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Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle¹

Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, USA; and
^{* 2}Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, USA

²Correspondence: Department of Pharmacology, Medical School, University of Minnesota, 312 Church St. SE, Minneapolis, MN 55455, USA. E-mail: walseth{at}mail.ahc.umn.edu

SPECIFIC AIM

How cADPR regulates intracellular Ca²⁺ ([Ca²⁺]_i) levels in response to agonist stimulation in airway smooth muscle is poorly understood as are the mechanisms through which CD38 may be regulated in response to agonist stimulation to bring about cADPR-induced [Ca²⁺]_i mobilization. The present study was designed to characterize the role of the CD38/cADPR signaling pathway in Ca²⁺ mobilization under stimulation by different agonists in airway smooth muscle.

PRINCIPAL FINDINGS

1. [Ca²⁺]_i mobilization through the CD38/cADPR signaling pathway is agonist specific in airway smooth muscle
We examined the effects of 8Br-cADPR in TSM cells stimulated with various agonists to determine the contribution of the CD38/cADPR pathway on the [Ca²⁺]_i responses to each. TSM cells were preincubated with HBSS (control) or 100 µM 8Br-cADPR in HBSS for 15 min, perfused with "0" Ca HBSS, and stimulated with one of the following: 100 nM ACh, 1 µM ACh, 100 nM ET-1, or 50 µM histamine. The net peak [Ca²⁺]_i responses to both 100 nM and 1 µM ACh as well as 100 nM ET-1 were significantly attenuated whereas those to 50 µM histamine were not (Fig. 1 ). Attenuated [Ca²⁺]_i responses to 100 nM ACh, 1 µM ACh, and 100 nM ET-1 were 74, 66, and 79% of controls, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 1. [Ca2+]i responses to 100 nM ACh (A, n=914 for control and 770 for 8Br-cADPR), 1 µM ACh (B, n=290 for control and 245 for 8Br-cADPR), 100 nM ET-1 (C, n=361 for control and 501 for 8Br-cADPR), or 50 µM histamine (D, n=100 for control and 107 for 8Br-cADPR) in ASM cells and effects of 100 µM 8Br-cADPR. Values are shown as net peak [Ca2+]i responses. Note significant attenuation of [Ca2+]i responses by 8Br-cADPR to ACh and ET-1 but not histamine. Results depicted as mean ± SE. *Significant difference compared with controls (P<0.0001).

2. Recruitment of the CD38/cADPR signaling pathway under muscarinic receptor activation results from stimulation of G_i-type G-proteins
The effects of muscarinic receptor subtype-specific antagonists on [Ca²⁺]_i responses to ACh were examined, as were effects of 8Br-cADPR on the residual [Ca²⁺]_i responses. TSM cells preexposed to the M₃ selective antagonist 4-diphenylacetoxy-N-(2-chloroethyl)piperidine (4-DAMP, 100 nM) for 15 min were subsequently perfused with "0" Ca HBSS and exposed to 1 µM ACh in the continued presence of 100 nM 4-DAMP. ACh stimulation did not elicit an [Ca²⁺]_i response in these cells (data not shown). Cells were preexposed 15 min to methoctramine, an M₂ selective antagonist, perfused with "0" Ca HBSS, and stimulated with 100 nM or 1 µM ACh in the continued presence of 100 nM methoctramine. In the presence of methoctramine, [Ca²⁺]_i responses to 100 nM or 1 µM ACh were 76 and 87% of controls, respectively (Fig. 2 A, B). To examine the effects of 8Br-cADPR on the portion of the [Ca²⁺]_i responses not sensitive to methoctramine, cells were preincubated with 100 nM methoctramine and 100 µM 8Br-cADPR for 15 min, then stimulated with 1 µM or 100 nM ACh in the continued presence of methoctramine. 8Br-cADPR had no additional effect on [Ca²⁺]_i responses to either 100 nM or 1 µM ACh (Fig. 2A, B ). In cells stimulated with 1 µM ACh, the inhibitory effect of 8Br-cADPR alone was greater than the effects of both methoctramine alone or 8Br-cADPR and methoctramine combined (Fig. 2B ). This was not the case in responses to 100 nM ACh, where responses under all three conditions were similarly attenuated (Fig. 2A ).

