Departments of Medicine, Pediatrics, and Bioengineering, University of Illinois at Chicago and Veterans Affairs Chicago Health Care System, Chicago, Illinois 60612
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ABSTRACT |
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The intracellular mechanisms whereby the inhibitory neurotransmitter neuropeptide Y (NPY) decreases ciliary beat frequency (CBF) were investigated in cultured human tracheal and bronchial ciliated cells. CBF was measured by nonstationary analysis laser light scattering. NPY at 1 and 10 µM decreased CBF from a baseline of 6.7 ± 0.5 (n = 12) to 6.1 ± 0.5 (P < 0.05) and 5.8 ± 0.4 (P < 0.01) Hz, respectively. Prior application of PYX-1, an NPY antagonist, prevented the decreases of CBF induced by both doses of NPY. Two broad protein kinase C (PKC) kinase inhibitors, staurosporine and calphostin C, also abolished the NPY-induced decrease in CBF. The NPY-induced decrease in CBF was abolished by GF 109203X, a novel PKC (nPKC) isoform inhibitor, whereas this decrease in CBF was not attenuated by Gö-6976, a specific inhibitor of conventional PKC isoforms. Because pretreatment with NPY did not block the stimulation of CBF by forskolin and pretreatment with forskolin did not abolish the NPY-induced inhibition of CBF, this NPY receptor-mediated signal transduction mechanism appears to be independent of the adenylate cyclase-protein kinase A (PKA) pathway. Inhibition of Ca2+-ATPase by thapsigargin also prevented the suppression of CBF induced by subsequent application of NPY. These novel data indicate that, in cultured human epithelia, NPY decreases CBF below its basal level via the activation of an nPKC isoform and Ca2+-ATPase, independent of the activity of PKA. This is consistent with the proposition that NPY is an autonomic efferent inhibitory neurotransmitter regulating mucociliary transport.
Gö-6976; GF 109203X; neuropeptide Y receptor subtype 1 (Y1); neuropeptide Y receptor subtype 2 (Y2); thapsigargin
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INTRODUCTION |
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IMPAIRED MUCOCILIARY clearance is an inherent pathological characteristic of patients with chronic obstructive lung disease, asthma, and cystic fibrosis. In each of these diseases, autonomic dysfunction of the airways has been implicated. Ciliary beat, the sole propelling mechanism responsible for mucociliary transport, appears to be regulated by the release of excitatory neurotransmitters from autonomic nerves (11, 42). These mediators stimulate ciliary beat frequency (CBF) predominantly via protein kinase A (PKA)-cAMP- or D-myo-inositol 1,4,5-trisphosphate-Ca2+-dependent intracellular pathways. The opposing inhibitory neural regulation of ciliary activity still awaits delineation (40). Neuropeptide Y (NPY), an inhibitory neuromodulator of the autonomic nervous system, has been found to colocalize with norepinephrine in the sympathetic neurons within the mucosa (19). Intra-arterial injection of NPY suppresses mucociliary wave activity in the rabbit maxillary sinus (4, 5). NPY has been shown in human neuroblastoma cell line to inhibit adenylate cyclase (33), to mobilize intracellular Ca2+ (20), and to activate protein kinase C (PKC) (1, 8, 25). Phorbol 12-myristate 13-acetate (PMA), a tumor-promoting factor with well-defined PKC action, has been shown to decrease CBF in cultured ovine (30) and rabbit (16) ciliated cells. Whether NPY decreases CBF by direct activation of PKC or via interactions with adenylate cyclase is unknown. Decreases of CBF either have been associated with hyperpolarization of the cell membrane or a substantial decrease in the basal intracellular Ca2+ concentration ([Ca2+]i) (38). We hypothesize that binding of NPY to NPY receptor subtype 2 (Y2) receptors results in an increase in PKC activity, independent of PKA, which in turn causes the Ca2+-ATPase pump to substantially decrease intracellular Ca2+, resulting in an associated suppression of CBF.
