATIF (n = 3–4 isolations with 3–4 replicates) were incubated in 100 nM of {alpha}1A-adrenoceptor antagonist KMD-3213 (kindly proved by Dr. Y. Kurashina and Kissei Pharmaceutical, Matsumoto City, Japan), {alpha}1B-adrenoceptor antagonist AH-11110A, {alpha}1D-adrenoceptor antagonist BMY-7378, or {alpha}2-adrenoceptor antagonist RX-821002 or 1 µM {beta}-adrenoceptor antagonist propranolol for 30 min before exposure to NE. The concentrations of the antagonists were within the selective concentration for these compounds (52, 53). All pharmacological agents were purchased from Calbiochem (San Diego, CA), Tocris (Ballwin, MO), or Sigma unless otherwise noted.

Statistical analysis. Data were analyzed using JMP (SAS Institute, Cary, NC) or SigmaStat (SPSS, Chicago, IL) and subjected to a Student's t-test, nonparametric t-test, or one-way ANOVA with a post hoc test, where appropriate, to determine significance between and among groups (P < 0.05). Data are presented as means ± SE. About 1.1% of all cells analyzed in 2-D cultures had a slow gradual increase in [Ca2+]ic over the entire period of data collection. Because these cells did not satisfy the defining criteria of a responding cell, they were considered to be nonresponders.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{alpha}-Adrenoceptor expression. The partial avian {alpha}1A-adrenoceptor sequence is aagagagaaa gccggggcct caagtccggc ctcaagacgg acaagtcaga ctcagagcaa gtgacgctcc gcatccaccg taaaaatgtc cctgcagaag gcggcggagt cagcagtgcc aagaataaga ctcacttctc agtgaggctg ctcaagtttt ctcgaga (GenBank AF548388). This sequence is 100% homologous with rat {alpha}1A-adrenoceptor. The partial avian {alpha}1B-adrenoceptor sequence is acaaaaaatc tggaagctgg tgttatgaaa gaaatgtcca actccaagga gctgacgtta cggatccatt acaggaacat tcatgaggac accttaaaca gcaacaaatc caagggtcac aattccagaa actccttagc tctcaaactt ttaaaattct ctaga (GenBank AY155438). This sequence is 85% homologous for rat {alpha}1B-adrenoceptor. All avian tissues analyzed expressed both the {alpha}1A- and {alpha}1B-adrenoceptor subtype mRNA (Fig. 1). The relative amounts of each adrenoceptor subtype within the tissues were not determined, only presence and absence of adrenoceptors. The avian {alpha}1D-adrenoceptor sequence lacked homology to the known rat sequence. Furthermore, the {alpha}1D-adrenoceptor antagonist did not affect the Ca2+ response to NE (see below); therefore, {alpha}1D-adrenoceptor expression was not further analyzed.



View larger version (71K):
[in this window]
[in a new window]
 
Fig. 1. Tissue from the flexor digitorum profundus (FDP) tendon in the toe (FDPdistal) and ankle region (FDPproximal), the extensor digitorum longus tendon in the toe (Extensordistal) and ankle region (Extensorproximal), the anterior cruciate ligament, and the medial and lateral menisci of 122-day-old White Leghorn chickens and tissue from the spleen, pectoralis major muscle, liver, kidney, heart, lung, cartilage from the tip of the metatarsal bone, metatarsal bone, FDP tendon, and brain of 52-day-old White Leghorn chickens express mRNA for {alpha}1A- and {alpha}1B-adrenoceptors. 18s rRNA (18s) was amplified as the internal standard. All reactions were run at 35 cycles to determine only the presence or absence of adrenoceptor expression.

