The Unexpected Role of the Endothelial Nitric Oxide Synthase at the Neurovascular Unit: Beyond the Regulation of Cerebral Blood Flow

Abstract

Nitric oxide (NO) is a highly versatile gasotransmitter that has first been shown to regulate cardiovascular function and then to exert tight control over a much broader range of processes, including neurotransmitter release, neuronal excitability, and synaptic plasticity. Endothelial NO synthase (eNOS) is usually far from the mind of synaptic neurophysiologists, who have focused most of their attention on neuronal NO synthase (nNOS) as the primary source of NO at the neurovascular unit (NVU). Nevertheless, the available evidence suggests that eNOS could also contribute to generating the burst of NO that, serving as volume intercellular messenger, is produced in response to neuronal activity in the brain parenchyma. Herein, we review the role of eNOS in both the regulation of cerebral blood flow and of synaptic plasticity and discuss the mechanisms by which cerebrovascular endothelial cells may transduce synaptic inputs into a NO signal. We further suggest that eNOS could play a critical role in vascular-to-neuronal communication by integrating signals converging onto cerebrovascular endothelial cells from both the streaming blood and active neurons.

Keywords: cerebrovascular endothelial cells; endothelial nitric oxide synthase; long-term potentiation; neurovascular coupling; neurovascular unit; nitric oxide; vascular-to-neuronal communication.

Figure 1

Schematic representation of the NVU. The penetrating arterioles depart from the pial arteries and are surrounded by the perivascular space, also known as Virchow–Robin space, which may house perivascular macrophages and other cell types, including mast cells and Mato cells [5,10]. Their wall includes 1–3 layers of VSMCs that determine their resistance to blood flow: an increase in the contracting state reduces blood perfusion, while a decrease in the contracting state increases blood perfusion to downstream capillaries. The outer limit of the perivascular space is lined by the astrocyte end-feet, which give rise to the glia limitans membrane. When the basal lamina fuses with the glia limitans, the perivascular space is obliterated, and the penetrating arterioles become intraparenchymal arterioles. In the capillary vessels, VSMCs are replaced by pericytes, which are contractile cells closely embedded in the vascular wall, and the outer wall is still contacted by the astrocytic end-feet. Intraparenchymal arterioles and capillaries receive intrinsic innervation from local interneurons and subcortical pathways. Modified from [26] (https://creativecommons.org/licenses/by/4.0/ (accessed on 18 June 2024)).

Figure 2

NO signaling at the NVU. Synaptic activity leads to extracellular Ca²⁺ influx through NMDARs, thereby stimulating CaM activity. Then, CaM promotes the physical association of PSD-95 to the PDZ motif in the NMDAR protein, and PSD-95 and CaM assemble into a trimeric complex with nNOS. Ca²⁺ entry through NMDARs can also activate eNOS, but it is still unclear whether this mechanism requires the involvement of PSD-95. nNOS and cNOS catalyze the 5-electron oxidation of L-arginine into L-citrulline and NO by using the cofactors NADPH and BH4. Once synthetized, NO regulates neurotransmitter release, promotes LTP, and increases local CBF by promoting VSMC and pericyte relaxation.

Figure 3

The mechanisms of NO-mediated vasorelaxation. NO can be produced by eNOS upon synaptic activation of NMDARs, although it is likely that any increase in endothelial [Ca²⁺]_i stimulates eNOS at the NVU [25,48]. Endothelium-derived NO may then diffuse to overlying VSMCs to induce relaxation by stimulating sGC activity, thereby resulting in GTP conversion into cGMP. In accordance, cGMP activates PKG, which in turn activates myosin light-chain phosphatase (MLCP) and inhibits myosin light-chain kinase (MLCK), thereby reducing the Ca²⁺-sensitivity of the contractile machinery. Moreover, PKG reduces VSMC excitability by activating large-conductance Ca²⁺-activated K⁺ channels (BK_Ca) and inhibiting voltage-gated Ca_V1.2 channels, thereby promoting VSMC hyperpolarization and preventing voltage-gated Ca²⁺ entry. The signal transduction pathway elicited by NO can be interrupted when cGMP is hydrolyzed into an inactive 5′-GMP metabolite by phosphodiesterase (PDE) activity.

Figure 4

The central role of eNOS in vascular-to-neuronal communication. Neuronal activity (possibly with the intermediation of astrocyte-derived signals) and blood-born mechanical signals (e.g., local changes in CBF evoked by the same neuronal activity or single-file transit of RBCs) lead to an increase in [Ca²⁺]_i in cerebrovascular endothelial cells, thereby leading to eNOS activation and NO release. NO serves as a volume intercellular messenger that can target multiple cell types within the NVU: (1) neurons, thereby supporting LTP; (2) mural cells, i.e., VSMCs and pericytes, thereby promoting vasorelaxation; and (3) astrocytes, thereby eliciting intracellular Ca²⁺ signals. Inspired by [18,19].