The role of cGMP in the regulation of rabbit airway ciliary beat frequency - PubMed (original) (raw)

The role of cGMP in the regulation of rabbit airway ciliary beat frequency

Luo Zhang et al. J Physiol. 2003.

Abstract

The involvement of cyclic guanosine 3',5'-monophosphate (cGMP) and cGMP-dependent protein kinase (PKG) and their interaction with the Ca2+-dependent mechanisms in the regulation of ciliary activity are not well understood. To investigate how cGMP regulates ciliary activity, changes in ciliary beat frequency (CBF) and intracellular calcium concentration ([Ca2+]i) of rabbit tracheal ciliated cells in response to 8-bromo-cGMP (Br-cGMP) were simultaneously quantified using digital, high-speed phase-contrast and fluorescence imaging. Br-cGMP induced a response in ciliary activity that could be separated into two parts. Firstly, Br-cGMP induced a concentration-dependent increase in the basal CBF that occurred without increasing the [Ca2+]i. This response was not affected by excessively buffering the [Ca2+]i with BAPTA but was abolished by KT5823, a PKG inhibitor. Secondly, Br-cGMP induced a series of transient increases in CBF that were superimposed on the sustained increases in CBF. These transient increases in CBF correlated with the stimulation of a series of transient increases in [Ca2+]i and were abolished by BAPTA, but were unaffected by KT5823. The magnitude of the transient increases in CBF and [Ca2+]i were not dependent on the concentration of Br-cGMP. The Ca2+-dependent changes in CBF induced by ionomycin or ATP were not affected by KT5823. From these results, we propose that cGMP increases CBF in two ways: firstly through a Ca2+-independent mechanism involving PKG, and secondly through a Ca2+-dependent mechanism following the stimulation of changes in [Ca2+]i. In addition, we suggest that the Ca2+-dependent stimulation of rabbit airway ciliary activity does not initially require PKG activation.

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Figures

Figure 1

Figure 1. The effects of 8-Bromo-cGMP (Br-cGMP) on the ciliary beat frequency (CBF) and [Ca2+]i of rabbit airway ciliated cells

Data for both CBF and [Ca2+]i are normalized with the initial basal value and are representative of multiple cells. A, 1 μM Br-cGMP induced a two-part response in the CBF consisting of a small increase in the basal CBF (basal CBF = 10.4 Hz) that reached a maximum level within 60 s and declined thereafter, and larger transient increases in CBF that were superimposed on the basal CBF. By contrast, Br-cGMP only induced a series of transient increases in the [Ca2+]i without affecting the basal [Ca2+]i (n = 10). B, 10 μM Br-cGMP induced a higher sustained increase in the basal CBF (basal CBF = 15.9 Hz) but the superimposed transient increases in CBF, as well as the transient increases in [Ca2+]i, were unchanged (n = 11). C, 100 μM Br-cGMP only induced a further increase in the sustained CBF increase (basal CBF = 14.1 Hz) (n = 11). A-C, in all cases, the sustained increases in CBF occurred in the absence of changes in [Ca2+]i while the transient changes in CBF and [Ca2+]i were tightly coupled.

Figure 2

Figure 2. The correlation of the initial changes in CBF (coloured dots) with changes in [Ca2+]i (grey line) induced by 10 (A and B) or 100 μM (C and D) Br-cGMP

A, after the addition of 10 μM Br-cGMP, the CBF (normalized with basal CBF) slowly increased (blue dots) from the basal CBF (17.9 Hz, left black dots) while the [Ca2+]i remained unchanged. Approximately 30 s after the addition of Br-cGMP, the first transient increase in [Ca2+]i occurred and this was strongly correlated with a transient increase in CBF (red dots). B, the normalized CBF plotted against the corresponding [Ca2+]i for the time period illustrated in A. The vertical distribution of the blue dots, which represent the initial CBF increase induced by Br-cGMP, indicate that this phase of the response occurs independently of the [Ca2+]i. The inclined distribution of the red dots, which represent the CBF during the transient increase in [Ca2+]i, indicate that the CBF is dependent on the [Ca2+]i during this phase of the response. C and D, similar temporal and correlative representations of the changes in CBF and [Ca2+]i induced by 100 μM Br-cGMP. The initial increase in CBF (blue dots) is larger (basal CBF = 14.8 Hz) but still independent of the [Ca2+]i. The transient changes in CBF (red dots) are dependent on the [Ca2+]i.