 
Response to NE. NE treatment increased [Ca2+]ic in both ATSC and ATIF cultured from the FDP of 42-day-old Cornish chickens to NE concentrations >0.1 µM (Fig. 2). About 1% of ATSC and 6% of ATIF spontaneously responded before NE treatment. Neither the overall percentage of responding cells to NE nor the pattern of the response was different between the two cell types except at high concentrations of NE (Fig. 2), which is surprising because ATSC are more closely associated with the blood vessels and nerves in tendon. NE induced an immediate Ca2+ transient in ATSC and ATIF (Fig. 2). Ca2+ oscillations also were observed in a small percentage of the cells in response to NE (Fig. 3). An oscillatory response was characterized by an initial Ca2+ transient that returned to a baseline followed by one or more secondary transients that usually had a smaller [Ca2+]ic peak (Fig. 3). The Ca2+ response to NE of tenocytes in FDP explants was relatively low compared with an ATP positive control (Fig. 4).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2. A: all doses of norepinephrine (NE) except 0.01 and 0.03 µM induced a significant increase over control (*P < 0.05). NE induced an immediate increase in intracellular Ca2+ concentration ([Ca2+]ic) that quickly returned to baseline (inset graph). There was no significant difference in response between the avian tendon surface epitenon cells (ATSC) and internal fibroblasts (ATIF) to NE. In the absence of NE (control), more ATIF spontaneously responded than ATSC (#P < 0.01). B: the increase in [Ca2+]ic over baseline did not vary among concentrations. Only 10 nM and 1 µM NE for ATIF and 10 nM and 10 µM NE for ATSC were significantly different from each other (*P < 0.05). At 10 µM NE, ATSC had a larger response than ATIF (#P = 0.004). [NE], NE concentration.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. NE induced an oscillatory Ca2+ response as depicted in the inset graph. The response to 1 µM NE was significantly greater than that for 10 nM NE in the ATIF (*P < 0.05). There was no difference between the response in ATSC and ATIF. NR, no oscillatory responses.

 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4. Tenocytes in FDP explants from 5- to 6-wk-old chickens responded to NE by increasing [Ca2+]ic. ATP also induced a Ca2+ response in the FDP from the opposing leg. All agents induced a significant (*P < 0.05) Ca2+ response compared with the control condition (cells that spontaneously responded in the absence of agonists).

 
Source of rise in intracellular Ca2+. To determine whether the rise in Ca2+ originated from intracellular stores, Ca2+ influx, or both, ATSC and ATIF were treated with 1 µM NE in the absence of exogenous Ca2+. For both cell types, the NE-induced Ca2+ response was moderately attenuated with 20–30% of the cells responding (Fig. 5). The overall increase in [Ca2+]ic over baseline was unaffected by the removal of extracellular Ca2+ (Fig. 5). In addition, <1% of the cells had an oscillatory response (data not shown).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5. The absence of extracellular Ca2+ (–Ca2+) reduced the overall percentage of cells responding to 1 µM NE (A) but not the overall increase in [Ca2+]ic over baseline (B). There was no difference in the Ca2+ response between ATSC and ATIF. A: *P < 0.05 vs. control. #P < 0.05 vs. +Ca2+.

 
Adrenoceptor subtype dependency. To determine the adrenoceptor subtype activated by NE, ATIF were treated with one of the following: KMD-3213, AH-11110A, BMY-7378, or RX-821002 (all at 0.1 µM) or 1 µM propranolol, for 30 min before exposure to 0.01, 0.03, 0.1, 0.3, or 1 µM NE. Only KMD-3213 significantly reduced the percentage of cells responding and the increase in [Ca2+]ic over baseline (Fig. 6, Table 1). In the presence of RX-821002, there was a reduced response to 0.03 µM NE (Table 1).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6. A: {alpha}1A-adrenoceptor antagonist KMD-3213 significantly reduced the response to 1 µM NE (no drug). The presence of other adrenoceptor antagonists did not affect the avian tendon cell Ca2+ response to 1 µM NE. The percentage of tenocytes responding to NE in the presence of an antagonist, except for KMD-3213, was greater than that for tenocytes that spontaneously responded in the absence of NE stimulation (control). B: the inhibitory action of {alpha}1A-adrenoceptor antagonist KMD-3213 on the Ca2+ response was significant at NE concentrations >300 nM.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Normalized increase in intracellular Ca2+ in the presence of {alpha}-adrenoceptor antagonist