PKC is a ubiquitous kinase consisting of three superfamilies. Most of
these isoforms in many cell types, including human airway smooth muscle
cells and lung tissues (14), have been identified by immunoblot
analysis. The conventional PKC (cPKC) isoforms are Ca2+ and diacylglycerol (DAG)
dependent (,
I,
II, and
) and the novel PKC
(nPKC) isoforms are Ca2+
independent and DAG dependent (
,
, µ,
, and
). Both
subtypes can be activated by PMA. The atypical PKC (aPKC) isoforms are Ca2+ and DAG independent. The
physiological functions of cPKC, nPKC, and aPKC isoforms are known.
Thus we questioned whether any of these isoforms were responsible for
the NPY-induced decrease in CBF.
The data herein demonstrate that the inhibition of CBF induced by NPY was abolished by an NPY antagonist and mimicked by the application of a Y2 agonist but not by a Y1 agonist. This surface receptor-mediated inhibition was dependent on the activation of nPKC isoforms rather than cPKC isoforms and possibly involved the activation of the Ca2+-ATPase pump. This mechanism found in the human airway ciliated cells may be unique, as the NPY-induced inhibition of CBF was completely independent of the adenylate cyclase pathway. Thus this novel intracellular inhibitory pathway appears to be independent of the excitatory adrenergic cAMP-dependent pathway regulating CBF, thereby providing an inhibitory intracellular counterpart for the excitatory messenger-induced intracellular stimulation of CBF.
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METHODS |
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The protocol for obtaining human tracheal and bronchial tissues for research was approved by the Institute Review Board of the University of Illinois at Chicago.
Human Cultured Tracheal and Bronchial Ciliated Cells
A total of eight tracheal or bronchial specimens from persons aged 16-56 yr were obtained within an 8-mo period. Each tracheal or bronchial tissue specimen contained 1-2 cartilage rings. Depending on the size of the tracheal or bronchial tissue, 12-32 ciliated epithelial samples were obtained.The human ciliated cell culture protocol was adapted from the protocol used for culturing bovine ciliated cells described in detail elsewhere (24). Briefly, human tracheal and bronchial segments were obtained either from the Illinois Organ Donor Bank or from tissues of the donor discarded following transplant surgery at University of Illinois at Chicago Hospital. The connective tissues were dissected from the underlying cartilage and the mucosa. The resulting tracheal or bronchial mucosae with the attached cartilage were cut into small pieces of ~20 mm2. They were digested for 11 h at 4°C using 0.1% protease (pronase) in medium 199 (GIBCO BRL, Gaithersburg, MD) containing 1% penicillin-streptomycin. Clusters of ciliated epithelial cells were harvested by gently shaking the mucosae with the cartilage in medium 199 containing 1% penicillin-streptomycin. The epithelial cell clusters were then dispersed in bronchial epithelial cell growth medium supplemented with bovine pituitary extract, penicillin, streptomycin, and fungizone (Clonetics). Approximately 0.5-0.75 ml of the cell suspensions was added to each chamber (Coverglass chamber, Nunc, Naperville, IL), which had been previously coated with collagen (Vitrogen 100, Collagen, Palo Alto, CA). The cells were incubated in air at 37°C and 100% humidity. The experiments reported herein were performed on ciliated epithelial cells cultured for 4-18 days.
Measurements of CBF in Tracheal Epithelial Cells
The nonstationary laser light scattering technique for measuring CBF described elsewhere (6) was adapted for in vitro experiments. Briefly, the culture chamber was placed on an inverted microscope stage. An attenuated He-Ne laser beam (Experimental Protocols
Eleven experimental conditions were performed at room temperature using 4- to 18-day-old cultures. Each experimental set consisted of at least seven experiments. Ciliated cells exhibiting vigorous beating were selected for the study. To ensure that the tracheal and bronchial specimens were responsive to NPY, at least one sample from each of the eight tracheal and bronchial specimens was used to confirm the CBF inhibitory responses to NPY. The protocols were designed according to the following four experimental aims.Determination of the responses of CBF to exogenous NPY and the NPY
receptor subtypes involved.
After the measurement of baseline CBF for 5 min, either Hanks'
balanced salt solution (HBSS) or 10 µM PYX-1 (Peninsula), a broad NPY
receptor antagonist, was added to the ciliated cells 5 min before the
cumulative applications of 1 and 10 µM human NPY (Peninsula). The NPY
receptor subtype responsible for the inhibition of CBF was delineated
by administration of specific Y1
and Y2 agonists.
