Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells

Abstract

Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O₂ and CO₂/H⁺ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 μM) had negligible effects on basal [Ca²⁺]_i in isolated rat type II cells. However, DA strongly inhibited [Ca²⁺]_i elevations (Δ[Ca^2+]_i) evoked by the P2Y2R agonist UTP (100 μM), an effect opposed by the D2/3R antagonist, sulpiride (1-10 μM). As expected, acute hypercapnia (10% CO₂; pH 7.4), or high K⁺ (30 mM) caused Δ[Ca²⁺]_i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca²⁺]_i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca²⁺]_i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca²⁺]_i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca²⁺]_i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.

Keywords: carotid body; dopamine; petrosal neurons; purinergic signalling; sulpiride; type II cells.

Figure 1

Dopamine attenuates purinergic signaling in type II cells. (A) Representative trace showing the reduction of the intracellular Ca²⁺ ([Ca²⁺]_i) response to UTP (100 µM) during application of DA (10 µM) in type II cells (blue trace); contrast the type I cell (red trace) which only responded to high K⁺. (B) Summary data of UTP-evoked integrated [Ca²⁺]_i (nM∙S) response before, during, and after DA perfusion (n = 8 dishes/group, 10–25 cells sampled per dish). In (B) 221 of the 298 type II cells showed a reduction in the UTP response in the presence of DA. (C) Mean duration (s) of the UTP-evoked [Ca²⁺]_i response in type II cells before, during, and after DA (10 µM) perfusion. Data were analysed using a one-way repeated measures analysis of variance (ANOVA) followed by Tukey’s post hoc test; ** signifies a p value of < 0.01. Values are means ± S.E.M.; n = 8 dishes.

Figure 2

Sulpiride, a D2/3R antagonist, reverses the inhibitory effect of dopamine on the UTP-evoked intracellular Ca²⁺ rise in type II cells. (A,D) Representative type I and type II cell traces showing the [Ca²⁺]_i response to UTP (100 µM), UTP + DA (10 µM), UTP + DA +,Sulpiride (SULP; 10 µM (A), 1 µM (D)), and UTP alone (after washout of DA and SULP). Note Sulpiride reversed the DA inhibition of UTP-evoked [Ca²⁺]_i response in the type II cell; the type I cell only responded to high K⁺. Summary data of the UTP-evoked integrated [Ca²⁺]_i (nM∙s) (B,E) and duration of the [Ca²⁺]_i responses (C,F) in type II cells before, during, and after exposure to DA, or DA plus Sulpiride (n = 3–5 dishes/group, 10–15 cells sampled per dish). In these experiments, 52 of the 101 cells showed both a reduction in the UTP-evoked response in the presence of DA and subsequent recovery of the response during co-application with Sulpiride. Data were analysed using a one-way repeated measures analysis of variance (ANOVA) followed by Tukey’s post hoc test; * signifies a p value of < 0.05. Values are means ± S.E.M.

Figure 3

Sulpiride potentiates crosstalk among type I and type II cells during hypercapnia and high K⁺ exposure. Representative traces showing sulpiride-mediated potentiation of type I cell [Ca²⁺]_i responses and the unveiling or potentiation of the delayed type II cell responses during stimulation with hypercapnia (A) and high K⁺ (D). Summary data of integrated [Ca²⁺]_i responses (nM∙s) to hypercapnia are shown for type II (B) and type I (C) cells; n = 5 separate cultures/group, 3–25 cells sampled per dish. Similar data for the high K⁺ stimulus are shown for type II (E) and type I (F) cells; n = 7 separate cultures/group, 3–25 cells sampled per dish). Data were analysed using a one-way repeated measures analysis of variance (ANOVA) followed by Tukey’s post hoc test; *, and ** signifies a p value of < 0.05 and 0.01, respectively. Values are means ± S.E.M.