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tendons are sympathetically innervated (2). Therefore, the purpose of this study was to determine whether avian tendon cells express adrenoceptors and respond to NE stimulation through a Ca2+ signal. Avian tendon cells expressed mRNA for both {alpha}1A- and {alpha}1B-adrenoceptors. Furthermore, ATSC and ATIF, as well as tenocytes in whole tendon explants, responded to NE by increasing [Ca2+]ic, demonstrating that these cells have functional adrenoceptors.

Tendon cells significantly responded to NE concentrations >0.1 µM. Physiological NE concentration can depend on nearness to adrenergic nerves and level of activity where concentrations are in the micromolar range (8). If the tissue is noninnervated, then the NE concentration depends on plasma concentrations. Basal plasma NE is ~2–3 nmol/l and can increase 10-fold during exercise (25, 30). Injury and inflammation can increase local NE release by locally activating nerves (13). In addition, ischemia can increase NE efflux and thus raise tissue concentrations to micromolar levels (45). Thus the response to NE within tendon cells in vitro is within physiological and/or pathophysiological ranges.

NE induced either an immediate or a biphasic Ca2+ signal. The majority of tenocytes had an immediate Ca2+ transient upon stimulation by NE that rapidly returned to basal levels. In a small percentage of avian tendon cells as well as in astrocytes (41) and hepatocytes (50), a biphasic Ca2+ response was also observed. This response is characterized by an initial Ca2+ transient followed by secondary Ca2+ oscillations. In Chinese hamster ovary cells transfected with {alpha}1A-adrenoceptor (29) and rat afferent arterioles (44), the Ca2+ response is characterized by an initial Ca2+ peak followed by a secondary sustained plateau increase in [Ca2+]ic. In UMR-106 cells (33), the secondary phase was characterized by a slow rise in [Ca2+]ic. This secondary oscillatory response in the avian cells is dependent on Ca2+ influx as illustrated by the loss of oscillations in the absence of exogenous Ca2+. Similar losses of the biphasic response were also seen in Chinese hamster ovary cells, afferent arterioles, and UMR-106 cells.

Because the source of Ca2+ can originate from intracellular stores or from Ca2+ influx, extracellular Ca2+ was removed from the bathing solution and cells were then stimulated with NE. The Ca2+ response in the absence of exogenous Ca2+ was attenuated but not completely abolished, indicating that NE induced the release of Ca2+ from both intracellular stores and influx of Ca2+ through ion channels in the plasma membrane. In addition, the NE-induced Ca2+ rise in tendon cells appeared to be mediated by {alpha}1A-adrenoceptor because a specific antagonist for this adrenoceptor reduced the overall percentage of cells responding to NE as well as the increase in [Ca2+]ic. The reduced response to 0.03 µM NE in the presence of RX-821002 indicated that there may be a small contribution from {alpha}2-adrenoceptors. There was a trend toward reduction in the percentage of cells responding to NE (Fig. 6) and in the rise in [Ca2+]ic (Table 1) in the presence of the {alpha}1B-, {alpha}1D-, and {alpha}2-adrenoceptor antagonists (not significant by 1-way ANOVA). This may reflect, in the case of AH-11110A and BMY-7378, partial inhibition of {alpha}1A-adrenoceptor population by these compounds. Although these agents are the most selective {alpha}1B- and {alpha}1D-adrenoceptor antagonists available, they are not fully "specific." BMY-7378 has a 267-fold selectivity for {alpha}1D-adrenoceptors over the {alpha}1A- and {alpha}1B-adrenoceptors (see Ref. 53). AH-11110A has only a 32- and 26-fold selectivity for {alpha}1B-adrenoceptors over the {alpha}1A- and {alpha}1D-adrenoceptors, respectively (see Ref. 53). KMD-3213 is 56- and 583-fold selective for {alpha}1A- over {alpha}1D- and {alpha}1B-adrenoceptors, respectively (see Ref. 53). RX-821002 and propranolol have 1,000-fold or more selectivity for {alpha}2- and {beta}-adrenoceptors, respectively, over {alpha}1-adrenoceptors. Thus, given the limitations of available antagonists, our data suggest that {alpha}1A-adrenoceptors are primarily responsible for NE activation of avian tendon fibroblasts, with a potential small contribution by {alpha}2-adrenoceptors.

