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.

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.

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).

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).

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).

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.

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).

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).