Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky
Submitted 12 April 2005 ; accepted in final form 15 July 2005
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ABSTRACT |
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gastrin-releasing peptide; pulmonary stress; small cell lung cancer; vagal sensory neuron
Most sensory inputs arising from lung structures are conducted in vagus nerves and their branches (10, 22). Cell bodies of these sensory nerves reside in nodose and intracranial jugular ganglia. Morphological evidence has shown that 75% of vagal pulmonary afferent nerves are nonmyelinated (C-) fibers (2). Activation of these C-fiber sensory endings by chemical irritants is known to elicit pulmonary chemoreflex (apnea, bradycardia, and hypotension), cough, chest pain, bronchospasm, and hypersecretion of mucus (10, 22). Furthermore, a number of neuropeptides, such as tachykinins and calcitonin gene-related peptide (CGRP), are synthesized in the cell bodies of these C-fiber afferents and released from nerve-endings in the airways upon activation. Tachykinins and CGRP are known to have potent effects on a number of target cells in the airways (e.g., airway smooth muscle, mast cells, and others). Thus sustained stimulation of these sensory endings can also induce neurogenic inflammation in the airways (5, 34). More importantly, recent studies have clearly demonstrated that pulmonary epithelial neuroendocrine cells, the precursor cells of SCLC, are extensively innervated by branches of these sensory endings expressing high levels of tachykinins (1, 7). However, the potential effects of these peptides on the vagal pulmonary C-fiber endings have not been investigated.
In light of the existing knowledge described above and the information that is currently lacking, the present study was carried out to investigate 1) whether intravenous infusion of bombesin or GRP enhances the pulmonary chemoreflex sensitivity in intact rats and, if so, 2) whether the effect of these peptides is caused by a direct sensitization of the vagal pulmonary C-fiber afferents. The excitability of these isolated sensory neurons was determined by using the fura-2-based Ca2+ imaging and the whole cell patch-clamp recording techniques.
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MATERIALS AND METHODS |
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Measurements of Cardiorespiratory Responses
Sprague-Dawley rats (360420 g) were initially anesthetized with an intraperitoneal injection of -chloralose (100 mg/kg) and urethane (500 mg/kg) dissolved in a 2% borax solution; supplemental doses of
-chloralose (
10 mg/kg/h) and urethane (
50 mg/kg/h) were injected intravenously to maintain abolition of pain reflexes elicited by paw pinch. The right femoral artery, the left jugular vein, and the right femoral vein were cannulated for recording arterial blood pressure (ABP) and injection and infusion of various chemical agents, respectively. The jugular venous catheter was advanced until its tip was positioned just above the right atrium. The volume of each bolus injection was 0.1 ml, which was first injected into the catheter (
0.2 ml) and then flushed into the circulation by an injection of 0.3 ml of saline. A short tracheal cannula was inserted after a tracheotomy, and tracheal pressure was measured (Validyne MP 45-28) via a side port of the tracheal cannula. Body temperature was maintained at
36°C by means of a heating pad placed under the animal lying in a supine position. At the end of the experiment, the animal was killed by an injection of KCl (200 mg/kg iv).
Rats breathed spontaneously via the tracheal cannula. Respiratory flow was measured with a heated pneumotachograph and a differential pressure transducer (Validyne MP45-14) and was integrated to give tidal volume (VT). Respiratory frequency (f), expiratory duration, VT, minute ventilation, ABP, and heart rate were analyzed (Biocybernetics TS-100) on a breath-by-breath basis by using an online computer as described previously (23).
Identification and Culture of Vagal Pulmonary Neurons
Sensory neurons innervating the lungs and airways were identified by retrograde labeling from the lungs by using the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as described previously (20). Young adult Sprague-Dawley rats (150200 g) were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and intubated with a polyethylene catheter (PE-150) with its tip positioned in the trachea above the thoracic inlet. DiI was initially sonicated and dissolved in ethanol, diluted in saline (1% ethanol, vol/vol), and then instilled into the lungs (0.2 mg/ml; 0.2 ml x 2) with the animal's head tilted up at 30°.