Figure 3

Figure 3. The concentration-response relationship of CBF and [Ca2+]i in airway epithelial cells to Br-cGMP

A, the initial increase in CBF (○) in response to 1, 10 and 100 μM Br-cGMP was 1.13 ± 0.02 (n = 10), 1.23 ± 0.04 (n = 11) and 1.28 ± 0.05, (n = 11), respectively, and was significantly greater (all P < 0.001) with respect to the basal rate with the increasing Br-cGMP concentration. The CBF increase induced by 100 μM Br-cGMP was also significantly greater than that induced by 1 μM Br-cGMP (*_P_ < 0.05). The sustained increase in CBF (□) in response to 1, 10 and 100 μM Br-cGMP was 1.02 ± 0.02 (_n_ = 10), 1.17 ± 0.04 (_n_ = 11) and 1.24 ± 0.04 (_n_ = 11), respectively, and this was also significantly greater (all _P_ < 0.001) with the increasing Br-cGMP concentration. The sustained increase in CBF induced by 10 and 100 μM Br-cGMP was also significantly greater (**_P_ < 0.01, ***_P_ < 0.001) than that induced by 1 μM Br-cGMP. The transient increases in CBF (▵) in response to 1, 10 and 100 μM Br-cGMP were 1.60 ± 0.08 (_n_ = 10), 1.73 ± 0.08 (_n_ = 10) and 1.77 ± 0.06 (_n_ = 11), respectively, but these were not significantly increased with increasing Br-cGMP concentration. The starting basal CBF (12.8 ± 0.9 Hz, _n_ = 10; 11.9 ± 0.9 Hz, _n_ = 11; 12.4 ± 0.6 Hz, _n_ = 11) for each concentration of Br-cGMP was similar (_P_ > 0.05). B, the concentration-response relationship of the transient change in [Ca2+]i to Br-cGMP. The transient changes in [Ca2+]i (♦) in response to 1, 10 and 100 μM Br-cGMP were 83 ± 10 nM, (n = 10), 94 ± 10 nM (n = 9) and 92 ± 10 nM (n = 10), respectively, but were not significantly different with increasing Br-cGMP concentration (P > 0.05). The basal [Ca2+]i (30 ± 4 nM, n = 10; 36 ± 4 nM, n = 11; 32 ± 5 nM, n = 11) for each concentration was similar (P > 0.05). C, the concentration-response relationship of the frequency of the transient changes in [Ca2+]i or CBF oscillations (•) to Br-cGMP. The frequency in response to 1, 10 and 100 μM Br-cGMP was 0.67 ± 0.02 min−1 (n = 5), 0.82 ± 0.13 min−1 (n = 5) and 1.07 ± 0.14 min−1 (n = 4), and not significantly different with the increasing Br-cGMP concentration (P > 0.05).

Figure 4

Figure 4. The effect of buffering the [Ca2+]i with BAPTA in response to 100 μM Br-cGMP

A, a representative response of a ciliated cell pretreated with 20 μM BAPTA AM and exposed to Br-cGMP (bar). The BAPTA buffering prevented any changes in [Ca2+]i (grey line). However, the CBF (black dots) still increased (to an initial normalized CBF = 1.36) and remained sustained (at a normalized CBF = 1.32). Transient increases in [Ca2+]i or CBF did not occur. The basal CBF was 12.1 Hz. B and C, the effect of buffering the [Ca2+]i with BAPTA on the initial (B) and sustained (C) changes in CBF induced by Br-cGMP. In control experiments, without BAPTA treatment, the initial and sustained increases in CBF induced by Br-cGMP were 1.28 ± 0.05 (n = 11) and 1.24 ± 0.04 (n = 11) respectively. These changes were indistinguishable (P > 0.05) from the initial (1.32 ± 0.08, n = 6) and sustained (1.23 ± 0.05, n = 6) increases in CBF induced by Br-cGMP in the presence of BAPTA. The basal CBFs for cells with (12.4 ± 1.0, n = 6) or without BAPTA treatment (12.4 ± 0.6, n = 11) were similar (P > 0.05).

