HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/10/1831 01-0018fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by TEN BROEKE, R. Articles by NIJKAMP, F. P. Search for Related Content PubMed PubMed Citation Articles by TEN BROEKE, R. Articles by NIJKAMP, F. P.

Calcium sensors as new therapeutic targets for airway hyperresponsiveness and asthma¹

ROBERT TEN BROEKE^*, GERT FOLKERTS^*2, THEA LEUSINK-MUIS^*, HENK J. VAN DER LINDE^*, MATTEO VILLAIN^,, MICHAEL K. MANION, FRED DE CLERCK^*,¶, J. EDWIN BLALOCK and FRANS P. NIJKAMP^*

^* Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht, The Netherlands;
Department of Physiology and Biophysics, Center for Neuroimmunology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA;
Department of Medicinal Biochemistry, University of Geneva, Geneva, Switzerland; and
^¶ Janssen Research Foundation, Beerse, Belgium

²Correspondence: Faculty of Pharmacy, Department of Pharmacology and Pathophysiology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. E-mail: G.Folkerts{at}pharm.uu.nl.

SPECIFIC AIMS

In the present study, we used two calcium-like peptides (CALP1 and CALP2) to test the hypothesis that calcium channels in the epithelium play a role in regulating airway responsiveness by controlling [Ca²⁺]_i and, consequently, modulating the activity of constitutive NOS (cNOS). In vitro effects on airway responsiveness of both peptides, which have the necessary characteristics to define the role of calcium in airway hyperresponsiveness and serve as prototypical new therapeutic agents that target calcium sensors, were determined and the mechanism of action was elucidated by measuring [Ca²⁺]_i and NO production by epithelial cells.

PRINCIPAL FINDINGS

1. CALP1 but not CALP2 increases airway responsiveness to histamine in vitro
The effects of CALP on airway responsiveness in vitro to histamine were investigated in guinea pig tracheas, which can be stimulated on either the inside (effect on epithelium) or outside (direct effect on smooth muscle). CALP1 (10 µM) and CALP2 (30 µM) were applied intraluminally; after 10 min incubation, a histamine concentration-response curve was made. Neither of the two peptides had an effect on basal tone. However, CALP1 shifted the histamine concentration-response curve significantly upward (P<0.01, Fig. 1A ). Incubation with CALP2, however, had no effect on the histamine-induced contractions (Fig. 1A ). CALP2 (applied 5 min before CALP1 administration) completely blocked the CALP1-induced airway hyperresponsiveness (Fig. 1A ). To investigate the role of the epithelium, the same experiments were performed with epithelium-denuded tracheas. Histamine concentration-response curves made with these preparations demonstrated a significant enhancement of the maximal responses vs. those of intact controls due to the absence of the epithelium. The pD₂ value was increased from 4.2 ± 0.2 for controls to 5.5 ± 0.2 for denuded tracheas. Neither CALP1 nor CALP2 changed the airway responsiveness when applied to the inside of epithelium-denuded tracheas (Fig. 1B ). No difference in pD₂ values was observed between groups. Thus, in epithelium-denuded tracheas, CALP1 did not further increase the contractions elicited by histamine or change the sensitivity (pD₂) to histamine. Although CALP2 could prevent the CALP1-induced airway hyperresponsiveness, CALP2 did not alter the increased tracheal contraction in epithelium-denuded tissues. These results show that CALP1 and CALP2 modulate airway responsiveness epithelium dependently.

View larger version (16K): [in this window] [in a new window]   Figure 1. Concentration-response curves of intraluminal administered histamine made on isolated perfused guinea pig tracheal tubes. A) The histamine concentration-response curve was significantly (**P<0.01) shifted upward after intraluminal administration of CALP1 (•). Incubation with CALP2 had no effect on the histamine-induced smooth muscle contraction (), but CALP2 prevented the CALP1-induced airway hyperresponsiveness to histamine (, ##P<0.01 vs. CALP1 alone). B) The histamine concentration-response curve was shifted upward in tracheas denuded of epithelium (). Intraluminal administration of neither CALP1 (•) nor CALP2 () influenced the smooth muscle contraction to histamine. Data are presented as mean ± SE, n = 6. **,##P < 0.01 two-way ANOVA.