After 711 days, an interval previously determined to be sufficient for the dye to diffuse to the cell body, the animals were anesthetized with halothane inhalation and decapitated. The head was immediately immersed in ice-cold Hanks' balanced salt solution. Nodose and jugular ganglia were extracted and digested as described previously (20). Briefly, each ganglion was desheathed, cut into 10 pieces, placed in 0.125% type IV collagenase, and incubated for 1 h in 5% CO2 in air at 37°C. The ganglion suspension was centrifuged (150 g, 5 min) and supernatant-aspirated. The cell pellet was resuspended in 0.05% trypsin and 0.53 mM EDTA in Hanks' balanced salt solution for 5 min and centrifuged (150 g, 5 min); the pellet was then resuspended in a modified DMEM/F-12 solution [DMEM/F-12 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µM MEM nonessential amino acids] and gently triturated with a small-bore fire-polished Pasteur pipette. The dispersed cell suspension was centrifuged (500 g, 8 min) through a layer of 15% bovine serum albumin to separate the cells from the myelin debris. The pellets were resuspended in the modified DMEM/F-12 solution supplemented with 50 ng/ml 2.5S nerve growth factor, plated onto poly-L-lysine-coated glass coverslips, and then incubated overnight (5% CO2 in air at 37°C).
Calcium Imaging
Intracellular Ca2+ was monitored using the fluorescent Ca2+ indicator fura-2 AM (14). Cells were loaded with 5 µM fura-2 AM for 30 min at 37°C, then rinsed (3 times) with extracellular solution (ECS; containing in mM: 136 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES, pH at 7.4) and allowed to deesterify for at least 30 min before use. Ratiometric Ca2+ imaging was performed using a Zeiss fluorescence inverted microscope equipped with a variable filter wheel (Sutter Instruments, Novato, CA) and digital charge-coupled device camera (Princeton Instruments, Trenton, NJ). Dual images (340- and 380-nm excitation, 510-nm emission) were collected, and pseudocolor ratiometric images were monitored during the experiments by using the software Axon Imaging Workbench (Axon Instruments, Union City, CA). The imaging system was standardized with a two-point calibration, using a Ca2+-free standard and a Ca2+-saturated standard. Both standards contained 11 µM fura-2 [44 µl of 10 mM fura-2 Penta K+ salt, 8 ml of 20 mM HEPES-Na (pH 7.4), and 32 ml H2O] and were prepared as follows: standard, 18 ml of fura-2 and 1.98 ml of 10 mM EGTA-Na (pH 7.6); + standard, 18 ml of fura-2 and 1.98 ml of 10 mM CaCl2. The parameters used for the two-point calibration include the dissociation constant of fura-2 (Kd; 225 nM), the ratio values for the Ca2+-free and -saturated concentration standards (Rmin and Rmax, respectively), and the fluorescence intensity at 380-nm excitation for the Ca2+-free and -saturated concentration standards (Denmin and Denmax, respectively). Intracellular Ca2+ concentration ([Ca2+]i, in nM) was calculated according to the following equation: [Ca2+]i = Kd(R Rmin)/(Rmax R)(Denmin/Denmax). Typical Rmin, Rmax, and Denmin/Denmax values were 0.59, 2.54, and 2.27, respectively.
Electrophysiology
Whole cell perforated patch-clamp recording was performed as previously described (20). In short, the coverslip containing the attached cells was placed in the center of a small (0.2 ml) recording chamber that was perfused by gravity feed (VC-6 perfusion valve controller; Warner Instruments, Hamden, CT) with ECS and ECS containing bombesin or GRP at 2 ml/min. The chemical stimulants (capsaicin and ATP) were applied by a pressure-driven drug delivery system (ALA-VM8; ALA Scientific Instruments, Westbury, NY), with its tip positioned to ensure that the cell was fully within the stream of the injectate. The intracellular solution contained (in mM) 92 potassium gluconate, 40 KCl, 8 NaCl, 1 CaCl2, 0.5 MgCl2, 10 EGTA, and 10 HEPES, pH at 7.2. Recordings were made in the whole cell perforated-patch configuration (50 µg/ml gramicidin) using Axopatch 200B/pCLAMP 9.0 (Axon Instruments). The experiments were performed at room temperature (22°C). The data were acquired at 5 kHz and filtered at 2 kHz. Series resistance was compensated at
80%. The resting membrane potential was held at 70 mV.
Experimental Protocols
Four series of experiments were carried out.
Study series 1. The effects of bombesin and GRP on pulmonary chemoreflex sensitivity were determined in anesthetized, spontaneously breathing rats. Response to right atrial bolus injection of capsaicin (0.25 or 0.5 µg/kg), a potent and selective stimulant of C-fibers, was tested before, during, and 15 min after a 2-min constant infusion of bombesin (1 µg·kg1·min1; iv) or GRP (3 µg·kg1·min1 iv). The effects of bombesin and GRP on the response to ATP (0.2 or 0.25 mg/kg iv), another selective stimulant of C-fiber afferents, were also determined in a separate group of animals.
