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Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel
Pain 110 (2004) 616–627 www.elsevier.com/locate/pain
Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel Susan M. Carlton*, Shengtai Zhou, Junhui Du, Gregory L. Hargett, Guangchen Ji, Richard E. Coggeshall Department of Anatomy and Neurosciences, Marine Biomedical Institute, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069, USA Received 23 February 2004; received in revised form 14 April 2004; accepted 26 April 2004
Abstract Activation of peripheral somatostatin receptors (SSTRs) inhibits sensitization of nociceptors, thus having a short term or phasic effect [Pain 90 (2001) 233] as well as maintaining a tonic inhibitory control over nociceptors [J Neurosci 21 (2001) 4042]. The present study provides several lines of evidence that an important mechanism underlying SSTR modulation of nociceptors is regulation of the transient receptor potential vanilloid 1 ion channel (TRPV1, formerly the VR1 receptor). Double labeling of L5 dorsal root ganglion cells demonstrates that , 60% of SSTR2a-labeled cells are positive for TRPV1. Conversely, , 33% of TRPV1-labeled cells are positive for SSTR2a. In vivo behavioral studies demonstrate that intraplantar injection of 20.0 but not 2.0 mM octreotide (OCT, SSTR agonist) significantly reduces capsaicin (CAP, a ligand for TRPV1) -induced flinching and lifting/licking behaviors. This occurs through local activation of SSTRs in the injected hindpaw and is reversed following co-application of the SSTR antagonist cyclo-somatostatin (c-SOM). In vitro studies using a skin-nerve preparation demonstrate that activation of peripheral SSTRs on nociceptors with 20.0 mM OCT significantly reduces CAP-induced activity and can prevent CAP-induced desensitization. Furthermore, blockade of peripheral SSTRs with c-SOM dramatically enhances CAP-induced behaviors and nociceptor activity, demonstrating SSTR-induced tonic inhibitory modulation of TRPV1. Finally, TRPV1 does not appear to be under tonic opioid receptor control since the opioid antagonist naloxone does not change CAP-induced excitation and does not effect OCT-induced inhibition of CAP responses. These data strongly suggest that SSTRs modulate nociceptors through phasic and tonic regulation of peripheral TRPV1 receptors. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Somatostatin; Primary afferent; Hyperalgesia; Nociception; Capsaicin; VR1
1. Introduction Somatostatin is a biologically active compound with diverse effects. In regards to pain, it is a peripheral analgesic agent having a short term (phasic) effect (Carlton et al., 2001a; Heppelmann and Pawlak, 1997) and also maintains tonic control of peripheral nociceptors (Carlton et al., 2001b, 2003; Heppelmann and Pawlak, 1999). These effects are manifest by decreases in formalin-induced pain behaviors following application of a somatostatin receptor (SSTR) agonist, and increases following application of an SSTR antagonist. To elucidate mechanisms by which peripheral SSTRs maintain inhibitory control of nociceptors, we focus * Corresponding author. Tel.: þ1-409-772-2124; fax: þ 1-409-762-9382. E-mail address: [email protected] (S.M. Carlton).
on their interactions with the transient receptor potential vanilloid 1 (TRPV1) ion channel. The TRPV1 was originally cloned as a capsaicin (CAP) receptor, but also responds to heat, protons and inflammatory mediators (Caterina and Julius, 2001; Caterina et al., 1997; Chuang et al., 2004), and thus is capable of integrating nociceptive thermal and chemical stimuli (Tominaga et al., 1998). The TRPV1 plays an important role in normal or physiologic nociception (Caterina et al., 1997) and also in inflammatory (Caterina et al., 2000) and neuropathic (Hudson et al., 2001) pain. We hypothesize that SSTRs both phasically and tonically inhibit TRPV1 activation. Thus, the present study investigates whether: (1) SSTR and TRPV1 receptors are co-localized in primary sensory neurons, (2) activation of SSTRs by SSTR agonists reduces the pain behaviors and nociceptor activity induced by CAP administration (phasic control),
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.04.042
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and (3) blockade of SSTR function enhances CAP-induced pain behaviors and nociceptor activity (tonic control) and (4) opioid receptors are involved in these actions.
2. Materials and methods All procedures were approved by the University Animal Care and Use Committee and followed guidelines for the ethical treatment of animals (Zimmermann, 1983). 2.1. Anatomical studies The lumbar (L) 5 dorsal root ganglia (DRG) from male Sprague – Dawley (SD) rats (Harlan, Indianapolis, IN, n ¼ 3) were used to determine total neuron counts and the percentages of neurons that were (1) single-labeled for either SSTR2a or TRPV1 or (2) double-labeled for both receptors. Rats were deeply anaesthetized with sodium pentobarbital (70 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde and 0.1% picric acid in 0.1 M phosphate buffer (PB, pH 7.4) at 4 8C. Left and right L5 DRGs were removed and placed in 30% sucrose-PB for cryoprotection overnight at 4 8C. The ganglia were fast frozen in liquid N2 in a cryo-embedding compound and sectioned on a cryostat (Microm International, GmbH). Serial sections (8 mm) were cut through the short axis of the DRG and placed on chrome alum dipped 3-well, teflon printed slides (Electron Microscopy Sciences, Fort Washington, PA). Systematic random sampling ensured that all neurons had an equal chance of being analyzed (Coggeshall and Lekan, 1996). Briefly, after dissection, each DRG was measured along the long axis under a dissecting microscope and the length was divided by the thickness of the cryostat sections (8 mm) to determine the approximate number of sections in that DRG. Section separation (k in stereological literature) was determined by dividing the number of sections by the number of pairs of serial sections (6 – 7 pairs per ganglia in this study) chosen for analysis. Starting randomly between the first and the kth section (the Rth section in stereological literature), subsequent pairs were taken for analysis at R þ k; R þ 2k; etc. until the tissue in a DRG was exhausted. 2.1.1. Staining procedures Single- and double-labeling for SSTR2a and TRPV1 were performed using immunohistochemistry and fluorescence tags. For single-labeling, sections were incubated in guinea pig anti-SSTR2a (1:40,000, Gramsch Labs, GmBH) for 48 h at room temperature, rinsed in 0.1 M phosphate buffered saline (PBS, pH 7.4), placed in biotinylated antiguinea pig IgG (1:200, Vector Laboratories, Burlingame, CA) for 1 h, followed by incubation in Vectastain Elite ABC peroxidase reagent (avidin –biotin complex, Vector Laboratories) for 1 h. After rinsing in PBS, sections were incubated in fluorescein-labeled tyramide (1:75, TSA kit,
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Perkin – Elmer Life Science, Boston, MA) for 7 min followed by a wash in PBS. Following incubation for 20 min in 0.3% H2O2 in PBS (inactivates peroxidase introduced during the ABC reaction), sections were incubated in goat anti-VR1 (anti-TRPV1) (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA) for 48 h at room temperature. Following rinsing, sections were placed in Cyanin 3- (Cy3-) conjugated anti-goat IgG (1:400, Jackson ImmunoResearch, West Grove, PA) for 1 h. After final rinsing, slides were cover slipped with Vectashield mounting media (Vector Laboratories, Burlingame, CA). Controls for specificity included staining DRG sections as described above but omitting the primary antisera. Omission of the primary antisera resulted in no staining in the tissue. Absorption controls were performed for each antiserum where the appropriate peptide was incubated with the working dilution of antiserum (100 mg peptide/1 ml antisera) overnight at room temperature. Immunostaining with these solutions resulted in a complete lack of staining in the tissue. The TRPV1 antiserum was also incubated over night with the vanilloid receptor like-1 (VRL-1, TRPV2) peptide (Caterina et al., 1999) (ADI, San Antonio, TX), and this resulted in a staining pattern similar to that observed following staining with TRPV1 antiserum alone, indicating that this antiserum did not recognize or cross-react with VRL-1. In immunostained DRG sections, a neuron was determined to be labeled for either SSTR2a or TRPV1 by inspection, comparing the amount of fluorescence in the neuron to that in control tissues and surrounding large diameter, unlabeled neurons. Total neuron counts in the L5 DRG were obtained using pairs of sections that were stained with cresyl violet. The diameters of fluorescently labeled and cresyl violet stained DRG neurons were estimated by summing the length and width of those neurons with a visible nucleus and dividing by two. Whenever possible, at least 100 neurons were measured in each DRG. The data are presented as the percentage of the total and labeled population. 2.1.2. Stereological analysis Following staining, color digitized images of each section (24 bits per pixel), were captured using an Olympus BX51 microscope with an attached Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI). Fluorescein isothiocynate (FITC) and Cy3 filter cube sets were used to visualize the fluorescein and Cy3, respectively. Camera exposures were adjusted automatically by Spot software (version 3.1) and brightness and gamma corrections were made manually to achieve the best signal-to-noise ratios. The physical disector (pairs of sections) was used to achieve unbiased estimates of total, SSTR2a-, and TRPV1-labeled DRG neurons (Coggeshall, 1992). The size of most DRG sections and the magnification needed to identify labeled cells necessitated analysis of a randomly chosen subregion or fraction ðf Þ in each section in a pair. Once identified,
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images in the subregion in each pair were outlined from the computer screen onto clear mylar sheets including all labeled cell profiles and an appropriate number of fiduciary landmarks. The mylar sheets from a pair were matched with one another using the fiduciary landmarks, and all labeled profiles that appeared in both sections were disallowed. Labeled profiles remaining were ‘tops’ or ‘counts’ (identified as Q- in the stereological literature), and each top was then checked for double-labeling. Using ‘fractionator sampling’ (Gundersen et al., 1988; West and Gundersen, 1990) total numbers of cresyl violet stained neurons and numbers of SSTR2a- and TRPV1-labeled neurons were estimated in each DRG by multiplying counts Q- by the reciprocal of the fraction ð1=f Þ of each sampled pair. These neuronal counts ðNÞ for all pairs analyzed in a DRG were summed ðSNÞ: Total number of neurons in the entire DRG was then estimated by ðSN £ kÞ=2 (divided by two because each section in a pair was used as both reference and look-up sections) (Coggeshall, 1992). Percentages of single-labeled neurons were calculated by dividing the numbers of singled-labeled neurons by the total DRG cell number £ 100. Percentages of doublelabeled neurons were calculated by dividing numbers of double-labeled neurons by numbers of single-labeled SSTR2a or TRPV1 neurons £ 100.
the center of the hindpaw where the drugs were delivered. In some groups, PBS was injected before or after the drug of interest to keep volumes constant between groups. Each animal was used only once and the experimenter was unaware of which drug was being injected into an animal. 2.2.3.1. SSTR-mediated phasic control of CAP-induced nociception. To demonstrate that activation of SSTRs modulated TRPV1 activity, rats received intraplantar injections of the SSTR agonist octreotide (OCT, Sandostatin, 30 ml), 2 mM ðn ¼ 6Þ or 20 mM ðn ¼ 8Þ; followed 10 min later by a 20 ml cocktail injection of the same dose of OCT þ 0.05% CAP into the same place and spontaneous pain behaviors were measured. A control group ðn ¼ 8Þ received intraplantar injections of phosphate buffered saline (PBS, 30 ml), followed 10 min later by a 20 ml injection of 0.05% CAP alone into the same place. To demonstrate that OCT effects were receptor specific, a group of rats ðn ¼ 8Þ received a 30 ml injection of 20 mM OCT þ the SSTR antagonist cyclo-somatostatin (c-SOM, 1.3 mM) followed 10 min later by a 20 ml injection of 0.05% CAP. To demonstrate that OCT produced inhibition of CAP-induced behaviors through a local effect in the hindpaw, another group of rats ðn ¼ 9Þ received an injection of OCT (30 ml, 20 mM) into one hindpaw followed 10 min later by a 20 ml injection of 0.05% CAP in the contralateral hindpaw.
