Prolonged increase in ciliary beat frequency after short-term purinergic stimulation in human airway epithelial cells

Abstract

Stimulation of ovine airway epithelial cells with 10 microM ATP for 1 min at 25 degrees C transiently increased both cytoplasmic calcium (fura-2 epifluorescence microscopy) and ciliary beat frequency (CBF; differential interference contrast microscopy) with a similar time course. Identical purinergic stimulation of human airway epithelial cells at 25 or 35 degrees C, however, lead to an increase in CBF that outlasted the calcium transient at least 20 min. While a nitric oxide synthase inhibitor had no effect, pre-treatment of human cells with inhibitors of cAMP-dependent kinase (PKA), 10 microM myristoylated PKA-inhibitory peptide and 1 microM KT-5720, as well as an inhibitor of adenylyl cyclase, 1 mM SQ22536, blocked the prolonged, but not calcium-coupled CBF increase. Addition of PKA inhibitors after purinergic stimulation only partially reduced CBF from its elevated plateau. Prolonged CBF increases did not depend on adenosine production as 10 microM UTP had an effect similar to ATP and 8-sulphophenyl-theophylline did not block them. After increasing human CBF in a PKA-dependent manner to a stable plateau with forskolin (10 microM), ATP caused only a transient, calcium-coupled CBF increase. Calcium transients were necessary for both short-term and prolonged CBF changes as ATP failed to produce CBF increases after emptying calcium stores with 1 microM thapsigargin. These data suggest that in human, but not ovine airway epithelial cells, ATP-induced calcium transients activate a signalling cascade including adenylyl cyclase and PKA. The resulting prolonged CBF stimulation does not rely only on PKA activity, suggesting that the decay of CBF is influenced by ciliary phosphatase activity.

Figure 1. CBF and [Ca²⁺]_i responses to short-term ATP exposure in ovine and to short-term ATP or UTP exposure in human tracheal epithelial cells

Simultaneous CBF and [Ca²⁺]_i recordings of single tracheal epithelial cells loaded with fura-2. [Ca²⁺]_i is represented by the original fura-2 ratiometric data obtained from its fluorescence emissions at excitations of 340 and 380 nm. A, representative recording from an ovine airway epithelial cell reveals a strictly calcium-coupled, transient increase in CBF upon short-term stimulation with ATP. B, recording from a representative human airway epithelial cell shows an initial calcium-coupled, and later prolonged increase in CBF upon short-term exposure to ATP. C, similar to ATP, a representative human airway epithelial cell responds with an initial calcium-coupled, and later prolonged increase in CBF upon short-term exposure to UTP.

Figure 11. Influence of late PKG inhibition with KT-5823 and late PKA inhibition with PKI_14–22 on CBF and [Ca²⁺]_i responses to short-term ATP exposure in human tracheal epithelial cells

A and B, the [Ca²⁺]_i and CBF responses to short-term ATP exposure of representative submerged human tracheal epithelial cells (date- and culture-matched) are shown. PKG inhibition during the prolonged CBF increase with 1 μm KT-5823 (B) did not significantly change the CBF decline towards baseline compared to control (A). C and D, in another pair of cells, PKA inhibition during the prolonged CBF increase with 10 μm PKI_14–22 (D) significantly accelerated the CBF decline towards baseline compared with control (C), but CBF was still significantly higher than baseline at the end of the experiment.

Figure 2. CBF and [Ca²⁺]_i responses to long-term ATP, UTP or ATP plus NECA exposure in human tracheal epithelial cells

A, a representative human airway epithelial cell shows an initial calcium-coupled, and later prolonged increase in CBF upon long-term exposure to ATP, similar to the response upon short-term ATP exposure (as shown in Fig. 1B). B, another cell shows also an initial calcium-coupled, and later prolonged increase in CBF upon long-term exposure to UTP, similar to the response upon short-term UTP exposure (as shown in Fig. 1C). C, the exposure to both ATP plus 5-(N-ethylcarboxamido)-adenosine (NECA) yielded a calcium response similar to ATP alone. CBF, however, increased to a higher level and had a tendency to increase further after its initial peak as opposed to exposure to ATP alone, where CBF had a tendency to slowly decay towards baseline.

Figure 3. Influence of adenosine receptor blockage on CBF and [Ca²⁺]_i responses to short-term ATP exposure in human tracheal epithelial cells

CBF and [Ca²⁺]_i responses to short-term ATP exposure of a representative submerged human tracheal epithelial cell are shown. Blockage of adenosine receptors with 100 μm 8-sulphophenyl-theophylline (8-SPT) does not accelerate the decline of the prolonged CBF increase towards baseline but actually slightly increases both CBF and [Ca²⁺]_i.