Study series 2. The role of vagal C-fiber afferents in the effects of bombesin and GRP on the pulmonary chemoreflex sensitivity was evaluated after perineural capsaicin treatment (PNCT) of both vagi, which produced a selective and reversible blockade of the C-fiber conduction in the vagus nerve. PNCT was applied as previously described (23). Briefly, cotton strips soaked in capsaicin solution (250 µg/ml) were wrapped around a 2- to 3-mm segment of the isolated cervical vagi for 20 min and then removed. The success of PNCT was verified in each animal by the abolition of capsaicin (1 µg/kg iv)-induced reflex responses.
Study series 3.
To determine whether the capsaicin-induced Ca2+ transient in pulmonary chemosensitive neurons was altered by the presence of bombesin or GRP, we determined the Ca2+ transient evoked by capsaicin (30 or 50 nM; 15 s) before, during, and 15 min after a pretreatment with bombesin (1 nM; 5 min) or GRP (3 nM; 5 min).
Study series 4. To investigate the effects of bombesin and GRP on the sensitivity of isolated pulmonary chemosensitive neurons, we determined capsaicin (0.3 or 1 µM; 14 s)-induced whole cell inward current before, during, and 1560 min after a pretreatment with bombesin (10 nM; 5 min) or GRP (30 nM; 5 min). The effect of bombesin on pulmonary sensory neurons responses to ATP (0.3 or 1 µM; 24 s) and current injection (10-pA increments from 0 to 100 PA; 230-s duration) were also determined in separate groups of neurons.
Chemicals. Fura-2 AM and DiI were purchased from Molecular Probes (Eugene, OR). DMEM/F-12, trypsin-EDTA, and 2.5S nerve growth factor were obtained from Invitrogen (Carlsbad, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Stock solution of capsaicin (1 mM) was prepared in a vehicle of 1% Tween 80, 1% ethanol, and 98% saline, and that of ATP (10 mM) was prepared in saline. Stock solutions of GRP (0.1 mM) and bombesin (1 mM) were both prepared in distilled H2O and kept in small aliquots at 80°C. The solutions of these chemicals at desired concentrations were prepared daily by dilution with saline on the basis of the animal's body weight in study series 1 and 2 or with ECS in study series 3 and 4. No detectable effect of the vehicles of bombesin and GRP was found in our preliminary experiments.
Statistical analysis. Data were analyzed using one-way analysis of variance (ANOVA). When ANOVA showed a significant interaction, pairwise comparisons were made with post hoc analysis (Fisher's least significant difference). Results were considered significant when P < 0.05. Data are means ± SE.
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RESULTS |
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Infusion of either bombesin (1 µg·kg1·min1 iv; 2 min) or GRP (3 µg·kg1·min1 iv; 2 min) caused an irregular, rapid, and shallow breathing and mild decreases in ABP and heart rate in the majority (24/29) of the animals studied (Fig. 1, B and E). During bombesin infusion, the respiratory depressor response to the same near-threshold dose of capsaicin (0.25 or 0.5 µg/kg) injected into the right atrium as a bolus was markedly augmented (Fig. 1, AC; Fig. 2, A and B); for example, the apneic ratio (apneic duration/average baseline expiratory duration) was increased significantly from 1.69 ± 0.23 at control to 3.88 ± 0.37 during the bombesin infusion (P < 0.05, n = 17) (Fig. 2B). The potentiating effect of bombesin was reversible in 15 min (apneic ratio: 1.47 ± 0.18). Similarly, infusion of GRP also significantly enhanced the response to the same dose of capsaicin (apneic ratio: 1.60 ± 0.23 at control, 3.41 ± 0.49 during GRP; P < 0.05, n = 7) (Fig. 1, DF; Fig. 2, C and D).
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Perineural capsaicin treatment (250 µg/ml; 20 min) of both cervical vagi caused only slight but not significant increases in baseline f, VT, ABP, and heart rate. However, it almost completely abolished the effect of a 2-min infusion of bombesin (n = 8) or GRP (n = 7) on the baseline breathing pattern (Fig. 4, B and E). In addition, the treatment completely blocked the potentiation of the pulmonary chemoreflex response to capsaicin (0.5 µg/kg iv) induced by infusion of either bombesin or GRP (Fig. 4).
