Katalin Bartus, Giti Garthwaite, & John Garthwaite
Wolfson Institute for Biomedical Research, University College London, Gower Street, London, UK

https://doi.org/10.36866/pn.67.25

Looking through a microscope at a histological section of brain, even at high magnification, the microvasculature usually passes unnoticed. Yet, dotted around are huge numbers of unimposing structures, only about 6 µm in diameter and mostly empty – the capillaries. When assembled in 3-dimensions, however, the capillaries form a complex labyrinth of tubing (Fig. 1A, B), any single part of which is maximally a typical cell diameter (25 µm) away from any neuronal or glial element. Of course, this plumbing arrangement is optimised for delivering and collecting dissolved gases needed for, or produced by, neuronal and glial cell metabolism. Now, it seems, the maze of microvessels also supplies its own dissolved gas, nitric oxide (NO), to affect the functioning of the brain.

The famous experiments of Robert Furchgott revealing the existence of an endothelium-derived relaxing factor (Furchgott & Zawadzki, 1980) and the subsequent identification of the factor as NO helped elevate the status of the endothelium from simply being the passive inner lining of blood vessels and introduced a new concept of cell-to-cell signalling that, in different ways, quickly became of importance to physiology and pathophysiology throughout the body. Endothelial cells manufacture NO from L-arginine using the eponymous endothelial NO synthase (eNOS) that is complexed with other proteins within specialised invaginations of the cell membrane. In the brain, eNOS is concentrated in the capillary network which lacks smooth muscle and is constituted largely of endothelial cells. The NO-synthesising enzyme within the neural tissue itself is mainly located in neurones (hence, nNOS) and is targeted to synapses where it binds to scaffold proteins that also link to the NMDA subclass of glutamate receptors found in most excitatory synapses. A third isoform, the inducible NO synthase, is not normally present but can be expressed in many different cell types during inflammatory conditions (Alderton et al. 2001).

The physicochemical properties of NO set it apart from other signalling molecules. Firstly, it lacks chemical specialisation, although it does possess an extra (unpaired) electron, making it a radical. Some other radicals are chemically reactive and can cause damage to cells, but NO is stable in physiological concentrations, which are probably around a nanomole or less. Secondly, like oxygen and carbon dioxide, NO diffuses very quickly through membranes, obviating the need for a specialised release mechanism and endowing it with the ability to act on neighbouring cells within microseconds of its manufacture. Finally, NO binds avidly to haem groups possessing vacant coordination sites. This property has been exploited to provide highly sensitive NO detectors within cells, initiating physiological NO signal transduction. The relevant haem groups are attached to proteins possessing intrinsic guanylyl cyclase activity and, unlike the haems of haemoglobin, they exclude oxygen, allowing NO to bind without becoming oxidised. The binding of NO triggers a conformational change in the protein that propagates to the active site, resulting in the formation of cyclic GMP (cGMP) from GTP. In this way, NO concentrations of 1 nM or less can be detected and transduced into greatly amplified cGMP concentrations (Garthwaite, 2005).

The conventional mechanism for NO formation in the brain is activation of synaptic NMDA receptors by the neurotransmitter glutamate. The physical association of nNOS with NMDA receptors and the high permeability of NMDA receptor channels to calcium ions, on which nNOS activity depends (via calmodulin), combine to explain this special relationship. NMDA receptors are well-known for their role in the initiation of synaptic plasticity and studies on many different areas of the central nervous system have found that NO participates in certain forms of long-term potentiation (e.g. in the hippocampus, cerebral cortex, cerebellum and spinal cord) or depression (e.g. in the cerebellum and striatum). Here, NO probably operates only very locally, its sphere of influence being limited to the dimensions of a single synapse (Garthwaite, 2005).

Evidence that NO made in blood vessels provides signals to neurones came from two related studies. The first was carried out on a synapse-free stretch of central white matter, the optic nerve, in which axons run from the retina to the visual centres of the brain (Fig. 1A, B). Our curiosity was raised by two unexpected observations. The first was that optic nerve axons are greatly enriched in NO receptors so that exposure to NO causes cGMP to accumulate in them to high levels. The second observation was that, through cGMP, exogenous NO causes the axons to depolarize by a few millivolts. For these findings to have any physio-logical meaning, there would need to be a local source of NO. Sure enough, when NO synthase activity was blocked, the axons hyperpolarized by a few millivolts, implying that NO was continuously being formed in sufficient amounts to keep them depolarized. The source was eventually identified as eNOS in endothelial cells. Moreover, increasing or decreasing the activity of eNOS caused opposite changes in the membrane potential of the axons, signifying a dynamic coupling between endothelial cell activity and axon function (Garthwaite et al. 2006). The voltage responses in the axons were brought about by alterations in the activity of a class of cyclic nucleotide-regulated ion channels (Fig. 1C). Among other functions, these channels help maintain reliable conduction during high frequency axonal firing.

The second study originated in trying to understand the role played by NO in synaptic plasticity in the hippocampus where we were puzzled to find that there needs to be a continuous low-level of endogenous NO in order for the synapses to become enduringly potentiated when exogenous NO is given at the same time as a weak synaptic stimulation. Again, eNOS present in blood vessels was pinpointed as the source of the continuous NO supply (Hopper et al. 2006). These findings lead to a picture in which NO derived from eNOS provides a global ‘enabling’ signal to the hippocampal synapses, priming them to respond to discrete nNOS-derived signals when NMDA receptors become active.

Although many details of the underlying mechanisms are missing, the notion that endothelium-derived NO affect brain neurones has circumstantial support from various quarters. For example, mice lacking eNOS show defective synaptic plasticity not only in the hippocampus but also in the cerebral cortex and striatum. Furthermore, they also exhibit various neurochemical and behavioural phenotypes (e.g. much reduced male aggression) and decreased neurogenesis (for references, see Garthwaite et al. 2006; Hopper & Garthwaite, 2006).

Many fascinating questions remain about the function of this line of communication. Are blood vessels persistently contributing to neuronal excitability in regions other than the optic nerve? Does the pathway provide a new link between the periphery and central nervous system and help explain, for instance, how hormones (e.g. oestrogens) or physical exercise, both of which increase eNOS activity, influence brain function? Could the loss of eNOS activity known to be caused by β-amyloid contribute to the symptoms of Alzheimer’s disease?

Acknowledgement

The work from our laboratory described in this article was funded by The Wellcome Trust.

References

Alderton WK, Cooper CE & Knowles RG (2001). Nitric oxide synthases: structure, function and inhibition. Biochem J 357, 593-615.

Furchgott RF & Zawadzki JV (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376.

Garthwaite G, Bartus K, Malcolm D, Goodwin DA, Kollb-Sielecka M, Dooldeniya C & Garthwaite J (2006). Signaling from blood vessels to CNS axons through nitric oxide. J Neurosci 26, 7730-7740.

Garthwaite J (2005). Dynamics of cellular NO-cGMP signaling. Front Biosci 10, 1868-1880.

Hopper RA & Garthwaite J (2006). Tonic and phasic nitric oxide signals in hippocampal long-term potentiation. J Neurosci 26, 11513-11521.