Figure 4

Depleting vesicular type I cell dopamine stores using reserpine enhances crosstalk from type I to type II cells. Representative traces showing intracellular Ca²⁺ responses of type I and type II cells from a control (A) and (C) reserpine-treated (C) ‘sister’ culture as a result of exposure to high K⁺ and UTP. (B) Integrated [Ca²⁺]_i in type II cells in response to high K⁺ depolarization of neighboring type I cells in control versus reserpine-treated sister cultures. Blue lines connect mean values from type II cells imaged on the same day from paired ‘sister’ cultures. (D) Percent (%) of type II cells displaying crosstalk events during from high K ⁺ depolarization of type I cells in sister cultures. In B, * denotes significant difference between control and reserpine-treated cells, evaluated using Student’s t-test (* = p < 0.05). In D, a one-way ANOVA followed by Tukey’s post hoc test was used; *** p < 0.001. Values are means ± S.E.M.; n = 8 dishes, 3–20 cells tested per dish.

Figure 5

Sulpiride enhances hypercapnia-evoked responses in type I cells, type II cells, and petrosal neurons during simultaneous Ca²⁺ imaging in functional cocultures. (A) Representative traces showing the [Ca²⁺]_i responses in a type I cell (red), type II cell (blue), and petrosal neuron (green), recorded simultaneously during hypercapnia (10% CO₂) in a carotid body coculture. (B) Typical phase contrast micrograph of a coculture showing a petrosal neuron (PN) adjacent to a type I/type II cell cluster; a solitary, isolated type II cell with its characteristic elongated shape is also shown (left). In A, note the marked potentiation of the hypercapnic responses in all 3 cell types when sulpiride was present, as well as the responses of the 3 cells to UTP. The UTP-evoked responses (dotted circle in A) are enlarged in (F) to reveal differences in response latency. Summary data of response latency (sec) of PN and type I cells relative to that in type II cell are shown in (G); note PN responses precede those in type I cells during direct stimulation of type II cells with UTP (*p < 0.05; n = 4 separate cultures). Histograms (C–E,H–J) show integrated [Ca²⁺]_i response (nM∙s) and mean duration of the [Ca²⁺]_i response during hypercapnia in type I cells (C,H), type II cells (D,I), and PN (E,J); n = 3 separate cultures/group. Data were analysed using a one-way repeated measures analysis of variance (ANOVA) followed by Tukey’s post hoc test; (*, **, and *** signifies a p value of < 0.05, 0.01 and 0.001, respectively. Values are means ± S.E.M.).

Figure 6

Model of the proposed interactions involving ATP and dopamine at the carotid body ‘tripartite’ synapse. During hypoxia or hypercapnia type I cells depolarize due to inhibition of Two-pore domain, Acid-Sensitive K⁺ (TASK) channels, leading to membrane depolarization, Ca²⁺ entry through voltage-gated Ca²⁺ channels (VGCC) and vesicular release of ATP and DA. ATP activates postsynaptic P2X2/3R on petrosal afferent terminals causing excitation. ATP also activates P2Y2R on adjacent type II glial cells, causing a rise in intracellular Ca²⁺ via the PLC-IP₃-Ca²⁺ pathway. This leads to Ca²⁺-dependent opening of pannexin (Panx)-1 channels which act as conduits for the release of ATP, thereby contributing directly to petrosal excitation. Co-released DA acts on autocrine-paracrine D2 receptors on the same or adjacent type I cells, leading to a negative feedback inhibition of neurotransmitter release. DA can also act on postsynaptic D2 receptors on petrosal terminals, leading to inhibition of action potential firing. DA may also activate sulpiride-sensitive (D2 and/or D3) receptors on type II cells, leading to a blunting of the ATP-P2Y2R -mediated rise in intracellular Ca²⁺ by an unknown mechanism. In this way, DA limits the ability of type II cells to contribute to the synaptic ATP pool via the mechanism of ‘ATP-induced ATP release’ involving Panx-1 channels. Omitted for clarity are paracrine signalling pathways involving other CB neurotransmitters including adenosine, generated in part from the breakdown of extracellular ATP.