In addition to stimulation of tenocytes in vitro, cells within whole tendon explants responded to NE. However, only a low percentage of the cells responded. This low responsiveness could be due to the anatomical location of the tenocytes within the FDP. Cells in closer proximity to blood vessels or nerves could have a greater response than those cells more isolated from the sources of NE. However, we found that epitenon surface cells, which are closely associated with blood vessels and nerves, did not respond differently from internal fibroblasts from the tendon midsubstance. The age of the tissue (5–6 wk old) could also account for the lower response. Cells in avian aortic smooth muscle respond to NE with the greatest Ca2+ response seen in 12- to 18-wk-old chickens and less in older (20–37 wk old) animals (42). Muscle from younger (5–8 wk old) chickens did not respond to NE with a Ca2+ transient. In addition, rat myocyte adrenoceptor coupling to L-type Ca2+ channels changes during development (37). Explant tissue may present fewer adrenoceptor-expressing cells. Alternatively, the explant model itself may decrease responsiveness to NE by reducing NE diffusion or increasing degradation.

The importance of adrenergic activation in tenocytes is unknown. Within most connective tissues, it is believed that sympathetic nerves control blood flow through the tissues, since most of the nerves are located in or around blood vessels (2, 9, 39). Adrenoceptors also may be involved in endocrine regulation of connective tissues. Both cartilage and meniscus have little vasculature and nerves but express the mRNA for {alpha}1A- and {alpha}1B-adrenoceptors (see RESULTS). Only about one-third of the meniscus is innervated and vascularized (16). Cartilage is aneural and avascular, relying on diffusion from the synovial fluid for nutrition, metabolites, and waste removal. However, the synovium is sympathetically innervated (27). NE is also secreted from cells in synovial tissue taken from arthritic knees (40). Thus NE or circulating adrenaline could diffuse from blood vessels into the synovial fluid and stimulate adrenoceptors in meniscus or cartilage cells. Finally, adrenoceptors may be involved in the response to healing after injury. In rheumatoid arthritis, tyrosine hydroxylase-positive nerve fibers were reduced, but the density of tyrosine hydroxylase-positive cells, which spontaneously secrete NE, was increased. Furthermore, NE (10–5 M) reduced the secretion of inflammatory cytokines in synoviocytes from patients with arthritis (40). Thus NE may play a role in modulating inflammation.

Sympathetic efferent nerves corelease neuropeptides and neurotransmitters. Neuropeptide Y is released with NE. In tendon, recent data have indicated that neuropeptides may be important in healing. Ackermann and colleagues (1) reported that in Achilles tendons, autonomic reinnervation was low up to 4 wk postrupture while the greatest increase occurred around 6–8 wk postrupture. Neuropeptide Y-positive nerve fibers were observed in the surrounding connective tissue and tendon proper, mainly in association with the vasculature. In addition, within the synovial fluid of arthritic joints, both sensory and autonomic neuropeptides were elevated (4, 26, 34). Neuropeptide Y also promotes wound healing and angiogensies (18). Thus autonomic neuropeptides may act locally alone or with NE to modulate the response to healing and perhaps promote the growth of new vasculature within the healing tendon.