2.2. Behavioral studies 2.2.1. Habituation Male SD rats (250 –300 g) were housed in groups of three in plastic cages with soft bedding under a reversed light/dark cycle. Following arrival at the University animal care facility, the animals were acclimated for at least 3 days before behavioral testing was initiated. Rats were habituated to behavioral testing by placing them on a wire screen platform in Plexiglas cages (8 £ 8 £ 18 cm3) for 1 h. Each rat was habituated twice before being placed in an experimental group. 2.2.2. Preparation of CAP To prepare CAP, 2 ml 100% ethanol were added to a bottle containing 1 g CAP (Fluka, Ronkonkoma, NY) and vigorously shaken. This mixture was added to a mixture of 9.3 ml saline and 0.7 ml Tween 80. This solution was stirred while heating until the alcohol evaporated and the total volume returned to 10 ml. The stock solution was diluted with vehicle to obtain a 0.05% CAP solution that was stored at 4 8C. Control experiments demonstrated that intraplantar injection of CAP vehicle alone did not produce any nociceptive behaviors. All other drugs were dissolved in PBS (pH 7.4). 2.2.3. Drug injections For intraplantar injections, a 28-gauge needle was attached to a 50 ml Hamilton syringe with PE20 tubing. For paw injections, the needle punctured the plantar skin distal to the foot pads and was guided into the subcutaneous space to
2.2.3.2. SSTR-mediated tonic control of CAP-induced nociception. To demonstrate that blockade of SSTRs released TRPV1 from tonic inhibition, rats (n ¼ 8 per group) received an intraplantar injection of the SSTR antagonist c-SOM (30 ml, 1.3 mM), followed 10 min later by a 20 ml injection of either PBS or 0.05% CAP into the same place and nociceptive pain behaviors were measured. To demonstrate that c-SOM produced its effects through blockade of local SSTRs in the hindpaw, a group of rats ðn ¼ 5Þ received an intraplantar injection of c-SOM (1.3 mM, 30 ml) into one paw followed 10 min later by a 20 ml injection of 0.05% CAP into the other paw. 2.2.4. Behavioral testing Spontaneous nociceptive behaviors resulting from CAP or c-SOM injections were assessed by counting the number of flinches and seconds an animal spent lifting and/or licking (L/L) the injected paw in 5 min intervals. A flinch was defined as a spontaneous, rapid jerk of the foot whether the foot was on the screen or held in the air. 2.3. Electrophysiological studies 2.3.1. In vitro skin-nerve recordings Single unit recordings from C-mechanoheat (CMH) sensitive fibers were obtained in glabrous skin using a modified in vitro skin-nerve preparation (Du et al., 2001; Kress et al., 1992; Reeh, 1986). Male SD rats (200 –300 g) were sacrificed with an overdose of CO2, and the glabrous
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skin of the hindpaw was dissected with the medial and lateral plantar nerves attached. The preparation was placed corium side up in an organ bath and superfused (15 ml/min, 34 8C) with an oxygen-saturated, modified synthetic interstitial fluid solution (SIF, in mM: NaCl, 123; KCl, 3.5; MgSO4, 0.7; CaCl2, 2.0; Na gluconate, 9.5; NaH2PO4, 1.7; Glucose, 5.5; Sucrose, 7.5; and HEPES, 10; pH 7.40 ^ 0.05). The plantar nerves were placed in a separate chamber containing a top layer of mineral oil and a bottom layer of SIF. The nerves were desheathed and teased apart on a mirror stage under a dissecting microscope. Small nerve bundles were repeatedly split with sharpened forceps until single unit activity was obtained. 2.3.2. Thermal stimulation A feedback-controlled lamp placed beneath the organ bath supplied radiant heat to the receptive field of each isolated unit. The beam was focused through the translucent bottom of the bath onto the epidermal surface of the skin. A thermocouple was placed in the corium above the light beam to measure intracutaneous temperature. A standard heat ramp was applied to each unit, starting from an adapting temperature of 34 8C and rising to 47 8C in 10 s (47 8C on the corium side was equivalent to 51 8C on the epidermal side). The temperature at which the second spike was elicited by the heat stimulus was defined as heat threshold. Choosing units that responded to this heat stimulus enhanced the probability that these units would also be capsaicin-sensitive. 2.3.3. Mechanical stimulation The mechanical threshold for each unit was determined using calibrated von Frey filaments (Stoelting, Inc., Wood Dale, IL). The filaments were applied to the receptive field on the corium side of the skin in a uniform fashion, starting with filaments delivering a relatively small amount of force and ascending (0.1 –166.7 mN) until an action potential could be consistently evoked. 2.3.4. Chemical stimulation To investigate responses of units to various drugs, a small plastic ring (5 mm diameter) was placed over the receptive field of each unit and the SIF in the ring was replaced with SIF containing 0.05% CAP alone, CAP þ the SSTR agonist OCT (20 mM), CAP þ the SSTR antagonist c-SOM (1.3 mM), CAP þ OCT þ the opioid antagonist naloxone (NAL, 2 mM) or CAP þ NAL. The concentrations of OCT, c-SOM and NAL were based on those used in a previous study (Carlton et al., 2001a). All drugs dissolved in SIF were buffered to pH 7.40 ^ 0.05.
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investigate the effect of the SSTR agonist OCT on CAPinduced activity, in a separate group of CMH fibers ðn ¼ 8Þ; 0.05% CAP was applied to the receptive field in the presence of 20 mM OCT for 2 min. To determine if OCT blocked CAP-induced desensitization, a phenomenon that often occurs following TRPV1 activation, units ðn ¼ 8Þ were exposed to sequential 0.05% CAP applications (CAP 1 and CAP 2) for 2 min each, with a 5 min intertrial interval; in a separate group of units ðn ¼ 6Þ; 20 mM OCT was applied with 0.05% CAP (CAP 1 þ OCT), followed 5 min later by 0.05% CAP alone (CAP 2). 2.3.4.2. SSTR-mediated tonic control of CAP-induced activity. To determine whether SSTRs maintained a tonic inhibitory control over TRPV1 receptors, 0.05% CAP ðn ¼ 8Þ; c-SOM ðn ¼ 18Þ or CAP þ c-SOM ðn ¼ 8Þ was applied for 2 min to the receptive fields in 3 different groups of nociceptors. 2.3.4.3. Opioid involvement. To rule out a tonic opioid control of TRPV1, CMH units ðn ¼ 6Þ were exposed to 0.05% CAP þ 2 mM naloxone (NAL, an opioid antagonist) or 0.05% CAP alone ðn ¼ 8Þ and activity monitored for 2 min. To rule out involvement of opioid receptors in inducing OCT effects, CMH units were monitored following exposure to 0.05% CAP þ 20 mM OCT þ 2 mM NAL ðn ¼ 8Þ or 0.05% CAP þ 20 mM OCT ðn ¼ 6Þ for 2 min. 2.3.5. Neurophysiological recordings Neural activity was recorded using a DAM80 Differential Amplifier (World Precision Instruments) attached to a monopolar gold wire electrode, with the reference electrode positioned nearby. Units were identified by manual probing of the glabrous skin with a blunt glass rod and if they had a clearly defined receptive field, they were studied in detail. Action potentials were analyzed on a Dell computer with a custom-made template-matching program that allowed discrimination of single unit activity based on the amplitude and wave form of each action potential (Forster and Handwerker, 1990). The conduction velocity of each unit was determined by monopolar electrical stimulation (0.1 ms duration, train frequency 1 Hz) at the most mechanosensitive site in the receptive field of each unit using a tefloncoated steel electrode (5 MV impedance, 250 mm shaft diameter). Conduction velocity was determined from the latency of the action potential and the distance from the stimulation electrode to the recording site (measured in millimeters). Units with a conduction velocity of less than 1.6 m/s were classified as C fibers. 2.4. Statistical analyses
2.3.4.1. SSTR-mediated phasic control of CAP-induced activity. To investigate responses of CMH fibers to CAP alone, background activity of units ðn ¼ 8Þ was recorded for 2 min, then 0.05% CAP was applied to the receptive field of the unit and recordings were made for another 2 min. To
All data were expressed as means ^ standard errors (SE) and evaluated using SigmaStat (Jandel Corporation). In the behavioral studies, the total sum of flinches or time spent L/L during 60 min were calculated and plotted as bar graphs.
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Differences between treatment groups were evaluated using a one-way analysis of variance (ANOVA) followed by the Tukey’s test. Time courses for nociceptive behaviors were constructed by plotting the mean number of flinches or time spent L/L in 5 min intervals as a function of time. For these outcomes the data were analyzed using a two-factor repeated measures ANOVA followed by a pairwise comparison using a Tukey’s test. In the electrophysiological studies, differences in discharge rates were evaluated with either a Friedman’s ANOVA, Kruskal – Wallis test followed by Dunn’s post hoc analysis, Wilcoxson signed rank test or two-way ANOVA followed by a Tukey’s test, where appropriate, P , 0:05 was considered significant.