Figure 5

Figure 5. The combined effect of buffering [Ca2+]i with BAPTA and inhibiting PKG activity with KT5823 on the response to Br-cGMP

A and B, two representative cells illustrate the combined effect of the pretreatment of cells with KT5823 and BAPTA AM on the changes in CBF (black dots) and [Ca2+]i (grey lines) induced by 100 μM Br-cGMP (bar). KT5823 and BAPTA AM treatment prevented any significant increases in the [Ca2+]i and CBF in response to Br-cGMP. The basal CBF of the cells was 17.7 Hz (A) and 12.5 Hz (B). C and D, summaries of the combined effects of KT5823 and BAPTA AM on the initial (C) and sustained (D) increases in CBF induced by 100 μM Br-cGMP. The increase in the initial CBF (1.09 ± 0.03, n = 7) induced by Br-cGMP in the presence of KT5823 and BAPTA AM was significantly lower than the increase induced in cells without treatment (1.32 ± 0.08, n = 6, *P < 0.05). Similarly, the sustained increase in CBF (0.93 ± 0.03, _n_ = 7) was significantly lower compared to cells without treatment (1.23 ± 0.05, _n_ = 6, ***_P_ < 0.001). The basal CBFs from each group were similar (12.4 ± 1.0 Hz, _n_ = 6 and 15.3 ± 1.2 Hz, _n_ = 7, _P_ > 0.05).

Figure 6

Figure 6. The effect of KT5823 treatment on changes in CBF and [Ca2+]i induced by 100 μM Br-cGMP

A, Br-cGMP (bar) induced a very small initial increase in CBF (black dots) that rapidly returned to the basal rate without inducing significant increases in [Ca2+]i (grey line). However, Br-cGMP still induced a series of transients in [Ca2+]i accompanied with transient increases in CBF. The basal CBF was 15.2 Hz. B and C, summaries of the effects of KT5823 treatment on Br-cGMP-induced transient changes in CBF (B) and the frequency of [Ca2+]i transients (C). KT5823 had no significant effect on the transient CBF (1.56 ± 0.08, n = 6 versus 1.77 ± 0.06, n = 11, P > 0.05) or the frequency of [Ca2+]i transients (0.74 ± 0.09, n = 4 versus 1.07 ± 0.14, n = 4, P > 0.05) as compared to controls. The basal CBFs from each group were similar (14.1 ± 0.9 Hz, n = 6 and 12.4 ± 0.6 Hz, n = 11, P > 0.05). D and E, summaries of the effects of KT5823 on initial (D) and sustained (E) CBF induced by Br-cGMP. Br-cGMP at 100 μM induced a significantly lower initial increase in CBF (1.08 ± 0.03, n = 6) compared to cells without KT5823 treatment (1.28 ± 0.05, n = 11, P < 0.05) and a significantly lower sustained CBF increase (0.97 ± 0.04, n = 6) compared to cells without KT5823 treatment (1.24 ± 0.04, n = 11, P < 0.001). *P < 0.05, ***P < 0.001.

Figure 7

Figure 7. The effect of KT5823 treatment on ionomycin-induced increases in CBF and [Ca2+]i

A, a representative trace (n = 5) of the simultaneous changes in CBF (black dots) and [Ca2+]i (grey line) in a ciliated epithelial cell in response to 1 μM ionomycin (bar). Ionomycin induced a rapid increase in CBF and [Ca2+]i. While the [Ca2+]i gradually returned to the basal level, the CBF remained sustained at an elevated level. B, following the pretreatment of cells with 2 μM KT5823, the response induced by 1 μM ionomycin was indistinguishable from the control response (n = 5).

Figure 8

Figure 8. The effect of KT5823 treatment on ATP-induced changes in CBF and [Ca2+]i

A, a representative trace of the simultaneous changes in CBF (black dots) and [Ca2+]i (grey line) in ciliated epithelial cells in response to 2 μM ATP (bar). ATP induced a rapid increases in CBF and [Ca2+]i which was followed by oscillations in both CBF and [Ca2+]i. The CBF oscillations occurred from an elevated minimum CBF while the [Ca2+]i oscillations occurred from a baseline that declined to the basal level. B, ATP induced a similar response in CBF and [Ca2+]i in cells that were pretreated with 2 μM KT5823 (n = 5).

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