View larger version (15K): [in this window] [in a new window]   Figure 2. Effect of methoctramine (M2), an M2 selective muscarinic receptor antagonist, on the [Ca2+]i response to ACh. 100 nM methoctramine attenuated the [Ca2+]i responses to 100 nM ACh (A, n=262, 120, 136, and 117 for control, M2, M2+8Br, and 8Br, respectively), and 1 µM ACh (B, n=191, 138, 145, and 152 for control, M2, M2+8Br, and 8Br, respectively); there was no additional attenuation by 8Br-cADPR (M2+8Br). Results shown as mean ± SE. *Significant difference compared with control (P<0.05).

CONCLUSIONS AND SIGNIFICANCE

Ca²⁺ mobilization by cADPR in a variety of cell types occurs through activation of ryanodine receptor (RyR) Ca²⁺ channels of the sarcoplasmic reticulum and is mechanistically independent of Ca²⁺ mobilization by IP₃. Modulators of RyR channels such as ryanodine, caffeine, and ruthenium red also affect cADPR mediated Ca²⁺ release. The cADPR signaling pathway has been implicated in a variety of cellular processes including secretion, immune cell regulation, cardiac function, cell proliferation, neuronal transmission, and myometrial contraction. In airway smooth muscle, previous studies have shown that cADPR induces [Ca²⁺]_i release and demonstrated a role for cADPR in muscarinic responses. Studies in other cell types have suggested that the CD38/cADPR signaling pathway results from G-protein activation. Understanding the mechanisms surrounding recruitment of the CD38/cADPR pathway in airway smooth muscle may have implications in conditions involving altered Ca²⁺ regulation such as asthma.

In this study, we provide evidence that the CD38/cADPR signaling pathway of [Ca²⁺]_i mobilization occurs in an agonist-specific manner in airway smooth muscle. [Ca²⁺]_i responses to ACh and ET-1 use cADPR to elicit a portion of the response, whereas histamine responses appear to be independent of the CD38/cADPR signaling pathway. The finding that cADPR is involved in the [Ca²⁺]_i responses to ACh and ET-1 is strengthened by the observation that responses to ACh and ET-1 are sensitive to ryanodine receptor blockade whereas those to histamine are not. We have also shown that recruitment of the CD38/cADPR pathway under muscarinic receptor activation occurs in a receptor subtype-specific manner. cADPR-mediated [Ca²⁺]_i responses to ACh stimulation occurs through activation of the M₂ muscarinic receptor subtype, as the responses sensitive to methoctramine are not further sensitive to 8Br-cADPR. M₂ muscarinic receptors are known to couple to G_i-type G-proteins, as are ET-1 receptors. These data suggest that utilization of the CD38/cADPR signaling pathway is coupled to G_i-type G-proteins.

The agonist specificity in recruiting the CD38/cADPR signaling pathway indicates complex mechanisms surrounding [Ca²⁺]_i regulation in airway smooth muscle. We propose a model of [Ca²⁺]_i mobilization in response to different agonists using a combination of different signaling pathways as well as different receptor subtypes using distinct signaling pathways in airway smooth muscle (Fig. 3 ). Responses to histamine are predominantly through activation of G_q-type G-proteins that couple to PLC-ß, resulting in IP₃ production and Ca²⁺ mobilization from IP₃ receptor channels. ACh responses result from activation of M₂ and M₃ muscarinic receptors, which couple to G_i and G_q-type G-proteins, respectively. The coupling under M₂ receptor activation is to CD38, resulting in cADPR production and release from RyR channels. M₃ receptor activation couples to PLC-ß, IP₃ production, and Ca²⁺ release from IP₃ receptor channels. Based on our findings and those of other investigators, we propose that stimulation by ET-1 results in activation of ETA and ETB receptors, which both couple to CD38, resulting in cADPR production and Ca²⁺ release from RyR channels, whereas ETA receptors also couple to PLC-ß, resulting in IP₃ production and release from IP₃ receptor channels.

View larger version (28K): [in this window] [in a new window]   Figure 3. Model depicting the CD38/cADPR signaling pathway of intracellular Ca2+ regulation in airway smooth muscle. In airway smooth muscle, recruitment of the CD38/cADPR signaling pathway is agonist specific. Activation of histamine receptors, M3 muscarinic receptors (M3AChR) or ETA receptors coupled to Gq-type G-proteins results in activation of PLC and IP3 production leading to Ca2+ release through IP3R. ETA, ETB, and M2AChR activation coupled to Gi-type G-proteins results in stimulation of CD38, with subsequent production of cADPR and Ca2+ release through RyR. 8Br-cADPR inhibits Ca2+ release through RyR.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0622fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0622fje

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