2. CALP2 but not CALP1 increases [Ca²⁺]_i and NO production in epithelial cells
It was reported recently that CALP1 inhibits calcium-permeable channels and that CALP can modulate [Ca²⁺]_i. Since CALP acts selectively on the epithelium, we examined the effects of CALP on [Ca²⁺]_i in epithelial cells. The basal [Ca²⁺]_i in epithelial cells was 22.5 ± 0.7 nM. CALP1 incubation (100 µM) had no effect on basal [Ca²⁺]_i (22.6±0.4 nM), whereas CALP2 (100 µM) increased the [Ca²⁺]_i (27.5±0.2 nM). These results show that CALP2 increases [Ca²⁺]_i in epithelial cells.

Produced by the calcium-dependent NO synthase in the epithelium, NO regulates smooth muscle tone. Since the effects of CALP on airway responsiveness were calcium and epithelium dependent, we investigated the role of NO in the responses to CALP in epithelial cells. Cells were stimulated with control solution, CALP1 (100 µM), or CALP2 (100 µM) and NO production was measured. Basal NO production was 15.2 ± 5.2 pmol. Incubation with CALP1 had almost no effect on NO production (21.8±4.6 pmol) whereas CALP2 markedly increased NO production (79.0±6.5 pmol). This demonstrates that incubation with CALP2, but not CALP1, enhances spontaneous NO production by epithelial cells. These results are consistent with our hypothesis that airway responsiveness is modulated by CALP effects on NO production by epithelial cells.

3. CALP1 decreases and CALP2 increases agonist-induced increase in [Ca²⁺]_i in epithelial cells
To further study the mode of action, the effects of CALP on agonist-induced increases in [Ca²⁺]_i were measured. Stimulation with bradykinin (BK, 30 µM) and acetylcholine (Ach, 30 µM) resulted in a biphasic increase in [Ca²⁺]_i, with a rapid initial increase lasting 1 min and a second phase leading to a sustained plateau. Stimulation with BK or Ach increased [Ca²⁺]_i in epithelial cells (Fig. 2A , B ). The sustained levels after stimulation were 3.3 ± 0.1 nM (BK, Fig. 2B ) and 24.9 ± 1.0 nM (Ach, Fig. 2D ), respectively. In CALP1-incubated cells, the BK- and Ach-induced increases in [Ca²⁺]_i were decreased. The initial peak was lower (Fig. 2A , C ), with no effect on the sustained plateau (Fig. 2B , D ). In CALP2-incubated cells, however, the initial peak was slightly higher (Fig. 2A , C ) and the sustained level of calcium in the cell (Fig. 2B , D ) after stimulation with BK and Ach was markedly higher. These results show that CALP1 decreases only the peak value of agonist-induced calcium influx, whereas CALP2 increases both the initial peak and the steady-state [Ca²⁺]_i after stimulation with agonists. The increase with CALP2 in combination with BK or Ach was higher than with CALP2 alone; therefore, CALP2 increases [Ca²⁺]_i further after stimulation with agonists.

View larger version (19K): [in this window] [in a new window]   Figure 2. Increase in [Ca2+]i in NCI-H292 bronchial epithelial cells after stimulation with bradykinin (BK) or acetylcholine (Ach). Before stimulation, cells were incubated with control solution (white bar), CALP1 (hatched bar), or CALP2 (black bar). See text for explanation. Data are presented as mean ± SE, n = 5. Similar results were seen on 3 separate days. ***P < 0.001 Student’s unpaired t test.

CONCLUSIONS

Whether calcium-dependent mechanisms have a role in airway responsiveness (and particularly in asthma) is largely unknown. We investigated these processes using two novel peptides, CALP1 and CALP2, which interact with calcium binding EF hand motifs and regulate calcium channels. We found that CALP1 caused an epithelium-dependent airway hyperresponsiveness in response to histamine in vitro. CALP2 had no effect on airway responsiveness, but blocked CALP1-induced airway hyperresponsiveness.

Since the degree of airway hyperresponsiveness correlates with the severity of the asthma, a better understanding of the pathogenesis of airway hyperresponsiveness has clinical implications. The finding that CALP1 and CALP2 modulate airway responsiveness could contribute significantly to resolving the pathology of airway hyperresponsiveness. It is tempting to speculate about the mechanism of action of CALP in modulation of airway responsiveness. Both peptides act specifically on the epithelium. This could mean that the targets of CALP are only in the epithelium and not in smooth muscle cells. This suggests, however, that targets other than CaM, which is present in both epithelial and smooth muscle cells, play a role in the CALP responses. It is likely that this difference between the two cell types arises from different types of calcium channels.

