1 Pediatric Research 2009 Vol: 65(3):274-278. DOI: 10.1203/PDR.0b013e3181973b22

Pulmonary Neuroendocrine Cell-Secreted Factors May Alter Fetal Lung Liquid Clearance

The neuroendocrine system is most active at birth and may play a role in the transition from fetal to postnatal life, in particular in the lungs' transition from fluid secretion to fluid absorption. Pulmonary neuroendocrine cells do release dopamine (DA), serotonin, and gastrin-releasing peptide but their effects on lung ion and fluid transport are poorly understood. Therefore, we studied their effects on fetal distal lung explants and primary cultures of fetal distal lung epithelium (FDLE). We show that DA, but neither serotonin nor gastrin-releasing peptide, alters ion and fluid transport, in a dose-dependent manner. DAs effects were abrogated by D1/D2 receptor blockers in FDLE but not in explants. Propranolol abrogated DAs effects in both models. DA increased intracellular cAMP levels in FDLE. Terbutaline, forskolin, and isobutylmethylxanthine did not increase short circuit current (Isc) in DA-treated cells, despite a further increase in cAMP. We conclude that at least one, but not all mediators released by pulmonary neuroendocrine cells alter distal lung epithelial ion transport.

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Figures
Figure 1: Neither serotonin nor GRP alters ion or fluid transport in the fetal distal lung. (A) Serotonin was added to FDLE monolayers (▴) and control monolayers received corresponding volumes of vehicle (□). Data (mean ± SEM) are presented as percent Isc relative to baseline Isc for each monolayer. (Raw baseline Isc values for vehicle and serotonin groups were 6.1 ± 0.32 μA/cm2 and 6.1 ± 0.31 μA/cm2, respectively). Serotonin had no effect on Isc in FDLE. (R = 941 ± 51.3 Ω · cm2, PD >1.0 mV, n = 10-11). (B) Fetal distal lung explants were treated with either 10−4 M serotonin or vehicle. Data are plotted as the size change relative to vehicle-treated control explants from the same lung, by the end of the treatment period. (Raw percent size change for vehicle-treated cells was 50.2 ± 3.90%). Serotonin had no effect on explant expansion when compared with explants treated with the vehicle alone. (n = 12). (C) GRP was added in increasing concentrations to FDLE (▴) and control monolayers were treated with vehicle (□). Data are presented as percent Isc relative to the baseline for each monolayer. (Raw baseline Isc values for vehicle and GRP groups were 4.7 ± 0.31 μA/cm2 and 4.5 ± 0.39 μA/cm2, respectively). GRP did not alter Isc in FDLE. (R = 891 ± 21.9 Ω · cm2, PD >1.0 mV, n = 8). (D) Fetal distal lung explants were treated with GRP (up to 10−6 M). GRP did not affect explant growth at any of the doses tested. Data are plotted as the size change relative to the vehicle control from the same lung, during treatment period. (Raw percent size change for vehicle-treated cells was 50.2 ± 2.07%). GRP had no effect on explant size. (n = 9-21). Figure 2: Dopamine increases ion transport across FDLE monolayers, and induces fluid absorption in fetal distal lung explants. (A) FDLE exposed to 10−4 M dopamine significantly increased Isc (*p < 0.01 for dopamine, with no significant change in control cells, by repeated measures ANOVA). Dopamine tended to increase both AIC (▪) and ASC (□) components of Isc. (R = 1732 ± 200.0 Ω * cm2, PD >1.0 mV, n = 10). (B) Dopamine increases Isc in FDLE in a dose-dependent manner, and this effect reached significance at the highest dose tested (*p < 0.05, dopamine vs. control). Raw baseline Isc values for vehicle (□) and dopamine (▴) groups were 5.7 ± 0.46 μA/cm2 and 5.9 ± 0.30 μA/cm2, respectively. (R = 1373 ± 112.5 Ω · cm2, PD >1.0 mV, n = 20-24). (C) Fetal distal lung explants were treated with increasing concentrations of dopamine. Dopamine treatment decreased the rate of expansion of lung explants, and this response was dose dependent. Raw percent size change 24-48 h for vehicle-treated cells was 46.6 ± 1.26%. (n = 12-24, *p < 0.01 relative to 0 M dopamine). Figure 3: Dopamine receptor blockers inhibit dopamine effects on FDLE monolayers but not on fetal distal lung explants. (A) FDLE in Ussing chambers were treated with dopamine receptor blockers (DRB: D1 dopamine receptor blocker SCH-23390 (10 μM) + D2 receptor blocker S(-)sulpiride (10 μM)), and then with dopamine. Data are reported as percent Isc after treatment, relative to the baseline value for each group. Dopamine receptor blockers inhibited the effect of dopamine in FDLE (*p < 0.05, DA vs. DA+ DRB, n = 10-11). (B) Fetal distal lung explants were treated with vehicle alone (V) or to dopamine (DA, 10−4 M) in the presence or absence of dopamine receptor blockers (DRB: D1 dopamine receptor blocker SCH-23390 (10 μM) + D2 receptor blocker S(-)sulpiride (10 μM)). Dopamine receptor blockers did not alter dopamine effects on distal lung explants. Raw percent size change for vehicle-treated cells was 53.3 ± 2.05%. (n = 8). Figure 4: Propranolol inhibits the effects of dopamine. (A) FDLE treated with propranolol (P, 10 μM) followed by dopamine. Data are reported as percent Isc after treatment, relative to the baseline value for each group. Propranolol inhibited the effect of dopamine (*p < 0.05, DA vs. DA+ P, n = 6-7). (B) Lung explants were treated with vehicle alone (V) or to dopamine (DA, 10−4 M) in the absence or presence of propranolol (P, 10 μM). Dopamine's effect was partially inhibited when propranolol was present. Raw percent size change for vehicle-treated cells was 64.8 ± 2.04%. (n = 16, *p < 0.01 relative to vehicle group, **p < 0.05 relative to dopamine group). Figure 5: Dopamine-treated FDLE do not further increase Isc in response to terbutaline, forskolin and IBMX. (A) FDLE monolayers were treated with dopamine (10−4 M) and then TFI (□, 100 μM terbutaline, 10 μM forskolin, 10 μM IBMX), or appropriate vehicle controls (▪). In control cells not exposed to dopamine, addition of TFI resulted in an increase in Isc (*p < 0.05). This increase was not observed in cells treated with dopamine before TFI treatment. (R = 1373 ± 112.5 Ω · cm2, PD >1.0 mV, n = 20-24). (B) FDLE monolayers were treated with dopamine (10−4 M) and then TFI (100 μM) terbutaline, 10 μM forskolin, 10 μM IBMX), or appropriate vehicle controls, similar to Ussing experiments, with different treatment times (see methods section). Cells were lysed and the lysate was assayed for cAMP levels. Measured cAMP levels were normalized to the protein concentration in each sample (n = 7-8). Dopamine treatment increased cAMP levels in FDLE (†p < 0.01, relative to vehicle), but not to the level seen after treatment. Addition of TFI resulted in large increases in cAMP levels in both cells treated with dopamine (**p < 0.01, relative to TFI dopamine), and control cells (*p < 0.01, relative to TFI vehicle).
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References
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