[Leu31,
Pro34]-NPY (Peninsula) at 1 and 10 µM was used to activate the
Y1 receptors, whereas NPY-(1636)
(Peninsular) at 1 and 10 µM was used to activate the
Y2 receptors.
Determination of the role of PKC and of PKC isoforms in the NPY-induced inhibition of CBF. The role of PKC in the NPY-induced responses of CBF was determined by four PKC inhibitors with different specificities, namely, 10 µM staurosporine, 10 µM calphostin C, 1 µM Gö-6976, and 10 µM GF 109203X. These four agents were obtained from Calbiochem. After the measurement of baseline CBF for a minimum of 5 min, one of these PKC inhibitors was applied to the ciliated cells before the cumulative applications of 1 and 10 µM NPY 5 min apart.
Determination of the role of adenylate cyclase in the NPY-induced decrease in CBF. Using a similar experimental design, the role of adenylate cyclase in the NPY-induced responses of CBF was determined in two experiments in which forskolin at 10 µM (Research Biochemicals International) was added either before or after the application of 10 µM NPY.
Determination of the role of Ca2+-ATPase in the NPY-induced decrease in CBF. Similarly, the role of Ca2+-ATPase in the NPY-induced inhibition of CBF was determined by applying 10 µM thapsigargin (Research Biochemicals International) 30 min before the cumulative applications of 1 and 10 µM NPY.
All mean (mean of means) data are expressed as arithmetic means ± SE. Geometric means are provided as necessary. ANOVA was applied to the raw data to determine the statistical significance of each experimental condition. ![]() |
RESULTS |
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Basal CBF of Human Tracheal Cultured Ciliated Cells
After 11 h of enzymatic digestion, layers of ciliated epithelial cells were harvested by gently shaking the mucosae in medium 199. When these epithelial cells were placed in the collagen-coated chamber, they attached to the collagen matrix within 24 h. Ciliated cells at the periphery of the epithelia began to migrate on the second day. They gradually dispersed to form a confluent monolayer of epithelial cells, an observation consistent with other reports of mammalian ciliated cell cultures (10). Vigorous beating of the cilia was observed for longer than 4 wk.Basal CBF of cultured human ciliated cells exhibited little
variability. The arithmetic mean of means of the basal CBF in 105 samples was 6.0 ± 0.2 Hz. There were no significant differences in
the intersample measurements (P > 0.006). These nonsignificant differences among tissues
were independent of the donor (P > 0.0065, regression analysis with donor tissues coded orthogonally).
These data imply that the cilia of these cultured human ciliated cells were beating at their intrinsic, basal autorhythmic rate (38). The
nature of the distribution of the basal rate of human CBF was
determined by grouping together all the individual measurements (n = 18,336) made in the 105 samples.
The distribution revealed in the 20-bin histogram (Fig.
1A)
was skewed to the right, possibly due to the transient spontaneous
oscillations of CBF (the high CBF measurements) observed in cultures
(29). A 20-bin histogram analysis of the log-transformed CBF (Fig.
1B) and
2 best-of-fit test
(
2 = 0.01) confirmed that CBF
values were log-normally distributed. This distribution had a mean CBF
of 5.9 ± 0.03 Hz, a median CBF of 4.9 Hz, and a geometric mean CBF
of 5.0 Hz.
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The temporal responses of CBF to the various agents used in the following series of experiments are first discussed with representative examples in each experimental condition. The arithmetic mean data of each experimental condition are then presented.
Responses of Exogenous NPY-Induced Decrease in CBF: Determination of the Receptor Subtypes
As expected, application of HBSS (control) did not change CBF compared with the baseline. In this example (Fig. 2A), subsequent application of 1 µM NPY immediately decreased CBF from a mean baseline of 5.0 ± 0.1 to 3.9 ± 0.2 Hz (means of all data in the corresponding measurement period). CBF did not return to baseline at the end of the measurement period. The second application of NPY at 10 µM decreased CBF to 4.0 ± 0.2 Hz. These inhibitory responses were reproducible in 12 of the 12 experiments.