3. Results 3.1. Anatomical studies Analysis of the L5 DRG demonstrated that many small to medium diameter cells were immunohistochemically stained for SSTR2a and TRPV1 (Fig. 1). The nucleus was often visible as a ‘dark hole’ in both labeled and unlabeled cells (Fig. 2) and labeled cells demonstrated diffuse cytoplasmic staining. The mean diameter of SSTR2alabeled cells was 26.3 ^ 3.8 mm (range 16– 40 mm); for TPRV1-labeled cells mean diameter was 26.1 ^ 4.0 mm (range 13– 39 mm) and for double-labeled cells the mean diameter was 26.3 ^ 3.8 mm (range 16 – 40 mm). Stereological counts indicated that 17.8 ^ 5.4% (mean ^ SE) of cells were single-labeled for SSTR2a and 31.9 ^ 7.9% were single-labeled for TRPV1 (Table 1). In the double-labeling studies, 60.0 ^ 5.6% of SSTR2a cells were also labeled for TRPV1; and 33.0 ^ 4.2% TRPV1-labeled cells were also labeled for SSTR2a. For the total population of L5 DRG cells (13,629 ^ 3,250), 11.0 ^ 5.2% were double-labeled.
Fig. 1. Histograms demonstrating the diameter frequencies for total neurons (A), single-labeled SSTR2a (B), single-labeled TRPV1 (C) and doublelabeled SSTR2a-TRPV1 (D) L5 DRG ðn ¼ 3Þ cells. Note that labeled populations are primarily small and medium diameter cells.
systemically but through activation of SSTRs localized in the injected hindpaw (Fig. 3E and F).
3.2. Behavioral studies 3.2.1. SSTR-mediated phasic control of CAP-induced nociception Intraplantar injection of 0.05% CAP induced both flinching and L/L behaviors. Pretreatment with 20 mM but not 2 mM OCT significantly reduced both of these CAPinduced behaviors (Fig. 3A and B, P , 0:05; one-way ANOVA). The time courses of these behaviors are shown in Fig. 3C and D and demonstrate that both the magnitude and the duration of the CAP-induced behaviors were reduced by OCT (P , 0:05; two-way repeated measures ANOVA). Injection of c-SOM with the OCT þ CAP blocked the inhibitory effects of OCT such that the flinching and L/L behaviors in these animals were no different from animals injected with CAP alone (Fig. 3E and F, P , 0:05; one-way ANOVA). Injection of 20 mM OCT into one hind paw and CAP into the contralateral paw did not reduce CAP-induced behaviors indicating that OCT was not producing its effect
3.2.2. SSTR-mediated tonic control of CAP-induced nociception Intraplantar injection of the SSTR antagonist c-SOM produced a little flinching but no L/L compared to that induced by CAP (Fig. 4A and B, P , 0:05; Kruskal – Wallis test). However, when c-SOM was co-injected with CAP, both flinching and L/L behaviors were significantly enhanced compared to either c-SOM or CAP alone, and the combined effect was more than additive (Fig. 4A and B, P , 0:05; Kruskal – Wallis test). The time courses of these behaviors are shown in Fig. 4C and D and demonstrate that co-application of c-SOM þ CAP resulted in a dramatic enhancement of both the magnitude and duration of the CAP-induced pain behaviors (P , 0:05; two-way repeated measures ANOVA). Injection of CAP into one hindpaw and c-SOM into the contralateral paw, did not significantly change the CAP-induced responses indicating that the c-SOM effect was produced through action on local SSTRs
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in the injected paw and not through a systemic action (Fig. 4E and F, P , 0:05; Kruskal– Wallis test). 3.3. Electrophysiological studies 3.3.1. SSTR-mediated phasic control of CAP-induced activity Application of 0.05% CAP to the receptive fields of CMH fibers for 2 min in the skin-nerve preparation resulted in an increased firing rate, with a mean frequency of 0.15 ^ 0.05 imp/s compared to background 0.02 ^ 0.01 imp/s (Fig. 5, P , 0:01; Wilcoxson test). In a separate group of units, co-application of 20 mM OCT with 0.05% CAP prevented the CAP-induced increase (0.04 ^ 0.02 imp/s compared to background, 0.01 ^ 0.00 imp/s), demonstrating that activation of SSTRs results in a short term (phasic) inhibition of TRPV1 receptors (Fig. 6). When sequential applications of CAP were made to the same fiber, both CAP exposures resulted in activity that was significantly higher than background (Fig. 7, *P , 0:05; Friedman’s ANOVA). However, activity evoked by the 2nd application (CAP 2, 0.10 ^ 0.05 imp/s) was significantly less than that evoked by the 1st application (CAP 1, 0.15 ^ 0.05 imp/s, þP , 0:05; Friedman’s ANOVA), demonstrating desensitization. In a separate set of units, co-application of OCT with CAP 1 prevented this desensitization such that responses to CAP 1 (0.09 ^ 0.02 imp/s) were not significantly different from background, but the response to CAP 2 (0.27 ^ 0.04 imp/s) was significantly higher when compared to CAP 1 (þP , 0:05; Friedman’s ANOVA) and to background (0.03 ^ 0.01, *P , 0:05; Friedman’s ANOVA).
Fig. 2. Fluorescent staining of L5 DRG cells: single-labeled SSTR2a cells appear green (A) and single-labeled TRPV1 cells appear red (B). When the images are merged, numerous double-labeled cells appear yellow (C). Arrows identify single-labeled cells, arrowheads identify double-labeled cells. Bar, 25 mm
3.3.2. SSTR-mediated tonic control of CAP-induced activity Application of either 0.05% CAP or 1.3 mM c-SOM to the receptive field of CMH fibers produced an increase in activity (580 ^ 132 and 305 ^ 57%, respectively, compared to background, Fig. 8). However, co-application of CAP þ c-SOM resulted in a dramatic increase in CAP-induced activity (1578 ^ 707% compared to background) which was significantly higher than CAP or c-SOM alone (*P , 0:05;
Table 1 Estimates of single- and double-labeled L5 DRG cells Animal
F10 F12 F13 Mean ^ SE N
SSTR2a single-labeled DRG cells, % of total
13.3 16.4 23.8 17.8 ^ 5.4 3
TRPV1 single-labeled DRG cells, % of total
24.8 30.4 40.4 31.9 ^ 7.9 3
SSTR2a and TRPV1 double-labeled DRG cells % of SSTR2a-labeled for TRPV1
% of TRPV1-labeled for SSTR2a
% of total cells double labeled
52.1 56.6 70.8 60.0 ^ 5.6 3
27.9 30.6 41.7 33.0 ^ 4.2 3
6.93 9.3 16.9 11.0 ^ 5.2 3
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Fig. 3. Intraplantar injection of 20 mM ðn ¼ 8Þ but not 2 mM ðn ¼ 6Þ OCT reduces CAP-induced flinching (A) and L/L (B, *P , 0:05 compared to all other groups, one-way ANOVA followed by a Tukey’s test). The time course indicates that both duration and magnitude of flinching (C) and L/L (D) are reduced by 20 mM OCT (*P , 0:05; two-way repeated measures ANOVA followed by a pairwise comparison using a Tukey’s test). OCT-induced inhibition of CAP pain behaviors is reversed by co-injection of the SSTR antagonist c-SOM so that behaviors in these rats ðn ¼ 8Þ are no different from the CAP alone group ðn ¼ 8Þ (E and F). OCT inhibits CAP pain behaviors through a local effect since animals injected with OCT in one hindpaw and CAP into the contralateral hindpaw (20 mM OCT ipsil, CAP contra, n ¼ 9) had nociceptive behaviors similar to those seen in animals injected with CAP alone (E and F). (OCT, CAP group shown for comparison purposes only, one-way ANOVA).
Kruskal – Wallis test), demonstrating that SSTRs maintain a tonic inhibitory influence over TRPV1 receptors. 3.3.3. Opioid involvement The excitation evoked by CAP alone (0.15 ^ 0.05 imp/s) and CAP þ NAL (0.21 ^ 0.06 imp/s) was significantly different from background (P , 0:05;
two-way ANOVA), but not significantly different from each other, indicating that opioid receptors do not maintain tonic inhibition over TRPV1 receptors (Fig. 9). Furthermore, the CAP þ OCT responses (0.09 ^ 0.02 imp/s) and the CAP þ OCT þ NAL responses (0.09 ^ 0.02 imp/s) were significantly different from background (P , 0:05; two-way ANOVA) but not different from each other, indicating that
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Fig. 4. Intraplantar injection of c-SOM alone ðn ¼ 8Þ produces mild flinching and L/L compared to the more robust effect of CAP ðn ¼ 8Þ (A and B, þP , 0:05; compared to c-SOM, PBS group, Kruskal–Wallis test). However, injection of both drugs (c-SOM þ CAP, n ¼ 8) results in dramatically increased behavioral responses that are more than additive (*P , 0:05; compared to PBS, CAP and c-SOM, PBS groups, Kruskal–Wallis test). Time courses show an increase in magnitude and duration of CAP-induced behaviors following release of TRPV1 from SSTR-induced inhibition (C and D, þP , 0:05; compared to c-SOM, PBS group; *P , 0:05 compared to PBS, CAP and c-SOM, PBS groups, two-way repeated measures ANOVA followed by a pairwise comparison using a Tukey’s test). c-SOM enhances CAP pain behaviors through a local effect since intraplantar CAP in one paw and 1.3 mM c-SOM into the contralateral hindpaw (CAP ipsil, c-SOM contra, n ¼ 5) produces nociceptive behaviors similar to those following CAP alone (E and F, *P , 0:05; different from all other groups, Kruskal– Wallis test).
activation of opioid receptors was not involved in the OCTinduced suppression of TRPV1 receptors (Fig. 9).
