NADPH oxidase contributes to angiotensin II signaling in the nucleus tractus solitarius

Abstract

Angiotensin II (AngII), acting through angiotensin type 1 (AT1) receptors, exerts powerful effects on central autonomic networks regulating cardiovascular homeostasis and fluid balance; however, the mechanisms of AngII signaling in functionally defined central autonomic neurons have not been fully elucidated. In vascular cells, reactive oxygen species (ROS) generated by the enzyme NADPH oxidase play a major role in AngII signaling. Thus, we sought to determine whether NADPH oxidase is present in central autonomic neurons and, if so, whether NADPH oxidase-derived ROS are involved in the effects of AngII on these neurons. The present studies focused on the intermediate dorsomedial nucleus of the solitary tract (dmNTS) because this region receives autonomic afferents via the vagus nerve and is an important site of AngII actions. Using double-label immunoelectron microscopy, we found that the essential NADPH oxidase subunit gp91phox is present in somatodendric and axonal profiles containing AT1 receptors. The gp91phox-labeled dendrites received inputs from large axon terminals resembling vagal afferents. In parallel experiments using patch clamp of dissociated NTS neurons anterogradely labeled via the vagus, we found that AngII potentiates the L-type Ca2+ currents, an effect mediated by AT1 receptors and abolished by the ROS scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride. The NADPH oxidase assembly inhibitor apocynin and the peptide inhibitor gp91phox docking sequence, but not its scrambled version, also blocked the potentiation. The results provide evidence that NADPH oxidase-derived ROS are involved in the effects of AngII on Ca2+ influx in NTS neurons receiving vagal afferents and support the notion that ROS are important signaling molecules in central autonomic networks.

Figure 1.

Electron micrographs showing gp91^phox subcellular distribution and coexpression with AT₁ receptors in neuronal perikarya in the dorsomedial NTS. A, B, In tissue processed only for immunoperoxidase labeling of gp91^phox, the reaction product (block arrows) is seen within and near the plasma membrane and also near mitochondria (m) and Golgi lamellae (Go) in somatic profiles. C, In dually labeled tissue, the gp91^phox immunogold (small arrows) has a comparable plasmalemmal and cytoplasmic distribution in a perikaryon that also contains immunoperoxidase identifying the AT₁ receptor (block arrows). The nuclei (Nu) in A-C are without immunolabeling for either marker. Scale bars, 0.5 μm.

Figure 2.

Dendritic and axonal distribution of gp91^phox and AT₁ receptors in dmNTS. A, Immunoperoxidase labeling for gp91^phox is seen along perisynaptic portions of the plasma membrane and near membranes of smooth endoplasmic reticulum (ser) and a multivesicular body (mvb) in a dendrite receiving input from an unlabeled axon terminal (ut). B, In dual-labeled tissue, immunogold gp91^phox (thin arrows) and immunoperoxidase (block arrows) labeling for the AT₁ receptor are seen within the cytoplasm of a dendritic profile receiving an asymmetric synapse (curved arrow) from an unlabeled terminal (ut). The unlabeled terminal is large and also contacts a small, unlabeled dendritic profile (ud). C, Immunogold labeling (small arrows) for gp91^phox and immunoperoxidase labeling (block arrows) for the AT₁ receptor are localized in an axon terminal forming an asymmetric synapse (curved arrow) with a small unlabeled dendrite (ud). A mitochondrion (m) is seen near portions of the plasmalemma showing AT₁ receptor labeling. D, A dually labeled small dendrite (dd) with diffuse immunoperoxidase labeling of AT₁ receptor and a cluster of immunogold (small arrows) labeling for gp91^phox receives an excitatory synapse (curved arrow) from an unlabeled terminal (ut) and a symmetric synapse (black pentagon) from a dually labeled terminal (l-te). The immunoperoxidase labeling in the terminal is light and diffusely distributed (white block arrow) around a large dense-core vesicle and a mitochondrion (m) near the presynaptic membrane specialization. Astrocytic processes (asterisk) are identified in C and D, and one mitochondrion (m) is indicated near the immunolabeled plasma membrane in C. Scale bars, 0.5 μm.

Figure 3.