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After pretreatment of the ciliated cells with 10 µM PYX-1, an NPY receptor antagonist, the NPY-induced decrease in CBF was attenuated (Fig. 2B). PYX-1 by itself did not induce any changes in CBF.
To determine the specific NPY receptor subtypes responsible for the
decrease in CBF induced by human NPY, specific
Y1 and Y2 agonists were used to mimic
the NPY-induced responses. Neither dose of
[Leu31,
Pro34]-NPY, a
Y1 agonist, caused any change in
CBF (Fig. 2C). NPY-(1636), a
Y2 agonist, induced characteristic
decreases of CBF similar to those of human NPY in terms of both the
temporal nature and the magnitude of the responses (Fig.
2D). In this example, the arithmetic
mean CBF of 4.8 ± 0.01 Hz was decreased by 1 and 10 µM
NPY-(16
36) to 4.3 ± 0.1 and 4.6 ± 0.1 Hz, respectively.
The arithmetic mean of means of CBF of all the samples of
each experimental condition (Fig. 3)
demonstrated a trend similar to the individual examples shown in Fig.
2,
A-D.
NPY at 1 and 10 µM decreased CBF from a baseline of 6.7 ± 0.5 to
6.1 ± 0.5 (P < 0.05) and 5.8 ± 0.4 Hz (P < 0.01),
respectively (unbalanced ANOVA compared with baseline using the raw
data). Such decreases were completely prevented by prior application of
PYX-1. PYX-1 by itself did not change CBF compared with HBSS, nor was
there any difference compared with the overall baselines.
[Leu31,
Pro34]-NPY had no effect on
the mean CBF derived from eight samples. CBF were decreased by 1 and 10 µM NPY(1636) from 5.2 ± 0.4 to 4.7 ± 0.3 and 4.6 ± 0.3 Hz, respectively. These decreases of CBF were comparable to those
induced by NPY.
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Responses of NPY-Induced Decrease in CBF: Role of PKC
Staurosporine at 10 µM, a nonspecific PKC inhibitor (Fig. 4A), caused a slight increase in CBF in some samples (~1 Hz). The stimulatory responses of CBF induced by staurosporine appear to be consistent with its potential to inhibit other protein kinases as well as to induce transient increases of [Ca2+]i at high concentrations. Thus it is likely that the stimulation of CBF observed in this example was due to an increase in [Ca2+]i. Staurosporine prevented NPY from inducing a decrease in CBF. These data are consistent with our hypothesis that the NPY-induced suppression of CBF was mediated through a PKC-dependent mechanism.
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To confirm that the NPY-induced inhibition of CBF was due to the activation of PKC, a more specific PKC inhibitor, calphostin C, was used. Calphostin C competes at the DAG and PMA binding site of the cysteine-rich regulatory domain of the PKC. Ten micromolar calphostin C (Fig. 4B) by itself did not change CBF. Calphostin C predictably and completely abolished the NPY-induced decrease of CBF. This provides additional evidence that the activation of PKC was involved in the NPY-induced suppression of CBF.
To determine which PKC isoforms were responsible for the observed NPY-induced, PKC-dependent inhibition of CBF, two additional experiments were performed using specific cPKC and nPKC isoform inhibitors. Gö-6976, a cPKC isoform inhibitor (13), was used to determine whether NPY-induced inhibition of CBF was mediated through the Ca2+ and DAG-dependent cPKC isoforms. As is evident in the example in Fig. 4C, the decreases of CBF induced by the subsequent applications of NPY were preserved. The mean CBF values were 4.3 ± 0.1 Hz (means of all data from 700 to 1,050 s) and 3.5 ± 0.05 Hz (mean of all data from 1,100 to 1,600 s), respectively, for 1 and 10 µM NPY compared with the basal mean CBF of 4.7 ± 0.1 Hz (mean of all data from 1 to 300 s).