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To identify the sensory neurons that give rise to vagal pulmonary C-fibers mediating the pulmonary chemoreflexes, we selected neurons isolated from nodose and jugular ganglia based on the following criteria for this and the next study series: 1) labeled with DiI, 2) diameter <30 µm, and 3) responding to capsaicin (20). Data obtained from nodose and jugular neurons were pooled because no detectable difference was found between them. Application of capsaicin (30 or 50 nM; 15 s) evoked a reversible Ca2+ transient in these small- to medium-size pulmonary sensory neurons. A 5-min treatment with either bombesin (1 nM) or GRP (3 nM) caused no detectable Ca2+ transient in these neurons (Fig. 5, A and C). However, during bombesin treatment, the peak response of the capsaicin-evoked Ca2+ transient was significantly increased from 65.6 ± 11.4 nM at control to 109.3 ± 21.7 nM during bombesin (P < 0.05, n = 13) (Fig. 5, A and B). This augmented response to capsaicin gradually declined but remained higher than control after washout for 15 min (81.6 ± 12.3 nM). Similarly, pretreatment with GRP also significantly enhanced the capsaicin-evoked Ca2+ transient (55.7 ± 6.0 nM at control, 147.1 ± 50.7 nM during GRP, 120.1 ± 39.5 nM after washout; P < 0.05, n = 7) (Fig. 5, C and D).
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In voltage-clamp mode, capsaicin (0.3 or 1 µM; 14 s) evoked a mild whole cell inward current in small- to medium-size (11.529.2 pF) pulmonary sensory neurons (Fig. 6A). Perfusion of bombesin (10 nM; 5 min) or GRP (30 nM; 5 min) alone failed to stimulate these sensory neurons. However, during the bombesin treatment, the response to the same concentration of capsaicin was markedly enhanced (Fig. 6, A and B); e.g., the capsaicin-evoked whole cell inward current density increased significantly from 10.4 ± 2.4 pA/pF at control to 20.8 ± 5.3 pA/pF during bombesin (P < 0.05, n = 9), which gradually declined after washout for 1560 min (14.1 ± 3.2 pA/pF). In a separate group of pulmonary sensory neurons, pretreatment with GRP also significantly potentiated the inward current induced by capsaicin (15.2 ± 2.9 pA/pF at control to 22.2 ± 5.3 pA/pF during GRP; P < 0.05, n = 6) (Fig. 6, C and D).
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DISCUSSION |
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Bombesin is a 14-amino acid bioactive peptide first isolated from frog skin. One of its major mammalian homologs, namely, BLPs, is GRP, a 27-amino acid peptide that is most closely related to bombesin, sharing all but 1 of the last 10 amino acids at the COOH-terminal end (13). BLPs have been shown to contribute to diverse biological functions in the central nervous system and peripheral tissues, including regulation of homeostasis, body temperature, metabolism, and behavior, and secretion of gastrointestinal, adrenal, and pituitary hormones (15, 19). These peptides also have important developmental and growth effects. They are potent mitogens for SCLC cells as well as normal bronchial epithelial cells (3, 11, 19, 33). On the other hand, SCLC cells are known to synthesize and secret GRP and other BLPs (11, 31, 36), and GRP has been suggested as a good serum marker of human SCLC (25). Indeed, BLPs have been considered as the prototypical autocrine growth factors in SCLC (15, 24). We have now, for the first time, demonstrated that GRP and bombesin enhance the sensitivity of pulmonary chemoreflex in vivo and the excitability of isolated pulmonary chemosensitive neurons in vitro. These sensory endings are known to release tachykinins upon stimulation. Substance P, the major type of tachykinin in human lung, has been shown to induce rapid mobilization of Ca2+ from internal stores of SCLC cell lines and to promote clonal growth of these cells (12, 21, 35). On the other hand, (D-Arg1,D-Trp5,7,9,Leu11)substance P, a potent antagonist of the tachykinin receptor, can exert a pronounced inhibitory action on both tumor growth in vivo and signal transduction pathways of SCLC cells in vitro (21, 32, 35). Together, the information described above suggests that the effects of BLPs on these sensory endings may be involved in the deteriorating process of this aggressive lung cancer, although direct evidence has not been established.
