Regulation of calcium channels and exocytosis in mouse adrenal chromaffin cells by prostaglandin EP3 receptors

Abstract

Prostaglandin (PG) E(2) controls numerous physiological functions through a family of cognate G protein-coupled receptors (EP1-EP4). Targeting specific EP receptors might be therapeutically useful and reduce side effects associated with nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors that block prostanoid synthesis. Systemic immune challenge and inflammatory cytokines have been shown to increase expression of the synthetic enzymes for PGE(2) in the adrenal gland. Catecholamines and other hormones, released from adrenal chromaffin cells in response to Ca(2+) influx through voltage-gated Ca(2+) channels, play central roles in homeostatic function and the coordinated stress response. However, long-term elevation of circulating catecholamines contributes to the pathogenesis of hypertension and heart failure. Here, we investigated the EP receptor(s) and cellular mechanisms by which PGE(2) might modulate chromaffin cell function. PGE(2) did not alter resting intracellular [Ca(2+)] or the peak amplitude of nicotinic acetylcholine receptor currents, but it did inhibit Ca(V)2 voltage-gated Ca(2+) channel currents (I(Ca)). This inhibition was voltage-dependent and mediated by pertussis toxin-sensitive G proteins, consistent with a direct Gβγ subunit-mediated mechanism common to other G(i/o)-coupled receptors. mRNA for all four EP receptors was detected, but using selective pharmacological tools and EP receptor knockout mice, we demonstrated that EP3 receptors mediate the inhibition of I(Ca). Finally, changes in membrane capacitance showed that Ca(2+)-dependent exocytosis was reduced in parallel with I(Ca). To our knowledge, this is the first study of EP receptor signaling in mouse chromaffin cells and identifies a molecular mechanism for paracrine regulation of neuroendocrine function by PGE(2).

Fig. 1.

PGE₂ inhibits I_Ca in mouse adrenal chromaffin cells. A, peak amplitude of I_Ca is plotted against time in a representative cell. The cell was voltage-clamped in the whole-cell configuration and stimulated with a 20-ms step depolarization from −80 to +20 mV every 10 s. Application of PGE₂ (100 nM) (indicated by horizontal bar) produced robust, reversible inhibition of I_Ca. Inset, the voltage command (top) and three representative current traces before (ctl), during (PGE₂), and after washout of PGE (wash). B, Log₁₀ concentration-response curve plotting percentage of inhibition of I_Ca to varying concentrations of PGE₂. Each cell was exposed to three increasing concentrations of PGE₂, with 10 nM being common to all experiments (n = 4–16 cells). The indicated fit was to a Boltzmann function with a Hill slope of 1 (see Materials and Methods) and yielded an EC₅₀ of 5.5 nM.

Fig. 2.

The inhibition of I_Ca by PGE₂ is voltage-dependent and mediated by pertussis toxin-sensitive G proteins. A, the percentage of inhibition of I_Ca produced by 100 nM PGE₂ or 100 μM ATP for control cells (left) and cells treated with 300 ng/ml pertussis toxin (PTX) for ∼24 h before whole-cell recording of I_Ca. Control and pertussis toxin-treated cells were from the same cultures, and recordings were alternated on the same day. PTX treatment significantly reduced the inhibition by PGE₂ (**, p < 0.002) and ATP (*, p < 0.05). B, the inhibition of I_Ca by PGE₂ was voltage-dependent. Top, the voltage command for the prepulse facilitation protocol. Cells were stimulated by two identical test pulses (P1 and P2, 20-ms step to +10 mV, separated by 300 ms), but the second pulse (P2) was preceded by a 50-ms step to +120 mV. Three representative currents are superimposed (bottom trace), showing I_Ca before (ctl), during application of 100 nM PGE₂ (PGE₂), and after washout (wash). The prepulse to +120 mV reversed most of the inhibition of I_Ca produced by PGE₂. C, mean data from six experiments like that shown in B. Bar chart summarizes the mean peak amplitude of I_Ca during the first pulse (P1, no prepulse) and the second pulse (P2, with prepulse) (*, p < 0.05; n = 6). D, the percentage inhibition by PGE₂ of I_Ca elicited by P1 (without a prepulse) and P2 (with a prepulse) (***, p < 0.001; n = 6).

Fig. 3.

PGE₂ does not alter peak nicotinic acetylcholine receptor currents or resting [Ca²⁺]_i in mouse chromaffin cells. A, representative recording of nAChR currents evoked by two applications of carbachol (100 μM) in the absence (left) or presence (right) of 100 nM PGE₂. Drug application is indicated by the horizontal bars. Cells were voltage-clamped at −80 mV in the perforated whole-cell recording configuration. B, bar chart showing that PGE₂ had no effect on the mean amplitude of the nAChR currents evoked by carbachol (n = 8 cells). C, ratiometric imaging of Fura-2-loaded chromaffin cells. Inset, a representative recording from a single cell plotting estimated [Ca²⁺]_i against time (sampling rate, 0.5 Hz). The cell was exposed to 1 μM PGE₂ for 3 min and then to 50 mM KCl to depolarize the membrane and elicit Ca²⁺ entry through voltage-gated Ca²⁺ channels (positive control). The main chart shows mean [Ca²⁺]_i before (control), during (PGE₂), and after washout (wash) of 1 μM PGE₂ and the response to 50 mM KCl (n = 9 cells from seven independent experiments).