A structural analog of staurosporine, GF 109203X (bisindolylmaleimide I), was used to determine whether the NPY-induced inhibition of CBF was mediated through the Ca2+-independent and DAG-dependent nPKC isoforms. GF 109203X, a purported inhibitor of the nPKC isoforms, was designed to bind to the ATP site at the catalytic domain of the PKC. Pretreatment of the cells with GF 109203X prevented any decrease in CBF by subsequent applications of NPY (Fig. 4D). These differential data derived from pretreatment with Gö-6976 and GF 109203X suggest that different PKC isoforms have different physiological roles in mammalian ciliated cells. This provides an alternate explanation for the observation that PMA can elicit different intracellular actions (21).
The mean CBF values for all samples in each experimental condition (Fig. 5) were consistent with the examples shown in Fig. 4, A-D. None of the four PKC inhibitors had any effect on the mean basal CBF. The NPY-induced decreases of CBF were abolished by three of four PKC inhibitors, namely staurosporine, calphostin C, and GF 109203X. In the presence of the specific cPKC inhibitor Gö-6976, NPY at 1 and 10 µM significantly inhibited CBF from a baseline of 5.7 ± 0.3 to 5.1 ± 0.4 (P < 0.05) and 4.6 ± 0.3 Hz (P < 0.05), respectively (Fig. 5). These data suggest that NPY inhibited CBF via an nPKC rather than the cPKC pathway.
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Responses of NPY-Induced Decreases of CBF: Role of Adenylate Cyclase.
NPY has been shown to decrease adenylate cyclase activity in some cell types (33). To ascertain that NPY-induced inhibition of CBF is independent of the adenylate cyclase pathway, two experiments were performed: 1) pretreatment of the ciliated cells with 10 µM forskolin before the application of 10 µM NPY and 2) determination of the effect of 10 µM forskolin following the application of 10 µM NPY.Application of 10 µM forskolin caused an immediate and sustained stimulation of CBF (Fig. 6A). CBF was increased from a mean baseline of 3.7 ± 0.04 Hz (mean of all data from 1 to 900 s) to 5.2 ± 0.05 Hz (mean of all data from 1,000 to 2,000 s) following the application of forskolin. Subsequent application of 10 µM NPY caused an immediate decrease in the forskolin-stimulated CBF to 4.7 ± 0.03 Hz (mean of all data from 2,000 to 3,000 s), a value lower than the forskolin-stimulated CBF but higher than the baseline. To ensure that the preactivation of adenylate cyclase did not interfere with the binding of NPY, in a separate experiment, CBF was suppressed by prior administration of NPY (Fig. 6B). Ten micromolar NPY predictably decreased CBF from a baseline of 6.7 ± 0.1 Hz (mean of all data from 1 to 480 s) to 4.7 ± 0.06 Hz (mean of all data from 500 to 1,200 s). Subsequent application of forskolin to this preparation immediately elevated CBF to 7.2 ± 0.1 Hz (mean of all data from 1,250 to 1,700 s).
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The mean data of these two experiments are shown in Fig. 7. In the experiments in which forskolin was applied before the administration of NPY, 10 µM forskolin increased the mean CBF from a baseline of 4.9 ± 0.4 to 6.2 ± 0.3 Hz (P < 0.01). This elevated CBF decreased to 5.8 ± 0.3 Hz following subsequent application of 10 µM NPY (P < 0.05 compared with forskolin stimulation). When the experimental sequence was reversed, the initial application of NPY decreased CBF from a baseline of 7.7 ± 1.2 to 6.6 ± 0.7 Hz (P < 0.01). The subsequent application of 10 µM forskolin elevated the NPY-induced decrease in CBF to 7.3 ± 0.5 Hz (P < 0.05 compared with NPY inhibition). The NPY-induced decrease in CBF was preserved, independent of the presence of forskolin. Similarly, the forskolin-induced stimulation of CBF was also preserved, independent of the presence of NPY. This indicates that the intracellular mechanisms transducing the NPY-induced suppression of CBF were independent of the intracellular mechanisms transducing the stimulation of CBF via adenylate cyclase-PKA-dependent mechanisms.
