Tomas C. Bellamy

On the activation of soluble guanylyl cyclase by nitric oxide

Soluble guanylyl cyclase (sGC) is the major cellular receptor for the intercellular messenger nitric oxide (NO) and mediates a wide range of physiological effects through elevation of intracellular cGMP levels. Critical to our understanding of how NO signals are decoded by receptive cells and translated into a useful physiological response is an appreciation of the molecular and kinetic details of the mechanism by which NO activates sGC. It is known that NO binds to a haem prosthetic group on the receptor and triggers a conformational change that increases the catalysis of cGMP synthesis by several hundred-fold. The haem is covalently attached to sGC at His-105 of the β1 subunit, and it was thought previously that activation of sGC by NO occurs in two steps: binding of NO to the haem to form a biliganded state and then rupture of the bond to His-105 triggering an increase in catalytic activity. A recent investigation of the kinetics of sGC activation [Zhao, Y., Brandish, P. E., Ballou, D. P. & Marletta, M. A. (1999) Proc. Natl. Acad. Sci. USA, 96, 14753–14758], however, proposed an additional mechanism by which NO regulates sGC activity, namely, by influencing the rate of cleavage of the His-105 bond. The existence of a second (unidentified) NO-binding site on the enzyme was hypothesized and suggested to be fundamental to cellular NO-signal transduction. Here, we show that it is unnecessary to postulate any such additional mechanism because the results obtained are predicted by the simpler model of sGC activation with a single NO-binding event.

Differential Sensitivity of Guanylyl Cyclase and Mitochondrial Respiration to Nitric Oxide Measured Using Clamped Concentrations

Nitric oxide (NO) signal transduction may involve at least two targets: the guanylyl cyclase-coupled NO receptor (NOGCR), which catalyzes cGMP formation, and cytochrome c oxidase, which is responsible for mitochondrial O2 consumption and which is inhibited by NO in competition with O2. Current evidence indicates that the two targets may be similarly sensitive to NO, but quantitative comparison has been difficult because of an inability to administer NO in known, constant concentrations. We addressed this deficiency and found that purified NOGCR was about 100-fold more sensitive to NO than reported previously, 50% of maximal activity requiring only 4 nm NO. Conversely, at physiological O2concentrations (20–30 μm), mitochondrial respiration was 2–10-fold less sensitive to NO than estimated beforehand. The two concentration-response curves showed minimal overlap. Accordingly, an NO concentration maximally active on the NOGCR (20 nm) inhibited respiration only when the O2concentration was pathologically low (50% inhibition at 5 μm O2). Studies on brain slices under conditions of maximal stimulation of endogenous NO synthesis suggested that the local NO concentration did not rise above 4 nm. It is concluded that under physiological conditions, at least in brain, NO is constrained to target the NOGCR without inhibiting mitochondrial respiration.

Interactions between Purkinje neurones and Bergmann glia

Throughout the development of the cerebellar cortex, Purkinje neurones interact closely with Bergmann glial cells, a specialized form of astrocyte. This review summarizes the intimate developmental, anatomical and functional relationships between these two cell types, with particular emphasis on recent discoveries regarding glutamate release from climbing and parallel fibres as a pathway for signalling synaptic activity to Bergmann glia

The receptor-like properties of nitric oxide-activated soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO), and so mediates a wide range of effects (e.g. vasodilatation, platelet disaggregation and neural signalling) through the accumulation of cGMP and the engagement of various downstream targets, such as protein kinases and ion channels. Until recently, our understanding of sGC functioning has been derived exclusively from studies of the enzyme in tissue homogenates or in its purified form. Here, NO binds to the haem prosthetic group of sGC, triggering a conformational change and a large increase in catalytic activity. The potency (EC50) of NO appears to be about 100–200 nM. The rate of activation of sGC by NO is rapid (milliseconds) and, in the presence of excess substrate, cGMP is formed at a constant rate; on removal of NO, sGC deactivates slowly (seconds–minutes). Recent investigation of the way that sGC behaves in its natural environment, within cells, has revealed several key differences. For example, the enzyme exhibits a rapidly desensitizing profile of activity; the potency of NO is 45 nM for the minimally-desensitized enzyme but becomes higher with time; deactivation of sGC on removal of NO is 25-fold faster than the fastest estimate for purified sGC. Overall, within cells, sGC behaves in a way that is analogous to the way that classical neurotransmitter receptors operate. The properties of cellular sGC have important implications for the understanding of NO-cGMP signalling. For example, the dynamics of the enzyme means that fluctuations in the rate of NO formation, even on subsecond time scale, will result in closely synchronized sGC activity in neighbouring cells; desensitization of sGC provides an economical way of generating a cellular cGMP signal and, in concert with phosphodiesterases, provides the basis for cGMP signal diversity, allowing different targets (outputs) to be selected from a common input (NO). Thus, despite exhibiting only limited molecular heterogeneity, cellular sGC functions in a way that introduces speed, complexity, and versatility into NO-cGMP signalling pathways.