During flexor tendon healing, the epitenon surface cells and even synovial sheath cells, where present, initiate the repair process (21, 24, 28, 36). In addition, blood vessels, and possibly nerves, may aid in healing (36). In avian tendons subjected to trauma, collagen synthesis was decreased in partially devascularized tendons compared with those tendons allowed to heal with the blood supply intact (6). It has also been shown that vascular adventitial fibroblast proliferation was induced by prolonged stimulation of {alpha}1-adrenoceptors by NE in the intact vascular wall, and this growth was further augmented after injury (52). In addition, NE and growth factors synergized to promote migration of adventitial fibroblasts (53). Thus, in a similar fashion to adventitial fibroblasts, the migration of tendon cells into a wound and the growth of new tissue during repair could be augmented by adrenergic stimulation. Indeed, stimulation of the mitotic pathway in most cells by growth factors and certain G protein-coupled agonists involves a rise in intracellular Ca2+ early in the signaling pathway. Therefore, activation of adrenoceptors, mainly {alpha}1, with a concomitant rise in [Ca2+]ic may be involved in a maintenance function as well as a healing response in tendon.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by National Institutes of Health Grants AR-38121 (to A. J. Banes) and AR-62584 (to J. E. Faber), the Hunt Foundation (A. J. Banes), and Flexcell International Corporation.


    ACKNOWLEDGMENTS
 
We thank Dr. Donald Kirkendall for assistance with the statistics.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Banes, Dept. of Orthopaedics, Univ. of North Carolina at Chapel Hill, 2340C Biomolecular Research Bldg. CB no. 7055, Chapel Hill, NC 27599-7055 (E-mail: Albert_Banes{at}med.unc.edu)

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. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Ackermann PW, Ahmed M, and Kreicbergs A. Early nerve regeneration after Achilles tendon rupture—a prerequisite for healing? A study in the rat. J Orthop Res 20: 849–856, 2002.[CrossRef][ISI][Medline]

2. Ackermann PW, Li J, Finn A, Ahmed M, and Kreicbergs A. Autonomic innervation of tendons, ligaments, and joint capsules. A morphologic and quantitative study in the rat. J Orthop Res 19: 372–378, 2001.[CrossRef][ISI][Medline]

3. Anesini C and Borda E. Modulatory effect of the adrenergic system upon fibroblast proliferation: participation of {beta}3-adrenoceptors. Auton Autacoid Pharmacol 22: 177–186, 2002.[CrossRef][Medline]

4. Appelgren A, Appelgren B, Eriksson S, Kopp S, Lundeberg T, Nylander M, and Theodorsson E. Neuropeptides in temporomandibular joints with rheumatoid arthritis: a clinical study. Scand J Dent Res 99: 519–521, 1991.[ISI][Medline]

5. Banes AJ, Donlon K, Link GW, Gillespie Y, Bevin AG, Peterson HD, Bynum D, Watts S, and Dahners L. Cell populations of tendon: a simplified method for isolation of synovial cells and internal fibroblasts: conformation of origin and biologic properties. J Orthop Res 6: 83–94, 1988.[ISI][Medline]

6. Banes AJ, Enterline D, Bevin AG, and Salisbury RE. Effects of trauma and partial devascularization on protein synthesis in the avian flexor profundus tendon. J Trauma 21: 505–512, 1981.[ISI][Medline]

7. Banes AJ, Horesovsky G, Larson C, Tsuzaki M, Judex S, Archambault J, Zernicke R, Herzog W, Kelley S, and Miller L. Mechanical load stimulates expression of novel genes in vivo and in vitro in avian flexor tendon cells. Osteoarthritis Cartilage 7: 141–153, 1999.[CrossRef][ISI][Medline]

8. Bevan JD, Bevan RD, and Duckles SP. Adrenergic regulation of vascular smooth muscle. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: Am. Physiol. Soc., 1980, vol. II, chapt.18, p. 515–566.

