Aging causes a slowing in ciliary beat frequency, mediated by PKCε

Abstract

The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and is the third most common reason for hospitalization in the elderly. One reason that elderly people may be more susceptible to pneumonia is a breakdown in the lung's first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however, little is known about the mechanism(s) involved. We compared the CBF in BALB/c and C57BL/6 mice aged 2, 12, and 24 mo and found that CBF diminishes with age. Cilia in the mice at age 12 and 24 mo retained their ability to be stimulated by the β2 agonist procaterol. To help determine the mechanism of ciliary slowing, we measured protein kinase C alpha and epsilon (PKCα and PKCε) activity. There were no activity differences in PKCα between the mice aged 2, 12, or 24 mo. However, we demonstrated a significantly higher PKCε activity in the mice at 12 and 24 mo than the in the mice 2 mo of age. The increase in activity is likely due to a nearly threefold increase in PKCε protein in the lung during aging. To strengthen the connection between activation of PKCε and ciliary slowing, we treated tracheas of mice at 2 mo with the PKCε agonist 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with β2 agonists. The mechanisms for the slowing of baseline CBF have not been previously determined. In this mouse model of aging we were able to show that decreases in CBF are related to an increase in PKCε activity.

Keywords: DCP-LA; PKC epsilon; Sisson-Ammons video analysis (SAVA), lung; elderly; innate immunity; pneumonia; β2 agonists.

Fig. 1.

Ciliary beat frequency (CBF) diminishes with normal aging. A: tracheas from healthy BALB/c mice aged 2, 5, 12, and 24 mo were cut into rings, and CBF analysis was performed. The 12- and 24-mo-old animals had significant decreases in CBF compared with the 2- and 5-mo-old animals. B: tracheas from healthy C57BL/6 mice aged 2, 12, and 24 mo were cut into rings, and CBF analysis was performed. The young mice (2 mo) had a baseline CBF of 12.44 ± 0.29 Hz (n = 8); 12-mo baseline was 10.05 ± 0.47 Hz (n = 10); and 24-mo baseline was 9.46 ± 0.32 Hz (n = 9). The differences in CBF between the 2- and 12-mo (P < 0.01), 2- and 24-mo mice (P < 0.001), and 12- and 24-mo mice (P < 0.001) were statistically significant.

Fig. 2.

Aging cilia remain responsive to β₂ agonists. Baseline readings of CBF were measured from each age group, then the tracheal rings were treated with the β₂ agonist procaterol (100 nM) for 1 h. CBF was once again measured, and there were significant increases in each group. The animals at 24 mo were able to be stimulated only to the baseline levels of young mice.

Fig. 3.

PKCε activity is stimulated with aging. The tracheas from BALB/c mice aged 2, 12, and 24 mo were homogenized and PKCε (A) and PKCα (B) activity was determined. There was a significant increase in PKCε activity in the animals at 12 and 24 mo. There was no difference in PKCα activity.

Fig. 4.

PKCε mRNA and protein are increased with aging. A: quantitative PCR was used to measure PKCε mRNA expression in the tracheas from BALB/c mice. There was a marked increase in PKCε mRNA in the mice aged 12 and 24 mo compared with mice aged 2 mo. B: PKCε protein was measured from C57BL/6 mice by ELISA. There was an increase in PKCε protein in the mice aged 12 and 24 mo compared with mice at 2 mo.

Fig. 5.

DCP-LA, a PKCε agonist slows the cilia of young mice. Tracheal rings from animals aged 2 mo were treated with DCP-LA for 0–3 h. After 3 h of stimulation with DCP-LA, the rings were treated with procaterol (P). There was a significant increase in CBF with procaterol.