Immunohistochemical studies have shown that vagal C-fiber afferents innervate all levels of the respiratory tract from trachea to alveoli in various mammalian species (1, 4, 7). When these C-fiber endings are activated, action potentials conducted through the vagus nerves to the central nervous system elicit the pulmonary chemoreflex response and other cardiorespiratory reflex responses, such as bronchoconstriction, mucus hypersecretion, bronchial vasodilation, plasma extravasation, dyspneic sensation, and cough (10, 22). Although we did not attempt to measure these airway responses in the present study, they are known to be mediated through both the cholinergic reflex pathways and local axon reflex; the latter involves a release of tachykinins and CGRP from these sensory endings (5, 10, 22, 34). It is well documented that bronchopulmonary C-fibers can be stimulated by various endogenous substances such as H+, anandamide, and serotonin in normal lungs (9, 22); some of these substances are known to be secreted at high concentrations during airway inflammation. Furthermore, these C-fiber afferents can be activated by various inhaled irritants such as cigarette smoke, SO2, ozone, and acid aerosol and also by lung expansion (10, 22); expansion of the lung is a normal stimulus and can occur in many physiological conditions (e.g., increased tidal volume during exercise, sigh). Under normal conditions, these stimuli (endogenous chemicals, inhaled irritants, and lung expansion) may not generate a significant stimulatory effect on bronchopulmonary C-fibers. However, when the excitability of these afferent endings is enhanced by BLPs released locally from SCLC cells in the lungs, their stimulation thresholds will be lowered and they can, therefore, be activated by these stimuli, which may consequently contribute, at least in part, to the pathogenesis of pulmonary stresses in SCLC patients.
Pulmonary C-fibers are generally known to possess polymodal sensitivities (9, 10, 22). The two chemical stimulants applied in this study, capsaicin and ATP, are known to activate two different ligand-gated ion channels: transient receptor potential vanilloid 1 (TRPV1) and P2X3 purinoceptor, respectively (9, 14). Current injections stimulate these sensory neurons because of the activation of various voltage-dependent ion channels (9, 20). The possible mechanism underlying the enhanced excitability of pulmonary C neurons after bombesin and GRP pretreatment was not determined in the present study. However, it is known that the effect of bombesin and related peptides is mediated by three major subtypes of the mammalian bombesin receptors, designated BB1 (neuromedin B preferring), BB2 (GRP preferring), and BB3 (orphan bombesin receptor subtype-3) (13, 19). Although the expression of these bombesin receptors on the soma or terminals of pulmonary C-fiber afferents has not been documented, significant levels of GRP specific binding, as well as mRNA as determined by RT-PCR for all three mammalian bombesin receptors, were detected in normal human lung tissue (17, 26). All bombesin receptors are known to be G protein-coupled receptors that are coupled via Gq/11 to activation of phospholipase C and subsequently activate protein kinase C. The latter has been demonstrated to enhance the neuronal excitability by increasing the phosphorylation of various ion channels such as the TRPV1 (28, 37) and TTX-resistance Na+ channel (18, 29). In addition, stimulation of bombesin receptors is believed to be linked to the activation of cytosolic phospholipase A2, release of arachidonic acid, and production of prostaglandin E2 (19, 31). It is well documented that prostaglandin E2 enhances the excitabilities of pulmonary C-fiber afferents in vivo and isolated pulmonary vagal sensory neurons in vitro through the cAMP/protein kinase A transduction cascade (14, 16, 20).
Two different experimental approaches were employed in this study: pulmonary C-fiber chemoreflexes in vivo and isolated pulmonary chemosensitive neurons in vitro. Our results showed that intravenous infusion of bombesin or GRP elicited a rapid, shallow, and irregular breathing, accompanied by mild decreases in ABP and heart rate in the majority of the animals studied (e.g., Fig. 1, B and E), indicating the stimulation of pulmonary C-fibers. However, pretreatment with bombesin or GRP alone did not cause any detectable inward current in patch-clamp recording or any Ca2+ transient in the imaging study (e.g., Fig. 5, A and C). The discrepancy of the stimulatory effect of these two peptides between the in vivo and in vitro experiments was probably related to the different sensitivities of these two preparations (afferent nerve endings vs. the neuronal soma) and/or the different doses of bombesin/GRP applied in these two approaches. Nevertheless, the ratio of the effective doses between bombesin and GRP (1:3) was consistent in all of the study series, suggesting that the same subtype(s) of bombesin receptors was probably involved.
In summary, our results demonstrate that bombesin and GRP upregulate the pulmonary chemoreflex sensitivity in vivo and the excitability of isolated pulmonary capsaicin-sensitive neurons in vitro. These results indicate that BLPs, one of the major autocrine factors in SCLC, may enhance the sensitivity of pulmonary chemosensitive nerve endings and contribute to the development of the pulmonary stresses in SCLC patients.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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