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Responses of NPY-Induced Decrease in CBF: Role of Ca2+-ATPase
Activation of PKC has been speculated to be associated with the regulation of [Ca2+]i via the endoplasmic Ca2+-ATPase pumps. This association could either be an upregulation of the Ca2+-ATPase pumps leading to a decrease in [Ca2+]i or a downregulation of the Ca2+-ATPase pump leading to an increase in [Ca2+]i. When ciliated cells were pretreated with thapsigargin, a specific inhibitor of endoplasmic Ca2+-ATPase, a transient increase in CBF from a baseline of 4.8 Hz to a peak of 20 Hz was observed (Fig. 8). CBF returned to near baseline levels before the subsequent administration of NPY. In this example, neither of the subsequently applied doses of NPY caused CBF to decrease below baseline levels. These data suggest that NPY-induced suppression of CBF is caused by a decrease in [Ca2+]i resulting from an induced increase in activity of the Ca2+-ATPase pump.
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Thapsigargin (10 µM) caused a transient stimulation of CBF and totally abolished any NPY-induced decrease in CBF (Fig. 8). The mean CBF was stimulated from a baseline of 5.4 ± 0.4 to 6.5 ± 0.5 Hz (P < 0.01) in eight samples (Fig. 9). The stimulation of CBF compared with HBSS is consistent with the inhibition of the sequestration of [Ca2+]i by intracellular organelles by thapsigargin resulting in an elevated [Ca2+]i (28). The thapsigargin-induced increase in CBF (Fig. 9) was inhibited by the subsequent application of 1 and 10 µM NPY to 5.8 ± 0.5 and 5.9 ± 0.3 Hz, significantly lower than the thapsigargin-induced responses (P < 0.05) but higher than the baseline CBF.
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To further demonstrate that the NPY-induced decrease in CBF was
completely independent of the PKA pathways, three additional studies
were performed. They were 1)
inhibition of the PKA pathway using H-8
(n = 3),
2) inhibition of the PKA pathway
using HA-1004 (n = 4), and
3) inhibition of the PKA-dependent
nitric oxide (n = 3) pathway using 10 µM nitro-L-arginine methyl ester
(L-NAME). H-8 at 10 µM
[inhibition constant
(Ki) = 1.2 µM
for PKA inhibition] did not change basal CBF. In these tissues,
subsequent applications of the two doses of NPY suppressed CBF from a
mean baseline of 7.6 ± 0.6 to 7.0 ± 0.9 and 6.9 ± 0.6 Hz,
respectively. HA-1004 at 10 µM
(Ki = 2.3 µM
for PKA inhibition) also by itself did not change CBF. Predictably,
subsequent applications of 1 and 10 µM NPY decreased CBF from a mean
baseline of 7.2 ± 0.5 to 5.9 ± 0.5 and 6.7 ± 0.6 Hz,
respectively. Nitric oxide has been suggested to be a common messenger
whereby 2-adrenergic (17) and
inflammatory cytokines (18) stimulate CBF. Although it is unlikely that
the stimulatory nitric oxide pathway was involved with the
PKA-independent NPY inhibition of CBF,
L-NAME was used to inhibit
nitric oxide synthase. L-NAME at
10 µM did not have any effect on CBF by itself. Again, in the
presence of L-NAME, 1 and 10 µM NPY predictably decreased CBF from a mean baseline of 5.8 ± 0.5 to 5.03 ± 0.2 and 5.04 ± 0.7 Hz, respectively. The lack of
effect of H-8, HA-1004 (9), and
L-NAME (17) on the basal CBF is
consistent with previously reported measurements in mammalian ciliated
cells (16). These data further confirm that the NPY-induced suppression
of CBF was completely independent of the PKA pathways. Because H-8 also
inhibits the guanylate cyclase-protein kinase G (PKG) pathway, it is
reasonable to expect that the PKG pathway, which has been previously
implicated in regulation of CBF (12), was also not involved with the
NPY-induced inhibition of CBF.
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DISCUSSION |
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The intracellular mechanisms whereby NPY suppressed CBF in human ciliated cells below baseline can be explained by the following proposed model. There are specific Y2 receptors on the airway epithelial ciliated cells. Binding of NPY to Y2 receptor activates nPKC, which increases the activities of the Ca2+-ATPase pumps. This in turn causes a decrease in [Ca2+]i to a very low level. This decrease in [Ca2+]i is associated with a decrease in CBF. This model of the NPY pathway appears to be unique, as it activates a specific Ca2+-independent but DAG-dependent nPKC isoform that operates independently from the adenylate cyclase pathway.