Fig. 4.

EP receptor mRNA expressed in mouse adrenal tissue. A, RT-PCR was used to detect expression of the EP3 receptor in mouse adrenal tissue (left) and kidney tissue (right) that was isolated in parallel as a positive control. Top, primers common to all splice variants of the EP3 receptor were used. Bottom, primers selective for the splice variants were used. The forward primers for EP3α and EP3β are identical so the fragments run in the same lane (middle): the top band corresponds with the expected amplicon size of EP3α and the bottom band EP3β. B, in addition to EP3, EP1 (top), EP2 (middle), and EP4 (bottom) mRNA were amplified. All samples from A and B expressed the internal standard GAPDH (data not shown). Data shown are representative of three replicate experiments on tissue from three different mice.

Fig. 5.

Pharmacological evidence that EP3 receptors mediate the inhibition of I_Ca by PGE₂. A, the selective EP1/EP3 receptor agonist sulprostone inhibits I_Ca. Representative voltage command (top) and I_Ca (bottom) recorded in presence and absence of sulprostone (100 nM) obtained in the perforated whole-cell recording configuration. B, bar chart illustrating the mean percentage of inhibition of I_Ca produced by PGE₂ (100 nM) or sulprostone (100 nM). The inhibition produced by the two agonists was not significantly different. C and D, DG-041, a selective EP3 receptor antagonist, blocked the inhibition of I_Ca produced by PGE₂. C, experimental time course in a representative cell plotting peak amplitude of I_Ca against time. DG-041 (30 nM) was applied ∼2 min before PGE₂ (100 nM) and completely blocked the inhibition of I_Ca but had no effect on the inhibition produced by the P2Y receptor agonist ATP (100 μM). D, bar chart summarizing the percentage inhibition of I_Ca by the application of 30 nM DG-041 alone (DG) and in the presence of either 100 nM PGE₂ (DG + PGE₂; n = 9) or 100 μM ATP (DG + ATP; n = 4). DG-041 prevented the inhibition produced by PGE₂ but not that produced by ATP.

Fig. 6.

The inhibition of I_Ca produced by PGE₂ was abolished in cells isolated from EP3 receptor knockout mice. A, experimental time course plotting peak amplitude of I_Ca versus time from a representative cell isolated from an EP3 receptor knockout mouse [EP3(−/−) cells]. I_Ca was recorded in the conventional whole-cell configuration and elicited every 10 s with a 20-ms step depolarization from −80 to +20 mV. The cell was exposed first to 100 nM PGE₂ and subsequently to 100 μM ATP (to activate P2Y receptors), as indicated by the horizontal bars. PGE₂ had no effect on I_Ca recorded from EP3(−/−) chromaffin cells, whereas the inhibition produced by P2Y receptors remained intact. The inset shows three superimposed currents recorded before application of PGE₂ (ctl) during application of PGE₂ and during application of ATP. B, bar chart plotting the effects of PGE₂ and ATP on the mean peak amplitude of I_Ca in EP3(−/−) chromaffin cells (*, p < 0.05; n = 6). C, data obtained from wild-type and EP receptor knockout mice using perforated whole-cell recording. Left, mean percentage of inhibition of I_Ca produced by PGE₂ in cells isolated from wild-type (wt) (n = 15) versus EP3 receptor knockout mice (EP3(−/−) (n = 6) (***, p < 0.001). Right, percentage of inhibition of I_Ca produced by sulprostone (an EP1/EP3 selective agonist) in cells isolated from wild-type mice (wt) (n = 6) versus EP1 receptor knockout mice (EP1(−/−) (n = 4). (Wild-type data are from the same cells shown in Fig. 5B.).

Fig. 7.

Parallel inhibition of I_Ca and Ca²⁺-dependent exocytosis by PGE₂. Perforated whole-cell recording was used to measure I_Ca and Cm in chromaffin cells isolated from wild-type mice. A, voltage command (top), I_Ca (middle), and membrane capacitance (bottom) recorded from a representative cell. Two superimposed recordings are shown in the absence (control) and presence of 100 nM PGE₂. The stimulus (top) consisted of two step-depolarizations (100-ms duration) from −80 to +10 mV. A 1-kHz sine wave was superimposed on the holding potential to calculate membrane capacitance (see Materials and Methods for details) and this was interrupted during the step-depolarizations as indicated. B, peak amplitude of I_Ca in the presence of PGE₂ was normalized to control I_Ca amplitude in the same cell (□, control). Cells were separated into two groups based on the response of I_Ca to application of PGE₂: group 1 (■; n = 7 of 12 cells), in which PGE₂ significantly reduced the amplitude of I_Ca, and group 2 (▩; n = 5 of 12 cells), in which PGE₂ did not inhibit I_Ca (*, p < 0.05 comparing group 1 and group 2 in the presence of PGE₂). C, the change in membrane capacitance (ΔCm) in response to stimulation in the presence of PGE₂ was normalized to ΔCm in control conditions in the same cell. ■, (group 1) data from cells in which I_Ca was inhibited (7 of 12 cells), and the ▩ (group 2) show data from cells in which I_Ca was not inhibited. ΔCm was reduced in both groups but the inhibition was significantly greater in group 1 compared with group 2 (*, p < 0.05) (i.e., in those cells in which I_Ca was also reduced).