9. Bjurholm A, Kreicbergs A, Ahmed M, and Schultzberg M. Noradrenergic and peptidergic nerves in the synovial membrane of the Sprague-Dawley rat. Arthritis Rheum 33: 859–865, 1990.[ISI][Medline]

10. Bjurholm A, Kreicbergs A, Schultzberg M, and Lerner UH. Parathyroid hormone and noradrenaline-induced enhancement of cyclic AMP in a cloned osteogenic sarcoma cell line (UMR 106) is inhibited by neuropeptide Y. Acta Physiol Scand 134: 451–452, 1988.[ISI][Medline]

11. Boittin FX, Macrez N, Halet G, and Mironneau J. Norepinephrine-induced Ca2+ waves depend on InsP3 and ryanodine receptor activation in vascular myocytes. Am J Physiol Cell Physiol 277: C139–C151, 1999.[Abstract/Free Full Text]

12. Bylund DB, Eikenberg DC, Hieble JP, Langer SZ, Lefkowitz RJ, Minneman KP, Molinoff PB, Ruffolo RR, and Trendelenburg U. International union of pharmacology nomenclature of adrenoceptors. Pharmacol Rev 46: 121–136, 1994.[ISI][Medline]

13. Candipan RC, Hsium PT, Richard P, and Cooke JP. Vascular injury augments adrenergic neurotransmission. Circulation 89: 777–784, 1994.[Abstract]

14. Chen L, Xin X, Eckhart AD, Yang N, and Faber JE. Regulation of vascular smooth muscle growth by {alpha}1-adrenoreceptor subtypes in vitro and in situ. J Biol Chem 270: 30980–30988, 1995.[Abstract/Free Full Text]

15. Civantos Calzada B and Aleixandre de Artinano A. Alpha-adrenoceptor subtypes. Pharmacol Res 44: 195–208, 2001.[CrossRef][ISI][Medline]

16. Day B, Mackenzie WG, Shim SS, and Leung G. The vascular and nerve supply of the human mensicus. Arthroscopy 1: 58–62, 1985.[Medline]

17. Docherty JR. Subtypes of functional {alpha}1- and {alpha}2-adrenoceptors. Eur J Pharmacol 361: 1–15, 1998.[CrossRef][ISI][Medline]

18. Ekstrand AJ, Cao R, Bjorndahl M, Nystrom S, Jonsson-Rylander AC, Hassani H, Hallberg B, Nordlander M, and Cao Y. Deletion of neuropeptide Y (NPY) 2 receptor in mice results in blockage of NPY-induced angiogenesis and delayed wound healing. Proc Natl Acad Sci USA 100: 6033–6038, 2003.[Abstract/Free Full Text]

19. Elfvin LG, Holmberg K, Johansson J, and Aldskogius H. The innervation of the synovium of the knee joint in the guinea pig: an immunohistochemical and ultrastructural study. Anat Embryol (Berl) 197: 293–303, 1998.[CrossRef][ISI][Medline]

20. Faber JE, Yang N, and Xin X. Expression of {alpha}-adrenoceptor subtypes by smooth muscle cells and adventitial fibroblasts in rat aorta and in cell culture. J Pharmacol Exp Ther 298: 441–452, 2001.[Abstract/Free Full Text]

21. Gelberman RH, Manske PR, Vande Berg JS, Lesker PA, and Akeson WH. Flexor tendon repair in vitro: a comparative histologic study of the rabbit, chicken, dog, and monkey. J Orthop Res 2: 39–48, 1984.[Medline]

22. Graham RM, Perez DM, Hwa J, and Piascik MT. {alpha}1-Adrenergic receptor subtypes. Molecular structure, function, and signaling. Circ Res 78: 737–749, 1996.[Free Full Text]

23. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract]