Human CBF Measurements
Using a modified organ culturing technique, we obtained abundant human ciliated cells. These cells were stabilized in culture for a minimum of 4 days using a well-defined culture medium specifically developed for human tracheal and bronchial cultures (Clonetics specifications sheet). The advantage of this preparation was evidenced by the maintenance of the synchronous beating of the cilia for longer than 4 wk. The small intra- and intervariations among the ~20,000 baseline measurements obtained from 105 samples of CBF derived from 8 tissue specimens imply that the ciliated cells were operating using their intrinsic autorhythmic basal regulatory mechanism (38) rather than being in a stimulated state. Measurements of CBF from freshly harvested human epithelial cells obtained from nasal, tracheal, and bronchial brushing (9, 28, 39) were higher than the 6 Hz reported herein. CBF in brushings ranged between 11 and 14 Hz, with little variation among the samples derived from either the upper or the lower airways of the trachea and major bronchi (39). Our measurements in cultured human ciliated cells closely resemble recent reports on CBF made in cultured human epithelial cells under similar conditions (9).The log-normal distribution of CBF (Fig. 1) is consistent with the distribution of tracheal mucus transport rates in humans, which are also log-normally distributed (41). These distributions likely represent the outward expression of the intrinsic regulatory mechanisms controlling ciliary activity and mucus production so as to maintain effective mucociliary transport while having the ability to respond to an inhaled insult to effect its rapid removal.
NPY-Induced Signal Transduction Mechanisms
The decrease in CBF induced by NPY through Y2 receptors was ~20% less than the baseline levels. This change in magnitude is similar to that observed in cultures at room temperature following activation by other agents (9). At 37°C, the basal CBF, being twice that at room temperature, may undergo a greater decrease in CBF than that observed herein. It has been claimed that changes in ciliary beat cause a fourfold change in mucoiliary transport (32). However, in vivo CBF oscillates (42). How NPY reduces these oscillations is presently unknown. Mucociliary transport is dependent on both ciliary beat and mucus secretion. NPY has been shown to suppress serous cell secretion rather than the low basal level of mucus secretion (23). However, in association with norepinephrine, NPY inhibits mucus secretion (36). Whether NPY and norepinephrine together cause a greater decrease in CBF awaits further studies. Because NPY and norepinephrine are colocalized in the sympathetic nerves (19), it would be reasonable that, together, these agents would decrease CBF and mucus production in a commensurate manner.Signal transduction mechanisms induced by NPY have been mostly derived from neuronal tissues. In these excitable tissues, activation of Y1 receptors appears to inhibit the generation of cAMP and mobilization of intracellular Ca2+ (25). The resulting increase in [Ca2+]i has been suggested to activate nifedipine-sensitive L-type voltage-operated channels (3). In neuronal tissues, Y2 receptor activation has been reported to be linked to a decrease in cAMP and G protein activation, leading to an increase in [Ca2+]i (25), activation of Ca2+-sensitive BK type K+ channels (20), and consequent hyperpolarization of the cell membrane. Y3 receptors have been linked to the activation of a neuronal, nicotinic ACh current by a G protein-mediated mechanism dependent on a decrease in cAMP (27).
Activation of NPY receptors in nonneuronal, nonexcitable tissues appears to involve different cellular mechanisms. For example, NPY decreases transepithelial ion transport in the gut and intestinal epithelia (7, 26). In a chromaffin cell line, PC-12 (1), and in macrophages (8), NPY action is tightly coupled with a PKC mechanism. Although investigations into the differentiation of NPY subtype function in airway epithelia have been limited, it appears that Y2 receptor activation plays a predominant role in slowing down the mucociliary transport system (4). The data described herein are not only consistent with these findings, they are also consistent with the regulatory mechanisms of mammalian ciliary activity in the following aspects. The NPY-associated suppression of ciliary activity shown herein was induced via an adenylate cyclase-PKA-independent mechanism. An increase in cAMP has been associated with an increase in CBF (35, 38). It is notable, however, that a decrease in cAMP has not been shown to decrease CBF below baseline levels. Thus it is considered unlikely that the decrease in CBF induced by the activation of Y2 receptors in airway epithelia is mediated in a manner similar to that of the neuronal tissues, i.e., via reciprocal interactions between cAMP and Ca2+. In those tissues, a decrease in cAMP was coupled with a concomitant increase in [Ca2+]i. However, in airway epithelia, an increase in [Ca2+]i has been associated with an increase in CBF (35, 38). Thus the signal transduction mechanisms whereby NPY decreases CBF do not utilize the PKA inhibitory mechanism commonly employed in the excitable tissues to regulate their inhibitory cell functions.