4. Discussion The present study demonstrates that the majority of SSTR2a-expressing DRG cells also express TRPV1.
Evidence that SSTRs can phasically and tonically modulate TRPV1 activity is provided by in vivo behavioral studies and in vitro single unit recordings. Thus, the behavior studies demonstrate that activation of peripheral SSTRs with OCT significantly reduces CAP-induced nociceptive behaviors in a dose-dependent fashion, and this occurs through local activation of SSTRs in the hindpaw. In vitro studies demonstrate that activation of peripheral SSTRs on
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Fig. 5. In the skin-nerve preparation, application of CAP to the receptive field of CMH units significantly increases the firing rate compared to background. Application of 20 mM OCT þ CAP prevents the CAPinduced activity such that the mean activity is no different from background. **P , 0:01; Wilcoxson test.
identified nociceptors significantly reduces CAP-induced nociceptor activity. Furthermore, blockade of peripheral SSTRs either in vivo or in vitro dramatically enhances CAPinduced behaviors and nociceptor activity, respectively. Taken together, these data strongly suggest there is both a somatostatinergic phasic and tonic modulation of peripheral TRPV1 receptors. A previous study noted expression of SSTR2a in DRG cells, but the percentage of labeled cells was not reported (Schulz et al., 1998). Stereological counts in the present study indicate that approximately 18% of L5 DRG cells are positively labeled for SSTR2a which is in close agreement
Fig. 6. (A) Demonstration of the CAP-induced increase in CMH activity during a 2 min application of CAP to the receptive field. (B) When OCT is co-applied with CAP, there is a significant decrease in the CAP-induced activity. H1, H2, heat stimulus.
Fig. 7. Sequential applications of CAP result in increased activity compared to background, but desensitization occurs such that activity evoked by CAP 2 is significantly less than that evoked by CAP 1. Co-application of CAP 1 þ OCT prevents this desensitization from occurring in CAP 2. (*P , 0:05 compared to background,þP , 0:05 compared to CAP 1 or CAP 1 þ OCT, Friedman’s ANOVA).
with percentages of SSTR2a-labeled peripheral axons in rat digital nerve (Carlton et al., 2001a). There are currently five cloned SSTR subtypes (Patel et al., 1995). In addition to SSTR2a, subtypes SSTR2b (Schindler et al., 1999) and SSTR3 (Senaris et al., 1995) have also been localized in DRG cells. Therefore, it is likely that the percentage of TRPV1-expressing sensory neurons under SSTR control reported here is conservative. Also, OCT will activate SSTR2, SSTR3 and SSTR5 subtypes (Patel et al., 1995; Viollet et al., 1995). Thus we must take into consideration that modulation of TRPV1 may be occurring through activation of additional SSTR subtypes. When other selective SSTR agonists become available, the role of each SSTR subtype in TRPV1 modulation can be more precisely defined.
Fig. 8. Application of 0.05% CAP to the receptive field of CMH units results in moderate activity compared to background (which is normalized to 100%). Blocking SSTRs by application of 1.3 mM c-SOM results in mild activation, however, co-application of CAP þ c-SOM results in a dramatic increase in CMH activity that is more than additive (*P , 0:05; compared to CAP and c-SOM alone, Kruskal– Wallis test).
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Fig. 9. When CMH units are exposed to CAP þ 2 mM NAL, unit activity is not different compared to CAP alone, indicating that opioid receptors do not maintain a tonic inhibitory control over CAP-sensitive nociceptors. When CMH units are exposed to CAP þ OCT þ NAL, unit activity is not different compared to CAP þ OCT, indicating that opioid receptor activation does not contribute to the OCT-induced decrease in CAP excitation.
The expression of TRPV1 has been previously reported in DRG cells, and the percentages determined here (31%) are in general agreement with those previously published (Carlton and Hargett, 2002; Guo et al., 1999; Michael and Priestley, 1999). The co-expression of SSTR and TRPV1 in DRG cells has not been examined previously and the data demonstrate that approximately 60% of L5 DRG cells expressing SSTR2a also express TRPV1. Conversely, 33% of TRPV1-expressing cells also express SSTR2a. As stated above, these are conservative estimates given that 4 additional SSTR subtypes may be expressed in DRG cells. Regardless, the high degree of co-localization of these receptors indicates that SSTRs could maintain significant control over the excitability of TRPV1, a consequence which is borne out in our behavioral and single fiber studies. As additional probes for SSTRs become available, the percentage of SSTR-TRPV1 expressing DRG cells will undoubtedly increase. Peripheral injection of the SSTR agonist OCT results in a significant attenuation of CAP-induced flinching and L/L behaviors. In related studies it has been demonstrated that OCT produces phasic inhibition of formalin-induced flinching and L/L behaviors (Carlton et al., 2001a). Control studies demonstrate that OCT produces its phasic effect through local and not systemic actions since injection of OCT into one hindpaw and CAP into the contralateral hindpaw results in nociceptive behaviors similar to those seen in animals injected with CAP alone. Activation of SSTRs has been previously shown to attenuate thermal responses in vivo (Eschalier et al., 1991) and in vitro (Carlton et al., 2001a), as well as block thermal sensitization following exposure of nociceptors to inflammatory mediators such as bradykinin (Carlton et al., 2001a).
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Mechanical sensitization has also been reduced following SSTR activation (Corsi et al., 1997), however, this action is undoubtedly through modulation of receptors or channels other than TRPV1 since mechanical stimuli do not activate TRPV1. The in vivo results are confirmed in the in vitro recordings from nociceptors in that OCT significantly reduced CAP-induced activity in CMH units. These data corroborate an earlier report that OCT dose-dependently inhibited thermal responses of nociceptors (Carlton et al., 2001a). Thus, nociceptors are subject to phasic control by SSTRs, and this is through modulation of the TRPV1 function. The percentage of CAP-sensitive fibers that was inhibited by OCT was greater that than predicted by the doubling labeling studies presumably because other SSTR subtypes (not localized in the present study) are expressed by CAP-sensitive nociceptors. In addition, the data indicate that when SSTRs are blocked by c-SOM, CAP induces a 3– 4-fold increase in behavioral responses and single unit activity compared to that produced by CAP alone. Thus somatostatin exerts an impressive tonic modulation of TRPV1 receptors. This is in agreement with previous reports demonstrating that an SSTR antagonist dramatically enhances formalin pain behaviors and generates considerable nociceptor activity that can be blocked by SSTR agonists or somatostatin antibody (Carlton et al., 2001b). Based on our anatomical studies, we hypothesize that OCT is acting directly on SSTRs localized on nociceptors. However, it has been reported that somatostatin and its analogues bind to opioid receptors (Gulya et al., 1986; Maurer et al., 1982; Pugsley and Lippmann, 1978; Terenius, 1976), so somatostatin may also activate opioid receptors, which are present on nociceptors (Coggeshall et al., 1997; Stein et al., 1988). For example, NAL, a widely used opioid antagonist, prevented the analgesia that followed intraventricular injection of somatostatin (Rezek et al., 1978). In contrast, NAL did not effect responses of dorsal horn neurons to somatostatin (Randic and Miletic, 1978; Sandkuhler et al., 1990), nor did it reverse the inhibitory effects of OCT on thermal responses of nociceptors (Carlton et al., 2001a). Finally, SSTRs and opioids have been localized on different populations of DRG cells (Taddese et al., 1995). Therefore, it seems most likely that somatostatin and opioids produce their effects through different systems. In the present study NAL did not enhance CAP-induced activity indicating that opioids did not have a tonic control over TRPV1 function. Furthermore, NAL did not alter the OCT-induced inhibition of CAP-induced responses indicating that opioid receptors are not contributing to the SSTR-induced inhibition. There are several lines of evidence indicating that TRPV1 activation plays a key role in inflammatory pain, including neurogenic inflammation, so understanding how this activation can be modulated (both endogenously and exogenously) could have far reaching implications in pain
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management. The TRPV1 receptors are localized on nociceptors and convey sensitivity to capsaicin (CAP), heat and acid (Caterina et al., 1997; Tominaga et al., 1998). Intradermal injection of CAP is exquisitely painful in humans and produces nociceptive behaviors in rats, with a short lasting (, 30 min) inflammatory response characterized by redness, swelling and plasma extravasation. Numbers of TRPV1-expressing axons increase in the periphery 2 days after inflammation and may contribute to peripheral sensitization in the inflamed state (Carlton and Coggeshall, 2001). The percentage of TRPV1-labeled DRG cells also increases following inflammation as do the number of DRG cells double-labeled for TRPV1 and NF200, a marker for cells giving rise to myelinated A fibers (Amaya et al., 2003). A key finding in TRPV1 knock out mice is that, though they still respond to thermal stimuli, they do not develop inflammatory pain behaviors (Caterina et al., 2000; Davis et al., 2000). Activation of CAP-sensitive fibers produces the plasma extravasation and vasodilatation (Jancso´ et al., 1967; Szolcsa´nyi, 1996) that characterizes neurogenic inflammation. Hence, TRPV1 receptors are essential components underlying the tumor (swelling), rubor (redness), dolor (pain) and calor (heat) of inflammation. At this time, we cannot rule out that SSTR-induced decreases in CAP responses might involve Kþchannel opening, and/or Caþ2 channel closing (Koch et al., 1988). However, there are several lines of evidence that all 5 SSTRs are functionally coupled to inhibition of adenylate cyclase (AC) and the cAMP/protein kinase A (PKA) pathway via pertussis toxin sensitive GTP binding proteins (Patel et al., 1995; Tentler et al., 1997), and inhibition of AC is clearly the most prominent transduction pathway used by this family of receptors (Viollet et al., 1995). The inhibitory effect of SST on AC and the cAMP/PKA pathway may be the link that allows SSTRs to modulate TRPV1 function (Bhave et al., 2002; De Petrocellis et al., 2001). Analysis of this interaction will be the subject of future investigations. It has recently been suggested that TRPV1 receptors are under tonic inhibitory control mediated by the membrane lipid phosphatidylinosital-4,5-biphosphate (Chuang et al., 2004). In heterologous systems expressing TRPV1 and bradykinin or track A receptors, exposure of HEK cells to bradykinin or nerve growth factor releases TRPV1 from this inhibition, resulting in enhanced responses to acid and CAP. Hence, TRPV1 may be controlled by several mechanisms which may be differentially affected in various disease states. Compromising or disrupting this tonic inhibition may result in prolonged states of hypersensitivity. In summary, demonstration that the TRPV1 is under both phasic and tonic SSTR regulation provides insight to the cellular mechanisms affecting TRPV1 receptor function and may provide novel approaches for modulation of a receptor family that underlies sensitization of peripheral primary afferent fibers and inflammatory pain.
Acknowledgements The authors thank Vicki Wilson for her excellent secretarial assistance. This work was supported by NIH NS27910 and NS40700 to S.M.C. and NS101621 to R.E.C.