24. Harrison RK, Mudera V, Grobbelaar AO, Jones ME, and McGrouther DA. Synovial sheath cell migratory response to flexor tendon injury: an experimental study in rats. J Hand Surg 28A: 987–993, 2003.[ISI]

25. Hoizey G, Lukas-Croisier C, Frances C, Grulet H, Delemer B, Millart H, Devillier P, and Caron J. Study of diurnal fluctuations of plasma methoxyamines in healthy volunteers. Clin Endocrinol (Oxf) 56: 119–122, 2002.[CrossRef][ISI][Medline]

26. Holmlund A, Ekblom A, Hansson P, Lind J, Lundeberg T, and Theodorsson E. Concentrations of neuropeptides substance P, neurokinin A, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide in synovial fluid of the human temporomandibular joint. A correlation with symptoms, signs and arthroscopic findings. Int J Oral Maxillofac Surg 20: 228–231, 1991.[ISI][Medline]

27. Iwasaki A, Inoue K, and Hukuda S. Distribution of neuropeptide-containing nerve fibers in the synovium and adjacent bone of the rat knee joint. Clin Exp Rheumatol 13: 173–178, 1995.[ISI][Medline]

28. Jones ME, Mudera V, Brown RA, Cambrey AD, Grobbelaar AO, and McGrouther DA. The early surface cell response to flexor tendon injury. J Hand Surg 28A: 221–230, 2003.[ISI]

29. Kawanabe Y, Hashimoto N, Masaki T, and Miwa S. Ca2+ influx through nonselective cation channels plays an essential role in noradrenaline-induced arachidonic acid release in Chinese hamster ovary cells expressing {alpha}1A, {alpha}1B, or {alpha}1D-adrenergic receptors. J Pharmacol Exp Ther 299: 901–907, 2001.[Abstract/Free Full Text]

30. Kjaer M, Farrell PA, Christensen NJ, and Galbo H. Increased epinephrine response and inaccurate glucoregulation in exercising athletes. J Appl Physiol 61: 1693–1700, 1986.[Abstract/Free Full Text]

31. Kirischuk S, Tuschick S, Verkhratsky A, and Kettenmann H. Calcium signalling in mouse Bergmann glial cells mediated by {alpha}1-adrenoreceptors and H1 histamine receptors. Eur J Neurosci 8: 1198–1208, 1996.[ISI][Medline]

32. Koshimizu TA, Yamauchi J, Hirasawa A, Tanoue A, and Tsujimoto G. Recent progress in {alpha}1-adrenoceptor pharmacology. Biol Pharm Bull 25: 401–408, 2001.[CrossRef][ISI]

33. Kumagai H, Sakamoto H, Guggino S, Filburn CR, and Sacktor B. Neurotransmitter regulation of cytosolic calcium in osteoblast-like bone cells. Calcif Tissue Int 45: 251–254, 1989.[ISI][Medline]

34. Larsson J, Ekblom A, Henriksson K, Lundeberg T, and Theodorsson E. Concentration of substance P, neurokinin A, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide in synovial fluid from knee joints in patients suffering from rheumatoid arthritis. Scand J Rheumatol 20: 326–335, 1991.[ISI][Medline]

35. Leicht M, Greipel N, and Zimmer HG. Comitogenic effect of catecholamines on rat cardiac fibroblasts in culture. Cardiovasc Res 48: 274–284, 2000.[CrossRef][ISI][Medline]

36. Lindsay WK and Thomson HG. Digital flexor tendons: an experimental study. Part 1. The significance of each component of the flexor mechanism in tendon healing. Br J Plast Surg 12: 289–316, 1960.[Medline]

37. Liu QY, Karpinski E, and Pang PK. Changes in {alpha}1-adrenoceptor coupling to Ca2+ channels during development in rat heart. FEBS Lett 338: 234–238, 1994.[CrossRef][ISI][Medline]

