Intracellular Ca2+ oscillation frequency and amplitude modulation mediate epithelial apical and basolateral membranes crosstalk

Figures

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  Figure 1. Intracellular Ca2+ signaling in Malpighian (renal) tubule cells of the blood-feeding insect Rhodnius prolixus (A) R. prolixus ingest large blood meal volumes that can be up to 12 times the unfed (upper panel) body weight (lower panel shows a fed animal, scale bar = 1 cm). While the cellular component of the blood meal is stored in the midgut, the excess water and ions ingested are excreted through the Malpighian tubules. The post-prandial diuretic process is stimulated by the release of diuretic hormones, including serotonin. (B) Intracellular Ca2+ in Malpighian tubules was measured in selected regions of identical size as indicated with circular symbols of different colors along the length of the entire tissue sample. The measuring regions were placed in such as way to avoid including the lumen of the tubule. The numbers 1, 20 and 40 indicate the measuring region from the distal to the proximal side. (C) Heatmap of the serotonin-triggered intracellular Ca2+ oscillations measured along the length of the tubule (y axis) at the sites indicated by the multicolor circles in panel B over time (x axis). The intracellular Ca2+ oscillations are required to stimulate transcellular fluid…

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  Figure 2. Intracellular Ca2+ oscillation frequency and amplitude are proportional to K+ concentration in the bathing fluid (A) After stimulation with serotonin the Malpighian tubules secreted fluid at maximum rate when bathed in saline containing 2, 6, 8, 10, and 14.5 Mm K+ ([K+]e) while all other ions remain constant (p = 0.42, ANOVA F(4, 32) = 1.003). However, the secreted fluid (B) K+ concentration (p < 0.0001, linear regression F(1, 36) = 141.8) and (C) transepithelial K+ flux (p < 0.0001, linear regression F(1, 29) = 128.1) produced by the tubules directly correlated with K+ concentration in the bathing fluid. Serotonin-stimulated (1 μM, downward arrow) intracellular Ca2+ signaling produced by tubules exposed to (D) 14.5, (E) 8, or (F) 2 mM K+ displayed oscillation with a (G) frequency (first 5 min after serotonin stimulation, p < 0.0004, linear regression F(1, 13) = 22.51) and (H) amplitude (p < 0.0001, linear regression F(1, 101) = 38.72) that correlated with the K+ concentration in the bathing fluid. Asterisks indicate statistical significance.

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  Figure 3. Intracellular Ca2+ oscillation frequency and amplitude are proportional to bumetanide-modulated transepithelial ion flux (A) Na+:K+:2Cl- cotransporter blocker bumetanide reduces Malpighian tubule fluid secretion in a concentration-dependent manner. Sample trace of intracellular Ca2+ oscillation from serotonin-stimulated (1 μM, downward arrow) tubules exposed to (B) 10, (C) 5, and (D) 1 μM bumetanide. The intracellular Ca2+ oscillation (E) frequency and (F) amplitude.

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  Figure 4. Instantaneous reduction in bathing fluid K+ concentration ([K+]e) causes a reduction in intracellular Ca2+ oscillation frequency and amplitude (A) Sample traces of intracellular Ca2+ oscillation by Malpighian tubules initially bathed in 14.5 mM K+ and instantaneously switched to 2, 4, 6, 8, or 14.5 mM K+. (B) The intracellular Ca2+ oscillation frequency displayed by the Malpighian tubules directly correlates with magnitude of the change in [K+]e (p < 0.0001, linear regression F(1, 130) = 67.8). (C) The intracellular Ca2+ oscillation amplitude displayed by the Malpighian tubules correlates directly with magnitude of the change in [K+]e (p < 0.0001, linear regression F(1, 130) = 90.23). Asterisks indicate statistical significance.

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  Figure 5. Blocking intracellular Ca2+ oscillation with BAPTA-AM inhibits cellular K+ homeostasis (A) Secreted fluid produced by Malpighian tubules exposed to an instantaneous change in bath K+ concentration from 14.5 to 2 mM (downward arrow) in control and BAPTA-AM (300 μM) groups. BAPTA-treated tubules significantly reduced secretion rate after the reduction in bathing fluid K+ concentration (p = 0.019, repeated measures two-way ANOVA, Tukey’s multiple comparisons test, n = 9) while control preparations maintain constant secretion rate (p = 0.64, repeated measures two-way ANOVA, Tukey’s multiple comparisons test, n = 6). (B) Secreted fluid K+ concentration decreased in control preparations (p = 0.017, repeated measures two-way ANOVA, Tukey’s multiple comparisons test, n = 7) while K+ concentration in secreted fluid remained constant in BAPTA-treated preparations (p = 0.7, repeated measures two-way ANOVA, Tukey’s multiple comparisons test, n = 10). (C) K+ flux diminished in both control preparation (p = 0.024, repeated measures two-way ANOVA, Tukey’s multiple comparisons test, n = 7) and BAPTA-treated preparations (p = 0.034, repeated measures two-way ANOVA, Tukey’s multiple comparisons…

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  Figure 6. Experimentally generated intracellular Ca2+ oscillation modulate transepithelial K+ flux (A) Diagram of the calcium clamping apparatus. Flow of Ringer’s solution containing 20 and 0 mM Ca2+ is controlled with computer-controlled solenoid valves and driven by a peristaltic pump. The Malpighian tubule is placed in a custom-built chamber consisting of a bathing section that allows for superfusion with Ringer’s solution (right side) and a secretion-collecting section (left side, arrows indicate direction of fluid flow). The fluid secreted by the Malpighian tubules is collected at the open end of the tubule under paraffin oil and the volume is calculated. The secreted droplets are collected to measure K+ concentration. (B) Heatmap of experimentally triggered intracellular Ca2+ oscillations measured along the length of the tubule (x axis) over time (y axis) with the frequency indicated in mHz at the top. The lower panel shows that the increase in intracellular Ca2+ coincides with the timing of superfusion with Ringer’s solution containing 20 mM K+ (dashed bars). (C) Calcium-clamped Malpighian tubules (stimulated serotonin 1 μM) secrete fluid at high rates that are similar to control…