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Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel Susan M. Carlton*, Shengtai Zhou, Junhui Du, Gregory L. Hargett, Guangchen Ji, Richard E. Coggeshall Department of Anatomy and Neurosciences, Marine Biomedical Institute, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069, USA Received 23 February 2004; received in revised form 14 April 2004; accepted 26 April 2004
Abstract Activation of peripheral somatostatin receptors (SSTRs) inhibits sensitization of nociceptors, thus having a short term or phasic effect [Pain 90 (2001) 233] as well as maintaining a tonic inhibitory control over nociceptors [J Neurosci 21 (2001) 4042]. The present study provides several lines of evidence that an important mechanism underlying SSTR modulation of nociceptors is regulation of the transient receptor potential vanilloid 1 ion channel (TRPV1, formerly the VR1 receptor). Double labeling of L5 dorsal root ganglion cells demonstrates that , 60% of SSTR2a-labeled cells are positive for TRPV1. Conversely, , 33% of TRPV1-labeled cells are positive for SSTR2a. In vivo behavioral studies demonstrate that intraplantar injection of 20.0 but not 2.0 mM octreotide (OCT, SSTR agonist) significantly reduces capsaicin (CAP, a ligand for TRPV1) -induced flinching and lifting/licking behaviors. This occurs through local activation of SSTRs in the injected hindpaw and is reversed following co-application of the SSTR antagonist cyclo-somatostatin (c-SOM). In vitro studies using a skin-nerve preparation demonstrate that activation of peripheral SSTRs on nociceptors with 20.0 mM OCT significantly reduces CAP-induced activity and can prevent CAP-induced desensitization. Furthermore, blockade of peripheral SSTRs with c-SOM dramatically enhances CAP-induced behaviors and nociceptor activity, demonstrating SSTR-induced tonic inhibitory modulation of TRPV1. Finally, TRPV1 does not appear to be under tonic opioid receptor control since the opioid antagonist naloxone does not change CAP-induced excitation and does not effect OCT-induced inhibition of CAP responses. These data strongly suggest that SSTRs modulate nociceptors through phasic and tonic regulation of peripheral TRPV1 receptors. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Somatostatin; Primary afferent; Hyperalgesia; Nociception; Capsaicin; VR1
1. Introduction Somatostatin is a biologically active compound with diverse effects. In regards to pain, it is a peripheral analgesic agent having a short term (phasic) effect (Carlton et al., 2001a; Heppelmann and Pawlak, 1997) and also maintains tonic control of peripheral nociceptors (Carlton et al., 2001b, 2003; Heppelmann and Pawlak, 1999). These effects are manifest by decreases in formalin-induced pain behaviors following application of a somatostatin receptor (SSTR) agonist, and increases following application of an SSTR antagonist. To elucidate mechanisms by which peripheral SSTRs maintain inhibitory control of nociceptors, we focus * Corresponding author. Tel.: þ1-409-772-2124; fax: þ 1-409-762-9382. E-mail address: [email protected] (S.M. Carlton).
on their interactions with the transient receptor potential vanilloid 1 (TRPV1) ion channel. The TRPV1 was originally cloned as a capsaicin (CAP) receptor, but also responds to heat, protons and inflammatory mediators (Caterina and Julius, 2001; Caterina et al., 1997; Chuang et al., 2004), and thus is capable of integrating nociceptive thermal and chemical stimuli (Tominaga et al., 1998). The TRPV1 plays an important role in normal or physiologic nociception (Caterina et al., 1997) and also in inflammatory (Caterina et al., 2000) and neuropathic (Hudson et al., 2001) pain. We hypothesize that SSTRs both phasically and tonically inhibit TRPV1 activation. Thus, the present study investigates whether: (1) SSTR and TRPV1 receptors are co-localized in primary sensory neurons, (2) activation of SSTRs by SSTR agonists reduces the pain behaviors and nociceptor activity induced by CAP administration (phasic control),
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.04.042
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and (3) blockade of SSTR function enhances CAP-induced pain behaviors and nociceptor activity (tonic control) and (4) opioid receptors are involved in these actions.
2. Materials and methods All procedures were approved by the University Animal Care and Use Committee and followed guidelines for the ethical treatment of animals (Zimmermann, 1983). 2.1. Anatomical studies The lumbar (L) 5 dorsal root ganglia (DRG) from male Sprague – Dawley (SD) rats (Harlan, Indianapolis, IN, n ¼ 3) were used to determine total neuron counts and the percentages of neurons that were (1) single-labeled for either SSTR2a or TRPV1 or (2) double-labeled for both receptors. Rats were deeply anaesthetized with sodium pentobarbital (70 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde and 0.1% picric acid in 0.1 M phosphate buffer (PB, pH 7.4) at 4 8C. Left and right L5 DRGs were removed and placed in 30% sucrose-PB for cryoprotection overnight at 4 8C. The ganglia were fast frozen in liquid N2 in a cryo-embedding compound and sectioned on a cryostat (Microm International, GmbH). Serial sections (8 mm) were cut through the short axis of the DRG and placed on chrome alum dipped 3-well, teflon printed slides (Electron Microscopy Sciences, Fort Washington, PA). Systematic random sampling ensured that all neurons had an equal chance of being analyzed (Coggeshall and Lekan, 1996). Briefly, after dissection, each DRG was measured along the long axis under a dissecting microscope and the length was divided by the thickness of the cryostat sections (8 mm) to determine the approximate number of sections in that DRG. Section separation (k in stereological literature) was determined by dividing the number of sections by the number of pairs of serial sections (6 – 7 pairs per ganglia in this study) chosen for analysis. Starting randomly between the first and the kth section (the Rth section in stereological literature), subsequent pairs were taken for analysis at R þ k; R þ 2k; etc. until the tissue in a DRG was exhausted. 2.1.1. Staining procedures Single- and double-labeling for SSTR2a and TRPV1 were performed using immunohistochemistry and fluorescence tags. For single-labeling, sections were incubated in guinea pig anti-SSTR2a (1:40,000, Gramsch Labs, GmBH) for 48 h at room temperature, rinsed in 0.1 M phosphate buffered saline (PBS, pH 7.4), placed in biotinylated antiguinea pig IgG (1:200, Vector Laboratories, Burlingame, CA) for 1 h, followed by incubation in Vectastain Elite ABC peroxidase reagent (avidin –biotin complex, Vector Laboratories) for 1 h. After rinsing in PBS, sections were incubated in fluorescein-labeled tyramide (1:75, TSA kit,
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Perkin – Elmer Life Science, Boston, MA) for 7 min followed by a wash in PBS. Following incubation for 20 min in 0.3% H2O2 in PBS (inactivates peroxidase introduced during the ABC reaction), sections were incubated in goat anti-VR1 (anti-TRPV1) (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA) for 48 h at room temperature. Following rinsing, sections were placed in Cyanin 3- (Cy3-) conjugated anti-goat IgG (1:400, Jackson ImmunoResearch, West Grove, PA) for 1 h. After final rinsing, slides were cover slipped with Vectashield mounting media (Vector Laboratories, Burlingame, CA). Controls for specificity included staining DRG sections as described above but omitting the primary antisera. Omission of the primary antisera resulted in no staining in the tissue. Absorption controls were performed for each antiserum where the appropriate peptide was incubated with the working dilution of antiserum (100 mg peptide/1 ml antisera) overnight at room temperature. Immunostaining with these solutions resulted in a complete lack of staining in the tissue. The TRPV1 antiserum was also incubated over night with the vanilloid receptor like-1 (VRL-1, TRPV2) peptide (Caterina et al., 1999) (ADI, San Antonio, TX), and this resulted in a staining pattern similar to that observed following staining with TRPV1 antiserum alone, indicating that this antiserum did not recognize or cross-react with VRL-1. In immunostained DRG sections, a neuron was determined to be labeled for either SSTR2a or TRPV1 by inspection, comparing the amount of fluorescence in the neuron to that in control tissues and surrounding large diameter, unlabeled neurons. Total neuron counts in the L5 DRG were obtained using pairs of sections that were stained with cresyl violet. The diameters of fluorescently labeled and cresyl violet stained DRG neurons were estimated by summing the length and width of those neurons with a visible nucleus and dividing by two. Whenever possible, at least 100 neurons were measured in each DRG. The data are presented as the percentage of the total and labeled population. 2.1.2. Stereological analysis Following staining, color digitized images of each section (24 bits per pixel), were captured using an Olympus BX51 microscope with an attached Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI). Fluorescein isothiocynate (FITC) and Cy3 filter cube sets were used to visualize the fluorescein and Cy3, respectively. Camera exposures were adjusted automatically by Spot software (version 3.1) and brightness and gamma corrections were made manually to achieve the best signal-to-noise ratios. The physical disector (pairs of sections) was used to achieve unbiased estimates of total, SSTR2a-, and TRPV1-labeled DRG neurons (Coggeshall, 1992). The size of most DRG sections and the magnification needed to identify labeled cells necessitated analysis of a randomly chosen subregion or fraction ðf Þ in each section in a pair. Once identified,
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images in the subregion in each pair were outlined from the computer screen onto clear mylar sheets including all labeled cell profiles and an appropriate number of fiduciary landmarks. The mylar sheets from a pair were matched with one another using the fiduciary landmarks, and all labeled profiles that appeared in both sections were disallowed. Labeled profiles remaining were ‘tops’ or ‘counts’ (identified as Q- in the stereological literature), and each top was then checked for double-labeling. Using ‘fractionator sampling’ (Gundersen et al., 1988; West and Gundersen, 1990) total numbers of cresyl violet stained neurons and numbers of SSTR2a- and TRPV1-labeled neurons were estimated in each DRG by multiplying counts Q- by the reciprocal of the fraction ð1=f Þ of each sampled pair. These neuronal counts ðNÞ for all pairs analyzed in a DRG were summed ðSNÞ: Total number of neurons in the entire DRG was then estimated by ðSN £ kÞ=2 (divided by two because each section in a pair was used as both reference and look-up sections) (Coggeshall, 1992). Percentages of single-labeled neurons were calculated by dividing the numbers of singled-labeled neurons by the total DRG cell number £ 100. Percentages of doublelabeled neurons were calculated by dividing numbers of double-labeled neurons by numbers of single-labeled SSTR2a or TRPV1 neurons £ 100.
the center of the hindpaw where the drugs were delivered. In some groups, PBS was injected before or after the drug of interest to keep volumes constant between groups. Each animal was used only once and the experimenter was unaware of which drug was being injected into an animal. 2.2.3.1. SSTR-mediated phasic control of CAP-induced nociception. To demonstrate that activation of SSTRs modulated TRPV1 activity, rats received intraplantar injections of the SSTR agonist octreotide (OCT, Sandostatin, 30 ml), 2 mM ðn ¼ 6Þ or 20 mM ðn ¼ 8Þ; followed 10 min later by a 20 ml cocktail injection of the same dose of OCT þ 0.05% CAP into the same place and spontaneous pain behaviors were measured. A control group ðn ¼ 8Þ received intraplantar injections of phosphate buffered saline (PBS, 30 ml), followed 10 min later by a 20 ml injection of 0.05% CAP alone into the same place. To demonstrate that OCT effects were receptor specific, a group of rats ðn ¼ 8Þ received a 30 ml injection of 20 mM OCT þ the SSTR antagonist cyclo-somatostatin (c-SOM, 1.3 mM) followed 10 min later by a 20 ml injection of 0.05% CAP. To demonstrate that OCT produced inhibition of CAP-induced behaviors through a local effect in the hindpaw, another group of rats ðn ¼ 9Þ received an injection of OCT (30 ml, 20 mM) into one hindpaw followed 10 min later by a 20 ml injection of 0.05% CAP in the contralateral hindpaw.