Villain et al. recently described the effects of CALP on CaM and found that CALP1 activates CaM-dependent phosphodiesterase activity and that CALP2 inhibited phosphodiesterase activity. More recently, Manion et al. showed that CALP1 blocked glutamate receptor channels in neurons via CaM. In addition, CALP1 directly blocked a nonselective cation channel in Jurkat T cells. Consequently, CALP1 decreased apoptosis in the two cell types. Calcium inhibits the activity of a number of calcium-permeable channels via a negative feedback mechanism; it was concluded therefore that CALP1 blocked calcium influx and apoptosis through inhibition of calcium channel opening. Although CaM is involved in these processes, the latter suggests an indirect effect of CALP1 through the modulation of calcium concentrations in the cell. Recently, it was found that L-type calcium channels, which show calcium-sensitive inactivation, have EF hand and CaM binding motifs on the cytoplasmic side of the channel. When intracellular calcium binds these sites, it causes the channel to close or inactivate. L2 alveolar epithelial cells, HT29 epithelial cells and primary rabbit airway epithelial cells express voltage-dependent (L-type) calcium channels. If CALP influences calcium-permeable channels in epithelial cells, CALP should be able to enter the cell. Using a fluoresceinated derivative of CALP1, we observed that the peptide crosses the cell membrane and accumulates in the cytoplasm of epithelial cells.

The effects of CALP on [Ca²⁺]_i in airway epithelial cells were investigated and the results showed that agonist-induced increases in [Ca²⁺]_i were decreased by CALP1 and increased by CALP2. Therefore, we hypothesize that CALP1, via the inhibition of calcium influx through calcium channels in epithelial cells, decreases [Ca²⁺]_i. CALP2, however, opens calcium channels (or prevents the negative feedback of calcium, which results in closing of these channels) and increases [Ca²⁺]_i. This altered [Ca²⁺]_i due to CALP may influence the release of epithelium-derived relaxing factors, such as NO (Fig. 3 ).

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized action of CALP1 and CALP2 on airway responsiveness in the guinea pig trachea. See text for explanation. R, receptor; C, calcium channel; EC, epithelial cell; SM, smooth muscle.

NO was first discovered as a regulator of blood vessel tone, but it is now known that this molecule also modulates airway smooth muscle tone. Previously, we showed the importance of NO in guinea pig trachea and in an animal model of asthma. The NO is formed via the calcium-dependent, constitutive form of NOS, which is present in airway epithelium of humans and animals. Stimulation with agonists is followed by an increase in [Ca²⁺]_i in both smooth muscle cells and epithelial cells. This increase leads to contraction of smooth muscle; however, the increased [Ca²⁺]_i in the epithelial cell stimulates the production of epithelium-derived relaxing factors (NO) by the epithelium. NO relaxes airway smooth muscle cells indirectly through stimulation of guanylyl cyclase, which results in an increase in cGMP in smooth muscle, and directly through activation of K_ca channels in airway smooth muscle cells. In the case of NO deficiency (e.g., epithelial damage or dysfunction), this route of antagonism is disrupted, resulting in airway hyperresponsiveness. It is likely that in diseases such as asthma, the calcium-permeable channels in epithelial cells malfunction, which results in less release of epithelium-derived relaxing factors.

Thus, we hypothesized that CALP1 closes EF hand-containing calcium channels in epithelial cells and, as a result, inhibits calcium influx into epithelial cells. This decrease in [Ca²⁺]_i might lead to NO deficiency in epithelial cells, followed by airway hyperresponsiveness to agonists. However, CALP2 opens the EF hand-containing calcium channels and thereby increases [Ca²⁺]_i in epithelial cells, resulting in the production of NO (Fig. 3) . Results of experiments showing that CALP1 incubation has no effect on NO production and that CALP2 incubation spontaneously leads to NO production by guinea pig airway epithelial cells make it likely that such a mechanism exists.

In conclusion, we have found that changes in calcium concentrations in epithelial cells, leading to altered release of NO, play a key role in airway hyperresponsiveness. Thus, novel therapeutic approaches, such as the use of CALP2, bring new insights into the pathogenesis and treatment of airway hyperresponsiveness and asthma by targeting calcium sensors as regulators of calcium channels in epithelial cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0018fje ; to cite this article, use FASEB J. (June 8, 2001) 10.1096/fj.01-0018fje

This article has been cited by other articles:

				F. L. M. Ricciardolo, P. J. Sterk, B. Gaston, and G. Folkerts Nitric Oxide in Health and Disease of the Respiratory System Physiol Rev, July 1, 2004; 84(3): 731 - 765. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/10/1831 01-0018fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by TEN BROEKE, R. Articles by NIJKAMP, F. P. Search for Related Content PubMed PubMed Citation Articles by TEN BROEKE, R. Articles by NIJKAMP, F. P.