Properties of voltage-gated Ca²⁺ currents in second-order sensory NTS neurons. A, An isolated labeled NTS neuron was seen in bright field (left) or fluorescence (right). Scale bar, 20 μm. B, Representative traces of whole-cell, voltage-gated Ca²⁺ channel currents elicited from the holding potential (HP) of -60 mV to the stepping potential (SP) of -20 mV in a labeled NTS neuron. The L-type Ca²⁺ channel activator BayK (2 μm) was applied after the N-type Ca²⁺ channel blocker GVIA (400 nm) inhibited the transient Ca²⁺ current. The dashed line indicates zero level of the current. C, Percentage of changes in the amplitude of the L-type and transient Ca²⁺ currents, respectively, are shown in control (Ctrl) conditions and in the presence of BayK (2 μm) (n = 5), nifedipine (nifed) (1 μm) (n = 4), GVIA (400 nm) (n = 3), or Cd²⁺ (1 mm) (n = 3). ^*p < 0.05.

Figure 4.

AngII potentiates L-type Ca²⁺ currents in second-order sensory NTS neurons. A, Representative traces of the whole-cell Ca²⁺ current in control conditions, with AngII (2 μm), or after wash with the control buffer. The dashed line indicates zero level of the current. HP = -60 mV; SP = -20 mV. B, Percentage of changes in amplitudes of the L-type and transient Ca²⁺ currents in control (Ctrl) conditions, with AngII (2 μm) (n = 8), or after wash with the control buffer (n = 4). C, The concentration-response curve of the AngII-mediated effect on L-type Ca²⁺ currents. The EC₅₀ was 37.4 nm, and the Hill slope coefficient was 1.45. The dashed line is the fitted curve. D, The I-V curve of L-type Ca²⁺ currents in control (♦) conditions shows that this current is high-voltage threshold activated (n = 4). In the presence of AngII (100 nm) (□), the amplitude of the L-type Ca²⁺ current increased only at a range of negative stepping potentials, especially at -10 and -20 mV (n = 4). ^*p < 0.05.

Figure 5.

AngII potentiates L-type Ca²⁺ currents via AT₁ receptors and ROS. A, Representative traces of the Ca²⁺ currents from a labeled NTS neuron in control (Ctrl) conditions, with AngII (2 μm), or AngII (2 μm) plus losartan (L) (2 μm). B, Percentage of changes in the amplitude of L-type Ca²⁺ currents under the experimental conditions described in the A (n = 4). C, Representative traces of the Ca²⁺ currents from a labeled NTS neuron in control (Ctrl) conditions, with AngII (2 μm), or AngII (2 μm) plus MnTBAP (M) (30 μm). D, Percentage of changes in the amplitude of L-type Ca²⁺ currents under the experimental conditions described in C (n = 4). E, Representative traces of the Ca²⁺ currents in a labeled NTS neuron in control (Ctrl) conditions, with AngII (2 μm) or AngII (2 μm) plus apocynin (A) (1 mm). F, Percentage of changes of the amplitude of L-type Ca²⁺ currents under the experimental conditions described in E (n = 4). ^*p < 0.05. The dashed line indicates zero level of the current. HP = -60 mV; SP = -20 mV. Note that the voltage-gated Na⁺ current at the beginning of the depolarizing pulse is followed by the Ca²⁺ currents.

Figure 6.

The peptide gp91ds blocks the AngII-mediated potentiation of L-type Ca²⁺ currents. A, Representative traces of Ca²⁺ currents in a labeled NTS neuron in response to AngII (2 μm) or BayK (2 μm) in the presence of gp91ds (1 μm) in the pipette solution. B, Percentage of changes in the amplitude of L-type Ca²⁺ currents under the experimental conditions described in A (n = 4). C, Representative traces of Ca²⁺ currents in another labeled NTS neuron in response to AngII (2 μm) in the presence of scrambled peptide (1 μm) in the pipette solution. D, Percentage of changes in the amplitude of L-type Ca²⁺ currents in the NTS neurons under the experimental conditions described in C (n = 4). ^*p < 0.05. The dashed line indicates zero level of the current. HP = -60 mV; SP = -20 mV. Note that the voltage-gated Na⁺ current at the beginning of the depolarizing pulse is followed by the Ca²⁺ currents.