2.2. Behavioral studies 2.2.1. Habituation Male SD rats (250 –300 g) were housed in groups of three in plastic cages with soft bedding under a reversed light/dark cycle. Following arrival at the University animal care facility, the animals were acclimated for at least 3 days before behavioral testing was initiated. Rats were habituated to behavioral testing by placing them on a wire screen platform in Plexiglas cages (8 £ 8 £ 18 cm3) for 1 h. Each rat was habituated twice before being placed in an experimental group. 2.2.2. Preparation of CAP To prepare CAP, 2 ml 100% ethanol were added to a bottle containing 1 g CAP (Fluka, Ronkonkoma, NY) and vigorously shaken. This mixture was added to a mixture of 9.3 ml saline and 0.7 ml Tween 80. This solution was stirred while heating until the alcohol evaporated and the total volume returned to 10 ml. The stock solution was diluted with vehicle to obtain a 0.05% CAP solution that was stored at 4 8C. Control experiments demonstrated that intraplantar injection of CAP vehicle alone did not produce any nociceptive behaviors. All other drugs were dissolved in PBS (pH 7.4). 2.2.3. Drug injections For intraplantar injections, a 28-gauge needle was attached to a 50 ml Hamilton syringe with PE20 tubing. For paw injections, the needle punctured the plantar skin distal to the foot pads and was guided into the subcutaneous space to
2.2.3.2. SSTR-mediated tonic control of CAP-induced nociception. To demonstrate that blockade of SSTRs released TRPV1 from tonic inhibition, rats (n ¼ 8 per group) received an intraplantar injection of the SSTR antagonist c-SOM (30 ml, 1.3 mM), followed 10 min later by a 20 ml injection of either PBS or 0.05% CAP into the same place and nociceptive pain behaviors were measured. To demonstrate that c-SOM produced its effects through blockade of local SSTRs in the hindpaw, a group of rats ðn ¼ 5Þ received an intraplantar injection of c-SOM (1.3 mM, 30 ml) into one paw followed 10 min later by a 20 ml injection of 0.05% CAP into the other paw. 2.2.4. Behavioral testing Spontaneous nociceptive behaviors resulting from CAP or c-SOM injections were assessed by counting the number of flinches and seconds an animal spent lifting and/or licking (L/L) the injected paw in 5 min intervals. A flinch was defined as a spontaneous, rapid jerk of the foot whether the foot was on the screen or held in the air. 2.3. Electrophysiological studies 2.3.1. In vitro skin-nerve recordings Single unit recordings from C-mechanoheat (CMH) sensitive fibers were obtained in glabrous skin using a modified in vitro skin-nerve preparation (Du et al., 2001; Kress et al., 1992; Reeh, 1986). Male SD rats (200 –300 g) were sacrificed with an overdose of CO2, and the glabrous
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skin of the hindpaw was dissected with the medial and lateral plantar nerves attached. The preparation was placed corium side up in an organ bath and superfused (15 ml/min, 34 8C) with an oxygen-saturated, modified synthetic interstitial fluid solution (SIF, in mM: NaCl, 123; KCl, 3.5; MgSO4, 0.7; CaCl2, 2.0; Na gluconate, 9.5; NaH2PO4, 1.7; Glucose, 5.5; Sucrose, 7.5; and HEPES, 10; pH 7.40 ^ 0.05). The plantar nerves were placed in a separate chamber containing a top layer of mineral oil and a bottom layer of SIF. The nerves were desheathed and teased apart on a mirror stage under a dissecting microscope. Small nerve bundles were repeatedly split with sharpened forceps until single unit activity was obtained. 2.3.2. Thermal stimulation A feedback-controlled lamp placed beneath the organ bath supplied radiant heat to the receptive field of each isolated unit. The beam was focused through the translucent bottom of the bath onto the epidermal surface of the skin. A thermocouple was placed in the corium above the light beam to measure intracutaneous temperature. A standard heat ramp was applied to each unit, starting from an adapting temperature of 34 8C and rising to 47 8C in 10 s (47 8C on the corium side was equivalent to 51 8C on the epidermal side). The temperature at which the second spike was elicited by the heat stimulus was defined as heat threshold. Choosing units that responded to this heat stimulus enhanced the probability that these units would also be capsaicin-sensitive. 2.3.3. Mechanical stimulation The mechanical threshold for each unit was determined using calibrated von Frey filaments (Stoelting, Inc., Wood Dale, IL). The filaments were applied to the receptive field on the corium side of the skin in a uniform fashion, starting with filaments delivering a relatively small amount of force and ascending (0.1 –166.7 mN) until an action potential could be consistently evoked. 2.3.4. Chemical stimulation To investigate responses of units to various drugs, a small plastic ring (5 mm diameter) was placed over the receptive field of each unit and the SIF in the ring was replaced with SIF containing 0.05% CAP alone, CAP þ the SSTR agonist OCT (20 mM), CAP þ the SSTR antagonist c-SOM (1.3 mM), CAP þ OCT þ the opioid antagonist naloxone (NAL, 2 mM) or CAP þ NAL. The concentrations of OCT, c-SOM and NAL were based on those used in a previous study (Carlton et al., 2001a). All drugs dissolved in SIF were buffered to pH 7.40 ^ 0.05.
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investigate the effect of the SSTR agonist OCT on CAPinduced activity, in a separate group of CMH fibers ðn ¼ 8Þ; 0.05% CAP was applied to the receptive field in the presence of 20 mM OCT for 2 min. To determine if OCT blocked CAP-induced desensitization, a phenomenon that often occurs following TRPV1 activation, units ðn ¼ 8Þ were exposed to sequential 0.05% CAP applications (CAP 1 and CAP 2) for 2 min each, with a 5 min intertrial interval; in a separate group of units ðn ¼ 6Þ; 20 mM OCT was applied with 0.05% CAP (CAP 1 þ OCT), followed 5 min later by 0.05% CAP alone (CAP 2). 2.3.4.2. SSTR-mediated tonic control of CAP-induced activity. To determine whether SSTRs maintained a tonic inhibitory control over TRPV1 receptors, 0.05% CAP ðn ¼ 8Þ; c-SOM ðn ¼ 18Þ or CAP þ c-SOM ðn ¼ 8Þ was applied for 2 min to the receptive fields in 3 different groups of nociceptors. 2.3.4.3. Opioid involvement. To rule out a tonic opioid control of TRPV1, CMH units ðn ¼ 6Þ were exposed to 0.05% CAP þ 2 mM naloxone (NAL, an opioid antagonist) or 0.05% CAP alone ðn ¼ 8Þ and activity monitored for 2 min. To rule out involvement of opioid receptors in inducing OCT effects, CMH units were monitored following exposure to 0.05% CAP þ 20 mM OCT þ 2 mM NAL ðn ¼ 8Þ or 0.05% CAP þ 20 mM OCT ðn ¼ 6Þ for 2 min. 2.3.5. Neurophysiological recordings Neural activity was recorded using a DAM80 Differential Amplifier (World Precision Instruments) attached to a monopolar gold wire electrode, with the reference electrode positioned nearby. Units were identified by manual probing of the glabrous skin with a blunt glass rod and if they had a clearly defined receptive field, they were studied in detail. Action potentials were analyzed on a Dell computer with a custom-made template-matching program that allowed discrimination of single unit activity based on the amplitude and wave form of each action potential (Forster and Handwerker, 1990). The conduction velocity of each unit was determined by monopolar electrical stimulation (0.1 ms duration, train frequency 1 Hz) at the most mechanosensitive site in the receptive field of each unit using a tefloncoated steel electrode (5 MV impedance, 250 mm shaft diameter). Conduction velocity was determined from the latency of the action potential and the distance from the stimulation electrode to the recording site (measured in millimeters). Units with a conduction velocity of less than 1.6 m/s were classified as C fibers. 2.4. Statistical analyses
2.3.4.1. SSTR-mediated phasic control of CAP-induced activity. To investigate responses of CMH fibers to CAP alone, background activity of units ðn ¼ 8Þ was recorded for 2 min, then 0.05% CAP was applied to the receptive field of the unit and recordings were made for another 2 min. To
All data were expressed as means ^ standard errors (SE) and evaluated using SigmaStat (Jandel Corporation). In the behavioral studies, the total sum of flinches or time spent L/L during 60 min were calculated and plotted as bar graphs.
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Differences between treatment groups were evaluated using a one-way analysis of variance (ANOVA) followed by the Tukey’s test. Time courses for nociceptive behaviors were constructed by plotting the mean number of flinches or time spent L/L in 5 min intervals as a function of time. For these outcomes the data were analyzed using a two-factor repeated measures ANOVA followed by a pairwise comparison using a Tukey’s test. In the electrophysiological studies, differences in discharge rates were evaluated with either a Friedman’s ANOVA, Kruskal – Wallis test followed by Dunn’s post hoc analysis, Wilcoxson signed rank test or two-way ANOVA followed by a Tukey’s test, where appropriate, P , 0:05 was considered significant.
