Cyclic ADP-ribose, a putative Ca²⁺-mobilizing second messenger, operates in submucosal gland acinar cells

Abstract

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.

Flagellin/TLR5 signaling potentiates airway serous secretion from swine tracheal submucosal glands

“…]i. Moreover, we found that physiologically relevant, basic secretions from swine tracheal submucosal glands could be triggered by ϳ30 nM of ACh (16,17,31,38,39,44,45). The viability of all tested cells was confirmed by the occurrence of ionic responses after the subsequent addition of 30 nM of ACh.…”

“…The isolated glands were further dispersed enzymatically by incubating them with enzyme solution containing collagenase (200 U/ml) and DL-DTT (0.31 mg/ml) for 30 min at 37°C. After dispersion and a wash with centrifugation at 180 g, the cells were resuspended in a standard extracellular solution until use, as reported previously (16,17,31,38,39,44,45). In a series of experiments investigating the effect of blocking antibodies, anti-TLR 5 polyclonal antibody (10 g/ml) or nonspecific IgG (10 g/ml) was also preincubated with cells for 1 h at 37°C before use.…”

“…Membrane currents were monitored at two different holding potentials (Hps), i.e., 0 and Ϫ80 mV, which roughly corresponded to the Cl Ϫ and K ϩ equilibrium potential. The double current monitoring, i.e., alternate recording of the ionic currents corresponding to Hp of 0 and Ϫ80 mV, was accomplished by applying 200-ms voltage pulses of Ϫ80 mV at a frequency of 2 Hz to the pipette voltage of 0 mV (16,17,31,38,39,44,45 (16,17,31,38,39,44,45). The solutions included an extracellular (bath) solution of 120 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl 2, 1.2 mM CaCl2, 10 mM glucose, and 10 mM HEPES and an intracellular (pipette) solution of 120 mM KCl, 1.13 mM MgCl 2, 0.5 mM EGTA, 1 mM Na2ATP, 10 mM glucose, and 10 mM HEPES.…”

“…It has recently become known that CaCC composes a newly identified transmembrane protein with unknown function, 16, Ano 1 (TMEM16A), which exists in both the airway surface epithelium and submucosal glands (28,33). Concerning the electrophysiological characteristics of CaCC, we have reported that tyrosine kinase, cyclic-ADP ribose, and nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (cGK) are involved in the intracellular pathway of oscillatory ionic currents evoked by low doses of acetylcholine (ACh) (18,39,44).Toll-like receptors (TLRs) are known to be key proteins that recognize distinct pathogen-associated molecular patterns (PAMPs) and activate innate immune responses as well as antigen-specific adaptive immunity (1,22). It is also known that airway epithelial cells express several kinds of TLRs and that the activation of TLRs on epithelial cells induces the production of several cytokines, chemokines, and antimicrobial peptides (11,12,40).…”

“…It has recently become known that CaCC composes a newly identified transmembrane protein with unknown function, 16, Ano 1 (TMEM16A), which exists in both the airway surface epithelium and submucosal glands (28,33). Concerning the electrophysiological characteristics of CaCC, we have reported that tyrosine kinase, cyclic-ADP ribose, and nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (cGK) are involved in the intracellular pathway of oscillatory ionic currents evoked by low doses of acetylcholine (ACh) (18,39,44).…”