A decrease in intracellular
[Cl]i
has been shown to be associated with an increase in CBF (38).
Activation of PKC by phorbol ester (PMA) in tracheal epithelia
increases short-circuit current (2, 37), predominantly due to
Cl
secretion. PKC has also
been shown to phosphorylate the cystic fibrosis transmembrane
conductance regulator Cl
channel (22). Of the three different PKC isoform superfamilies, with
the exception of the aPKC in which the activation mechanism is unknown,
the activity of the membrane-associated PKC is potentiated by DAG.
These isoforms appear to be species and tissue specific. They may also
play different physiological roles. Thus it is possible that activation
of the cPKC isoform causes
Cl
efflux and increases
CBF, whereas activation of nPKC by agents such as NPY decreases CBF as
demonstrated herein.
We have employed CBF as a functional assay to determine specifically the role of nPKC upon activation of Y2 receptors. We used multiple PKC inhibitors to confirm the observed mechanisms and have performed preliminary studies to determine the optimal dose of the PKC inhibitors. The selected physiological doses appear to be higher than the doses commonly used in immunoblotting analysis for PKC. However, at these concentrations, none of the PKC inhibitors had any consequential effect on the basal CBF. This is consistent with the perception that dose-response curves differ depending on the assay and tissue or cell preparation under investigation. The dose-response curves and PKC isoform characterization studies are difficult to pursue due to the restricted availability of human tissues. An alternate approach is to passage and redifferentiate human tracheal or bronchial epithelial cell lines in such a way that the ciliogenesis mechanism is reactivated. This pioneering study opens this possibility for further characterization of this novel pathway.
Some PKC activators have been shown to stimulate ciliary activity in frog palate ciliated cells (21). The discrepancies of the PMA effect on CBF in mammals and amphibians could be due to either the activation of different PKC isoforms or phylogenetic differences. Of all the signal transduction mechanisms involved with the regulation of mammalian CBF, as yet only agents activating the PKC-dependent pathway have been demonstrated to cause a decrease in CBF (16, 30).
It is notable that, with the exceptions of major basic protein derived from sensitized eosinophils, endogenous atrial natriuretic factor, and intrinsic bacterial products such as procanin and 1-hydroxyphenazine, virtually all known neurotransmitters and intrinsic mediators either stimulate CBF or have little effect (35). Such mediators include autonomic neural agonists, nonadrenergic noncholinergic neuropeptides, purinergic agonists, and humoral and inflammatory metabolites. Thus the novel finding that NPY acts directly on ciliated cells to decrease CBF may suggest its potential for producing the impaired mucociliary function in persons with diseases of the airways.
In conclusion, NPY decreased CBF in human ciliated cells. In contrast to other cell types, in which NPY elicit the cellular responses by interacting with the adenylate cyclase complex or by activating the basolateral Ca2+-dependent K+ channels, the decrease in CBF induced by NPY in airway epithelium, thus far, appears to contain a unique mechanism in that the data reported herein indicate that NPY decreases CBF through an nPKC-dependent pathway. The specific cellular mechanisms elucidated could comprise an effector motor response resulting from the release of NPY from sympathetic inhibitory neural pathways (19, 34, 40).
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ACKNOWLEDGEMENTS |
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We thank Dr. W. T. Vigneswaran for obtaining the human tracheal tissues and Dr. F. J. Al-Bazzaz for sharing some of the human tracheal and bronchial tissues.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-46376 and the Medical Service of the Department of Veterans Affairs.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and present address of L. B. Wong: LiDon Technologies LLC, 2201 W. Campbell Park Dr., Chicago, IL 60612.
Received 26 February 1998; accepted in final form 6 May 1998.
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