3. Results 3.1. Anatomical studies Analysis of the L5 DRG demonstrated that many small to medium diameter cells were immunohistochemically stained for SSTR2a and TRPV1 (Fig. 1). The nucleus was often visible as a ‘dark hole’ in both labeled and unlabeled cells (Fig. 2) and labeled cells demonstrated diffuse cytoplasmic staining. The mean diameter of SSTR2alabeled cells was 26.3 ^ 3.8 mm (range 16– 40 mm); for TPRV1-labeled cells mean diameter was 26.1 ^ 4.0 mm (range 13– 39 mm) and for double-labeled cells the mean diameter was 26.3 ^ 3.8 mm (range 16 – 40 mm). Stereological counts indicated that 17.8 ^ 5.4% (mean ^ SE) of cells were single-labeled for SSTR2a and 31.9 ^ 7.9% were single-labeled for TRPV1 (Table 1). In the double-labeling studies, 60.0 ^ 5.6% of SSTR2a cells were also labeled for TRPV1; and 33.0 ^ 4.2% TRPV1-labeled cells were also labeled for SSTR2a. For the total population of L5 DRG cells (13,629 ^ 3,250), 11.0 ^ 5.2% were double-labeled.
Fig. 1. Histograms demonstrating the diameter frequencies for total neurons (A), single-labeled SSTR2a (B), single-labeled TRPV1 (C) and doublelabeled SSTR2a-TRPV1 (D) L5 DRG ðn ¼ 3Þ cells. Note that labeled populations are primarily small and medium diameter cells.
systemically but through activation of SSTRs localized in the injected hindpaw (Fig. 3E and F).
3.2. Behavioral studies 3.2.1. SSTR-mediated phasic control of CAP-induced nociception Intraplantar injection of 0.05% CAP induced both flinching and L/L behaviors. Pretreatment with 20 mM but not 2 mM OCT significantly reduced both of these CAPinduced behaviors (Fig. 3A and B, P , 0:05; one-way ANOVA). The time courses of these behaviors are shown in Fig. 3C and D and demonstrate that both the magnitude and the duration of the CAP-induced behaviors were reduced by OCT (P , 0:05; two-way repeated measures ANOVA). Injection of c-SOM with the OCT þ CAP blocked the inhibitory effects of OCT such that the flinching and L/L behaviors in these animals were no different from animals injected with CAP alone (Fig. 3E and F, P , 0:05; one-way ANOVA). Injection of 20 mM OCT into one hind paw and CAP into the contralateral paw did not reduce CAP-induced behaviors indicating that OCT was not producing its effect
3.2.2. SSTR-mediated tonic control of CAP-induced nociception Intraplantar injection of the SSTR antagonist c-SOM produced a little flinching but no L/L compared to that induced by CAP (Fig. 4A and B, P , 0:05; Kruskal – Wallis test). However, when c-SOM was co-injected with CAP, both flinching and L/L behaviors were significantly enhanced compared to either c-SOM or CAP alone, and the combined effect was more than additive (Fig. 4A and B, P , 0:05; Kruskal – Wallis test). The time courses of these behaviors are shown in Fig. 4C and D and demonstrate that co-application of c-SOM þ CAP resulted in a dramatic enhancement of both the magnitude and duration of the CAP-induced pain behaviors (P , 0:05; two-way repeated measures ANOVA). Injection of CAP into one hindpaw and c-SOM into the contralateral paw, did not significantly change the CAP-induced responses indicating that the c-SOM effect was produced through action on local SSTRs
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in the injected paw and not through a systemic action (Fig. 4E and F, P , 0:05; Kruskal– Wallis test). 3.3. Electrophysiological studies 3.3.1. SSTR-mediated phasic control of CAP-induced activity Application of 0.05% CAP to the receptive fields of CMH fibers for 2 min in the skin-nerve preparation resulted in an increased firing rate, with a mean frequency of 0.15 ^ 0.05 imp/s compared to background 0.02 ^ 0.01 imp/s (Fig. 5, P , 0:01; Wilcoxson test). In a separate group of units, co-application of 20 mM OCT with 0.05% CAP prevented the CAP-induced increase (0.04 ^ 0.02 imp/s compared to background, 0.01 ^ 0.00 imp/s), demonstrating that activation of SSTRs results in a short term (phasic) inhibition of TRPV1 receptors (Fig. 6). When sequential applications of CAP were made to the same fiber, both CAP exposures resulted in activity that was significantly higher than background (Fig. 7, *P , 0:05; Friedman’s ANOVA). However, activity evoked by the 2nd application (CAP 2, 0.10 ^ 0.05 imp/s) was significantly less than that evoked by the 1st application (CAP 1, 0.15 ^ 0.05 imp/s, þP , 0:05; Friedman’s ANOVA), demonstrating desensitization. In a separate set of units, co-application of OCT with CAP 1 prevented this desensitization such that responses to CAP 1 (0.09 ^ 0.02 imp/s) were not significantly different from background, but the response to CAP 2 (0.27 ^ 0.04 imp/s) was significantly higher when compared to CAP 1 (þP , 0:05; Friedman’s ANOVA) and to background (0.03 ^ 0.01, *P , 0:05; Friedman’s ANOVA).
Fig. 2. Fluorescent staining of L5 DRG cells: single-labeled SSTR2a cells appear green (A) and single-labeled TRPV1 cells appear red (B). When the images are merged, numerous double-labeled cells appear yellow (C). Arrows identify single-labeled cells, arrowheads identify double-labeled cells. Bar, 25 mm
3.3.2. SSTR-mediated tonic control of CAP-induced activity Application of either 0.05% CAP or 1.3 mM c-SOM to the receptive field of CMH fibers produced an increase in activity (580 ^ 132 and 305 ^ 57%, respectively, compared to background, Fig. 8). However, co-application of CAP þ c-SOM resulted in a dramatic increase in CAP-induced activity (1578 ^ 707% compared to background) which was significantly higher than CAP or c-SOM alone (*P , 0:05;
Table 1 Estimates of single- and double-labeled L5 DRG cells Animal
F10 F12 F13 Mean ^ SE N
SSTR2a single-labeled DRG cells, % of total
13.3 16.4 23.8 17.8 ^ 5.4 3
TRPV1 single-labeled DRG cells, % of total
24.8 30.4 40.4 31.9 ^ 7.9 3
SSTR2a and TRPV1 double-labeled DRG cells % of SSTR2a-labeled for TRPV1
% of TRPV1-labeled for SSTR2a
% of total cells double labeled
52.1 56.6 70.8 60.0 ^ 5.6 3
27.9 30.6 41.7 33.0 ^ 4.2 3
6.93 9.3 16.9 11.0 ^ 5.2 3
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Fig. 3. Intraplantar injection of 20 mM ðn ¼ 8Þ but not 2 mM ðn ¼ 6Þ OCT reduces CAP-induced flinching (A) and L/L (B, *P , 0:05 compared to all other groups, one-way ANOVA followed by a Tukey’s test). The time course indicates that both duration and magnitude of flinching (C) and L/L (D) are reduced by 20 mM OCT (*P , 0:05; two-way repeated measures ANOVA followed by a pairwise comparison using a Tukey’s test). OCT-induced inhibition of CAP pain behaviors is reversed by co-injection of the SSTR antagonist c-SOM so that behaviors in these rats ðn ¼ 8Þ are no different from the CAP alone group ðn ¼ 8Þ (E and F). OCT inhibits CAP pain behaviors through a local effect since animals injected with OCT in one hindpaw and CAP into the contralateral hindpaw (20 mM OCT ipsil, CAP contra, n ¼ 9) had nociceptive behaviors similar to those seen in animals injected with CAP alone (E and F). (OCT, CAP group shown for comparison purposes only, one-way ANOVA).
Kruskal – Wallis test), demonstrating that SSTRs maintain a tonic inhibitory influence over TRPV1 receptors. 3.3.3. Opioid involvement The excitation evoked by CAP alone (0.15 ^ 0.05 imp/s) and CAP þ NAL (0.21 ^ 0.06 imp/s) was significantly different from background (P , 0:05;
two-way ANOVA), but not significantly different from each other, indicating that opioid receptors do not maintain tonic inhibition over TRPV1 receptors (Fig. 9). Furthermore, the CAP þ OCT responses (0.09 ^ 0.02 imp/s) and the CAP þ OCT þ NAL responses (0.09 ^ 0.02 imp/s) were significantly different from background (P , 0:05; two-way ANOVA) but not different from each other, indicating that
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Fig. 4. Intraplantar injection of c-SOM alone ðn ¼ 8Þ produces mild flinching and L/L compared to the more robust effect of CAP ðn ¼ 8Þ (A and B, þP , 0:05; compared to c-SOM, PBS group, Kruskal–Wallis test). However, injection of both drugs (c-SOM þ CAP, n ¼ 8) results in dramatically increased behavioral responses that are more than additive (*P , 0:05; compared to PBS, CAP and c-SOM, PBS groups, Kruskal–Wallis test). Time courses show an increase in magnitude and duration of CAP-induced behaviors following release of TRPV1 from SSTR-induced inhibition (C and D, þP , 0:05; compared to c-SOM, PBS group; *P , 0:05 compared to PBS, CAP and c-SOM, PBS groups, two-way repeated measures ANOVA followed by a pairwise comparison using a Tukey’s test). c-SOM enhances CAP pain behaviors through a local effect since intraplantar CAP in one paw and 1.3 mM c-SOM into the contralateral hindpaw (CAP ipsil, c-SOM contra, n ¼ 5) produces nociceptive behaviors similar to those following CAP alone (E and F, *P , 0:05; different from all other groups, Kruskal– Wallis test).
activation of opioid receptors was not involved in the OCTinduced suppression of TRPV1 receptors (Fig. 9).