Figure 4. CBF responses to short-term ATP exposure in tracheal epithelial cells grown at the air-liquid interface

A, a representative ovine cell responds to 50 μm ATP, applied apically, with a transient increase in CBF, similar to ovine cells in submerged cultures (see Fig. 1A). B, in a representative human cell, on the other hand, CBF increases with two maxima and remains elevated above baseline for an extended period of time after short-term ATP exposure, similar to human cells in submerged culture (see Fig. 1B).

Figure 5. Neither physiological temperature nor a NOS inhibitor change CBF responses to short-term ATP exposure in human cells

A, [Ca²⁺]_i and CBF responses to short-term ATP exposure of a representative human cell measured at 35 °C. CBF reveals both a calcium-coupled and a prolonged increase after short-term ATP exposure, similar to cells measured at room temperature. B, representative human cell in submerged culture and measured at room temperature. Despite the presence of the NOS inhibitor 3-bromo-7-nitroindazole (5 μm), CBF shows an immediate rise and a delayed second peak, followed by a prolonged elevation of CBF, similar to control cells (Fig. 1B).

Figure 6. CBF and [Ca²⁺]_i responses to short-term ATP exposure after inhibition of PKA with PKI_14–22 in ovine and human tracheal epithelial cells

A, [Ca²⁺]_i and CBF responses to short-term ATP exposure in a representative ovine tracheal epithelial cell after pre-treatment with 10 μm of the PKA inhibitory peptide PKI_14–22 as outlined in Methods. The response of both signals and their coupling is not different from control cells (see Fig. 1A). B, in a representative human tracheal cell, however, the CBF response to short-term ATP exposure is now strictly calcium-coupled in contrast to control cells (see Fig. 1B).

Figure 7. CBF response to short-term ATP exposure after inhibition of PKA with PKI_14–22 in human tracheal epithelial cells grown at the air-liquid interface

The CBF response to short-term ATP exposure is reduced to a transient increase in a representative human cell that has been pretreated with 10 μm of the PKA inhibitory peptide PKI_14–22 as indicated in Methods.

Figure 8. CBF and [Ca²⁺]_i responses to short-term ATP exposure after inhibition of PKA with KT-5720, PKG with KT-5823, and adenylyl cyclase with SQ22536 in human tracheal epithelial cells

A, [Ca²⁺]_i and CBF responses to short-term ATP exposure of a representative submerged human tracheal epithelial cell after pretreatment with 1 μm of the PKA inhibitor KT-5720. The CBF response to short-term ATP exposure is mainly calcium-coupled, similar to pretreatment with PKI_14–22 (see Fig. 6B) and in contrast to control cells (see Fig. 1B). B, pretreatment with 1 μm of the PKG inhibitor KT-5823 has no effect on the prolonged CBF increase in response to short-term ATP exposure. Since both KT-5720 and KT-5823 are dissolved in DMSO, this solvent cannot explain the blocking ability of KT-5720. C, pretreatment with 1 mm of the adenylyl cyclase inhibitor SQ22536 inhibits the prolonged CBF increase upon short-tem exposure to ATP, similar to PKA inhibition.

Figure 9. CBF and [Ca²⁺]_i responses to short-term ATP exposure in the presence of forskolin in human tracheal epithelial cells

A, exposure of a representative human submerged tracheal epithelial cell to a direct activator of adenylyl cyclase, 10 μm forskolin, leads to a [Ca²⁺]_i-independent rise of CBF to a stable plateau above the original baseline (ΔCBF). ATP stimulation in the presence of forskolin reveals only a short, calcium-coupled CBF increase. B, after PKA-inhibition with 10 μm PKI_14–22, forskolin has no effect on CBF and ATP stimulation reveals again only a short, calcium-coupled rise in CBF.

Figure 10. Calcium requirements for prolonged CBF increases in response to short-term ATP exposure in human tracheal epithelial cells

A, release of calcium from intracellular stores is required for prolonged CBF increases in human cells after short-term ATP exposure. CBF and [Ca²⁺]_i responses to 1 μm thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, which results in both a gradual calcium and CBF rise are shown. ATP exposure after calcium depletion elicits neither a calcium transient nor a CBF increase. In fact, both calcium and CBF decrease slightly (arrow). B, extracellular calcium is not required for extended CBF increases in response to short-term ATP exposure. The CBF and [Ca²⁺]_i responses of a representative human tracheal cell exposed to ATP while bathed in calcium-free medium are shown.