38. Macrez-Lepretre N, Kalkbrenner F, Schultz G, and Mironneau J. Distinct functions of Gq and G11 proteins in coupling {alpha}1-adrenoreceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes. J Biol Chem 272: 5261–5268, 1997.[Abstract/Free Full Text]

39. McDougall JJ, Ferrell WR, and Bray RC. Spatial variation in sympathetic influences on the vasculature of the synovium and medial collateral ligament of the rabbit knee joint. J Physiol 503: 435–443, 1997.[Abstract]

40. Miller LE, Grifka J, Scholmerich J, and Straub RH. Norepinephrine from synovial tyrosine hydroxylase positive cells is a strong indicator of synovial inflammation in rheumatoid arthritis. J Rheumatol 29: 427–435, 2002. [Corrigenda. J Rheumatol 30: May, 2003, p. 1125.][ISI][Medline]

41. Muyderman H, Angehagen M, Sandberg M, Bjorklund U, Olsson T, Hansson E, and Nilsson M. {alpha}1-Adrenergic modulation of metabotropic glutamate receptor-induced calcium oscillations and glutamate release in astrocytes. J Biol Chem 276: 46504–46514, 2001.[Abstract/Free Full Text]

42. Qin ZL and Nishimura H. Ca2+ signaling in fowl aortic smooth muscle increases during maturation but is impaired in neointimal plaques. J Exp Biol 201: 1695–1705, 1998.[Abstract/Free Full Text]

43. Ruffolo RR and Hieble JP. {alpha}-Adrenoceptors. Pharmacol Ther 61: 1–64, 1994.[CrossRef][ISI][Medline]

44. Salomonsson M and Arendshorst WJ. Norepinephrine-induced calcium signaling pathways in afferent arterioles of genetically hypertensive rats. Am J Physiol Renal Physiol 281: F264–F272, 2001.[Abstract/Free Full Text]

45. Schomig A and Richardt G. The role of catecholamines in ischemia. J Cardiovasc Pharmacol 16, Suppl 5: S105–S112, 1990.

46. Schwab W, Bilgicyildirim A, and Funk RH. Microtopography of the autonomic nerves in the rat knee: a fluorescence microscopic study. Anat Rec 247: 109–118, 1997.[CrossRef][ISI][Medline]

47. Sherman BE and Chole RA. Effects of catecholamines on clavarial bone resorption in vitro. Ann Otol Rhinol Laryngol 110: 682–689, 2001.[ISI][Medline]

48. Takemiya T and Maeda J. The functional characteristics of tendon blood circulation in the rabbit hindlimbs. Jpn J Physiol 38: 361–374, 1988.[ISI][Medline]

49. Tanoue A, Koshimizu TA, Shibata K, Nasa Y, Takeo S, and Tsujimoto G. Insights into {alpha}1-adrenoceptor function in health and disease from transgenic animal studies. Trends Endocrinol Metab 14: 107–113, 2001.

50. Tordjmann T, Berthon B, Claret M, and Combettes L. Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist. EMBO J 16: 5398–5407, 1997.[Abstract/Free Full Text]

51. Varma DR and Deng XF. Cardiovascular {alpha}1-adrenoceptor subtypes: functions and signaling. Can J Physiol Pharmacol 78: 267–292, 2000.[CrossRef][ISI][Medline]

52. Zhang H and Faber JE. Trophic effect of norepinephrine on arterial intima-media and adventitia is augmented by injury and mediated by different {alpha}1-adrenoceptor subtypes. Circ Res 89: 815–822, 2001.[Abstract/Free Full Text]

53. Zhang H, Facemire CS, Banes AJ, and Faber JE. Different {alpha}-adrenoceptors mediate migration of vascular smooth muscle cells and adventitial fibroblasts in vitro. Am J Physiol Heart Circ Physiol 282: H2364–H2370, 2002.[Abstract/Free Full Text]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
287/4/C912    most recent
00099.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Wall, M. E.
Articles by Banes, A. J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Wall, M. E.
Articles by Banes, A. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.