4. Discussion The present study demonstrates that the majority of SSTR2a-expressing DRG cells also express TRPV1.
Evidence that SSTRs can phasically and tonically modulate TRPV1 activity is provided by in vivo behavioral studies and in vitro single unit recordings. Thus, the behavior studies demonstrate that activation of peripheral SSTRs with OCT significantly reduces CAP-induced nociceptive behaviors in a dose-dependent fashion, and this occurs through local activation of SSTRs in the hindpaw. In vitro studies demonstrate that activation of peripheral SSTRs on
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Fig. 5. In the skin-nerve preparation, application of CAP to the receptive field of CMH units significantly increases the firing rate compared to background. Application of 20 mM OCT þ CAP prevents the CAPinduced activity such that the mean activity is no different from background. **P , 0:01; Wilcoxson test.
identified nociceptors significantly reduces CAP-induced nociceptor activity. Furthermore, blockade of peripheral SSTRs either in vivo or in vitro dramatically enhances CAPinduced behaviors and nociceptor activity, respectively. Taken together, these data strongly suggest there is both a somatostatinergic phasic and tonic modulation of peripheral TRPV1 receptors. A previous study noted expression of SSTR2a in DRG cells, but the percentage of labeled cells was not reported (Schulz et al., 1998). Stereological counts in the present study indicate that approximately 18% of L5 DRG cells are positively labeled for SSTR2a which is in close agreement
Fig. 6. (A) Demonstration of the CAP-induced increase in CMH activity during a 2 min application of CAP to the receptive field. (B) When OCT is co-applied with CAP, there is a significant decrease in the CAP-induced activity. H1, H2, heat stimulus.
Fig. 7. Sequential applications of CAP result in increased activity compared to background, but desensitization occurs such that activity evoked by CAP 2 is significantly less than that evoked by CAP 1. Co-application of CAP 1 þ OCT prevents this desensitization from occurring in CAP 2. (*P , 0:05 compared to background,þP , 0:05 compared to CAP 1 or CAP 1 þ OCT, Friedman’s ANOVA).
with percentages of SSTR2a-labeled peripheral axons in rat digital nerve (Carlton et al., 2001a). There are currently five cloned SSTR subtypes (Patel et al., 1995). In addition to SSTR2a, subtypes SSTR2b (Schindler et al., 1999) and SSTR3 (Senaris et al., 1995) have also been localized in DRG cells. Therefore, it is likely that the percentage of TRPV1-expressing sensory neurons under SSTR control reported here is conservative. Also, OCT will activate SSTR2, SSTR3 and SSTR5 subtypes (Patel et al., 1995; Viollet et al., 1995). Thus we must take into consideration that modulation of TRPV1 may be occurring through activation of additional SSTR subtypes. When other selective SSTR agonists become available, the role of each SSTR subtype in TRPV1 modulation can be more precisely defined.
Fig. 8. Application of 0.05% CAP to the receptive field of CMH units results in moderate activity compared to background (which is normalized to 100%). Blocking SSTRs by application of 1.3 mM c-SOM results in mild activation, however, co-application of CAP þ c-SOM results in a dramatic increase in CMH activity that is more than additive (*P , 0:05; compared to CAP and c-SOM alone, Kruskal– Wallis test).
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Fig. 9. When CMH units are exposed to CAP þ 2 mM NAL, unit activity is not different compared to CAP alone, indicating that opioid receptors do not maintain a tonic inhibitory control over CAP-sensitive nociceptors. When CMH units are exposed to CAP þ OCT þ NAL, unit activity is not different compared to CAP þ OCT, indicating that opioid receptor activation does not contribute to the OCT-induced decrease in CAP excitation.
The expression of TRPV1 has been previously reported in DRG cells, and the percentages determined here (31%) are in general agreement with those previously published (Carlton and Hargett, 2002; Guo et al., 1999; Michael and Priestley, 1999). The co-expression of SSTR and TRPV1 in DRG cells has not been examined previously and the data demonstrate that approximately 60% of L5 DRG cells expressing SSTR2a also express TRPV1. Conversely, 33% of TRPV1-expressing cells also express SSTR2a. As stated above, these are conservative estimates given that 4 additional SSTR subtypes may be expressed in DRG cells. Regardless, the high degree of co-localization of these receptors indicates that SSTRs could maintain significant control over the excitability of TRPV1, a consequence which is borne out in our behavioral and single fiber studies. As additional probes for SSTRs become available, the percentage of SSTR-TRPV1 expressing DRG cells will undoubtedly increase. Peripheral injection of the SSTR agonist OCT results in a significant attenuation of CAP-induced flinching and L/L behaviors. In related studies it has been demonstrated that OCT produces phasic inhibition of formalin-induced flinching and L/L behaviors (Carlton et al., 2001a). Control studies demonstrate that OCT produces its phasic effect through local and not systemic actions since injection of OCT into one hindpaw and CAP into the contralateral hindpaw results in nociceptive behaviors similar to those seen in animals injected with CAP alone. Activation of SSTRs has been previously shown to attenuate thermal responses in vivo (Eschalier et al., 1991) and in vitro (Carlton et al., 2001a), as well as block thermal sensitization following exposure of nociceptors to inflammatory mediators such as bradykinin (Carlton et al., 2001a).
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Mechanical sensitization has also been reduced following SSTR activation (Corsi et al., 1997), however, this action is undoubtedly through modulation of receptors or channels other than TRPV1 since mechanical stimuli do not activate TRPV1. The in vivo results are confirmed in the in vitro recordings from nociceptors in that OCT significantly reduced CAP-induced activity in CMH units. These data corroborate an earlier report that OCT dose-dependently inhibited thermal responses of nociceptors (Carlton et al., 2001a). Thus, nociceptors are subject to phasic control by SSTRs, and this is through modulation of the TRPV1 function. The percentage of CAP-sensitive fibers that was inhibited by OCT was greater that than predicted by the doubling labeling studies presumably because other SSTR subtypes (not localized in the present study) are expressed by CAP-sensitive nociceptors. In addition, the data indicate that when SSTRs are blocked by c-SOM, CAP induces a 3– 4-fold increase in behavioral responses and single unit activity compared to that produced by CAP alone. Thus somatostatin exerts an impressive tonic modulation of TRPV1 receptors. This is in agreement with previous reports demonstrating that an SSTR antagonist dramatically enhances formalin pain behaviors and generates considerable nociceptor activity that can be blocked by SSTR agonists or somatostatin antibody (Carlton et al., 2001b). Based on our anatomical studies, we hypothesize that OCT is acting directly on SSTRs localized on nociceptors. However, it has been reported that somatostatin and its analogues bind to opioid receptors (Gulya et al., 1986; Maurer et al., 1982; Pugsley and Lippmann, 1978; Terenius, 1976), so somatostatin may also activate opioid receptors, which are present on nociceptors (Coggeshall et al., 1997; Stein et al., 1988). For example, NAL, a widely used opioid antagonist, prevented the analgesia that followed intraventricular injection of somatostatin (Rezek et al., 1978). In contrast, NAL did not effect responses of dorsal horn neurons to somatostatin (Randic and Miletic, 1978; Sandkuhler et al., 1990), nor did it reverse the inhibitory effects of OCT on thermal responses of nociceptors (Carlton et al., 2001a). Finally, SSTRs and opioids have been localized on different populations of DRG cells (Taddese et al., 1995). Therefore, it seems most likely that somatostatin and opioids produce their effects through different systems. In the present study NAL did not enhance CAP-induced activity indicating that opioids did not have a tonic control over TRPV1 function. Furthermore, NAL did not alter the OCT-induced inhibition of CAP-induced responses indicating that opioid receptors are not contributing to the SSTR-induced inhibition. There are several lines of evidence indicating that TRPV1 activation plays a key role in inflammatory pain, including neurogenic inflammation, so understanding how this activation can be modulated (both endogenously and exogenously) could have far reaching implications in pain
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management. The TRPV1 receptors are localized on nociceptors and convey sensitivity to capsaicin (CAP), heat and acid (Caterina et al., 1997; Tominaga et al., 1998). Intradermal injection of CAP is exquisitely painful in humans and produces nociceptive behaviors in rats, with a short lasting (, 30 min) inflammatory response characterized by redness, swelling and plasma extravasation. Numbers of TRPV1-expressing axons increase in the periphery 2 days after inflammation and may contribute to peripheral sensitization in the inflamed state (Carlton and Coggeshall, 2001). The percentage of TRPV1-labeled DRG cells also increases following inflammation as do the number of DRG cells double-labeled for TRPV1 and NF200, a marker for cells giving rise to myelinated A fibers (Amaya et al., 2003). A key finding in TRPV1 knock out mice is that, though they still respond to thermal stimuli, they do not develop inflammatory pain behaviors (Caterina et al., 2000; Davis et al., 2000). Activation of CAP-sensitive fibers produces the plasma extravasation and vasodilatation (Jancso´ et al., 1967; Szolcsa´nyi, 1996) that characterizes neurogenic inflammation. Hence, TRPV1 receptors are essential components underlying the tumor (swelling), rubor (redness), dolor (pain) and calor (heat) of inflammation. At this time, we cannot rule out that SSTR-induced decreases in CAP responses might involve Kþchannel opening, and/or Caþ2 channel closing (Koch et al., 1988). However, there are several lines of evidence that all 5 SSTRs are functionally coupled to inhibition of adenylate cyclase (AC) and the cAMP/protein kinase A (PKA) pathway via pertussis toxin sensitive GTP binding proteins (Patel et al., 1995; Tentler et al., 1997), and inhibition of AC is clearly the most prominent transduction pathway used by this family of receptors (Viollet et al., 1995). The inhibitory effect of SST on AC and the cAMP/PKA pathway may be the link that allows SSTRs to modulate TRPV1 function (Bhave et al., 2002; De Petrocellis et al., 2001). Analysis of this interaction will be the subject of future investigations. It has recently been suggested that TRPV1 receptors are under tonic inhibitory control mediated by the membrane lipid phosphatidylinosital-4,5-biphosphate (Chuang et al., 2004). In heterologous systems expressing TRPV1 and bradykinin or track A receptors, exposure of HEK cells to bradykinin or nerve growth factor releases TRPV1 from this inhibition, resulting in enhanced responses to acid and CAP. Hence, TRPV1 may be controlled by several mechanisms which may be differentially affected in various disease states. Compromising or disrupting this tonic inhibition may result in prolonged states of hypersensitivity. In summary, demonstration that the TRPV1 is under both phasic and tonic SSTR regulation provides insight to the cellular mechanisms affecting TRPV1 receptor function and may provide novel approaches for modulation of a receptor family that underlies sensitization of peripheral primary afferent fibers and inflammatory pain.
Acknowledgements The authors thank Vicki Wilson for her excellent secretarial assistance. This work was supported by NIH NS27910 and NS40700 to S.M.C. and NS101621 to R.E.C.
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