Short-term plasticity of Bergmann glial cell extrasynaptic currents during parallel fiber stimulation in rat cerebellum.

Bergmann glial cells (BGC) enclose the synapses of Purkinje neurons (PN) and interneurons in the molecular layer of the cerebellar cortex. During synaptic transmission, glutamate evokes inward currents in the glia by activation of Ca2+‐permeable aminohydroxymethylisoxazole propionic acid receptors (AMPAR) and electrogenic transporters. We describe the plasticity of BGC currents during paired‐pulse and repetitive stimulation of parallel fibers in cerebellar slices. Paired‐pulse facilitation (PPF) of BGC AMPAR currents was 4‐fold, twice that of PN PPF. Experiments with a low‐affinity AMPAR antagonist showed an increase in extrasynaptic glutamate concentration during the second pulse of the pair. PPF of glial transporter currents was 1.8‐fold, similar to synaptic PPF. Tetanic stimulation revealed that facilitation of BGC AMPAR currents is not sustained during high‐frequency stimulation, and substantial depression is observed after a few pulses. Consequently, Ca2+ influx through glial AMPARs would initially be facilitated but subsequently depressed, generating a transient Ca2+ influx in response to a sustained tetanus. This pattern of plasticity may be important in enabling Bergmann glial cell processes to detect and support synapses with high‐frequency input. Finally, a new current was observed in BGC during repetitive stimulation. It was blocked by NBQX and intracellular GDP‐β‐S, increased by glutamate uptake inhibition, had PPF similar to synaptic PPF, and was unaffected by an inhibitor of fast glial AMPAR currents. The evidence suggests that activation of neuronal AMPARs causes the release of a paracrine messenger to activate a G‐protein coupled receptor in the BGC.

Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP.

Physiologically, nitric oxide (NO) signal transduction occurs through soluble guanylyl cyclase (sGC), which catalyses cyclic GMP (cGMP) formation. Knowledge of the kinetics of NO‐evoked cGMP signals is therefore critical for understanding how NO signals are decoded. Studies on cerebellar astrocytes showed that sGC undergoes a desensitizing profile of activity, which, in league with phosphodiesterases (PDEs), was hypothesized to diversify cGMP responses in different cells. The hypothesis was tested by examining the kinetics ofcGMP in rat striatal cells, in which cGMP accumulated in neurones in response to NO. Based on the effects of selective PDE inhibitors, cGMP hydrolysis following exposure to NO was attributed to a cGMP‐stimulated PDE (PDE 2). Analysis of NO‐induced cGMP accumulation in the presence of a PDE inhibitor indicated that sGC underwent marked desensitization. However, the desensitization kinetics determined under these conditions described poorly the cGMP profile observed in the absence of the PDE inhibitor. An explanation shown plausible theoretically was that cGMP determines the level of sGC desensitization. In support, tests in cerebellar astrocytes indicated an inverse relationship between cGMP level and recovery of sGC from its desensitized state. We suggest that the degree of sGC desensitization is related to the cGMP concentration and that this effect is not mediated by (de)phosphorylation.

Pharmacology of the nitric oxide receptor, soluble guanylyl cyclase, in cerebellar cells

The nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), is commonly manipulated pharmacologically in two ways. Inhibition of activity is achieved using 1‐H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐l‐one (ODQ) which oxidizes the haem prosthetic group to which NO binds, while the compound 3‐(5‐hydroxymethyl‐2‐furyl)‐1‐benzylindazole (YC‐1) is considered an ‘allosteric’ activator. Knowledge of how these agents function and interact in a normal cellular environment is limited. These issues were addressed using rat cerebellar cells. Inhibition by ODQ was not simply competitive with NO. The rate of onset was ODQ concentration‐dependent and developed in two kinetic phases. Recovery from inhibition occurred with a half‐time of ∼5 min. YC‐1 slowed the rate at which sGC deactivated on removal of NO by 45 fold, consistent with YC‐1 increasing the potency of NO for sGC. YC‐1 also enhanced the maximal response to NO by 2 fold. Furthermore, when added to cells in which sGC was 90% desensitized, YC‐1 abruptly enhanced sGC activity to a degree that indicated partial reversal of desensitization. After pre‐exposure to YC‐1, sGC became resistant to inhibition by ODQ. In addition, YC‐1 rapidly reversed inhibition by ODQ in cells and for purified sGC, suggesting that YC‐1 either increases the NO affinity of the oxidized sGC haem or reverses haem oxidation. It is concluded that the actions of ODQ and YC‐1 on sGC are broadly similar in cells and purified preparations. Additionally, YC‐1 transiently reverses sGC desensitization in cells. It is hypothesized that YC‐1 has multiple actions on sGC, and thereby both modifies the NO binding site and enhances agonist efficacy.

Long-term depression of neuron to glial signalling in rat cerebellar cortex.

Bergmann glial cells enclose synapses throughout the molecular layer of the cerebellum and express extrasynaptic AMPA receptors and glutamate transporters. Accordingly, stimulation of parallel fibres leads to the generation of inward currents in the glia due to AMPA receptor activation and electrogenic uptake of glutamate. Elimination of AMPA receptor Ca2+ permeability leads to the withdrawal of glial processes and synaptic dysfunction, suggesting that AMPA receptor‐mediated Ca2+ signalling is essential for glial support of the neuronal network. Here we show that glial extrasynaptic currents (ESCs) exhibit activity‐dependent plasticity, specifically, long‐term depression during repetitive stimulation of parallel fibres at low frequencies (0.033–1 Hz) − conditions in which Purkinje neuron excitatory postsynaptic currents (EPSCs) remain stable. Both the rate of onset and the magnitude of ESC depression increased with stimulation frequency. Depression was reversible following brief periods of stimulation, but became increasingly persistent as the duration of repetitive stimulation increased. All glial currents − AMPA receptors, glutamate transporter and a recently discovered slow 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo[f]quinoxaline‐7‐sulphonamide (NBQX)‐sensitive current − were depressed. Increasing presynaptic release probability by doubling external Ca2+ concentration did not affect the time course of depression, suggesting that neither decreased release probability nor fatigue of release sites contribute to depression. Inhibition of glutamate uptake caused a dramatic enhancement of the rate of depression, implicating glutamate in the underlying mechanism. The strength of neuron to glial signalling in the cerebellum is therefore dynamically regulated, independently of adjacent synapses, by the frequency of parallel fibre activity.

Depression of parallel and climbing fiber transmission to Bergmann glia is input specific and correlates with increased precision of synaptic transmission.

In the cerebellar cortex, Bergmann glia enclose the synapses of both parallel and climbing fiber inputs to the Purkinje neuron. The glia express Ca2+‐permeable AMPA receptors, and the GLAST and GLT‐1 classes of glutamate transporter, which are activated by glutamate released during synaptic transmission. We have previously reported that parallel fiber to Bergmann glial transmission in rat cerebellar slices exhibits a form of frequency‐dependent plasticity, namely long‐term depression, following repetitive stimulation at 0.1–1 Hz. Here, we report that this form of plasticity is also present at the climbing fiber input, that climbing and parallel fibers can be depressed independently, that discrete parallel fiber inputs can also be depressed independently, and that depression is maintained when a distributed array of parallel fibers are stimulated (in contrast to several forms of synaptic plasticity at the Purkinje neuron). Depression of glutamate transporter currents does not correlate with a decrease in the stringency with which Purkinje neuron synapses are isolated. Rather, postsynaptic currents in Purkinje neurons decay more rapidly and perisynaptic metabotropic glutamate receptors are activated less effectively after stimulation at 0.2 and 1 Hz, suggesting that depression arises from a decrease in extrasynaptic glutamate concentration and not from impairment of glutamate clearance in and around the synapse. These results indicate that neuron‐glial plasticity is activity dependent, input specific and does not require spillover between adjacent synapses to manifest. They also argue against a withdrawal of the glial sheath from synaptic regions as the putative mechanism of plasticity.

High-Throughput Analysis of Calcium Signalling Kinetics in Astrocytes Stimulated with Different Neurotransmitters

Astrocytes express a wide range of receptors for neurotransmitters and hormones that are coupled to increases in intracellular Ca2+ concentration, enabling them to detect activity in both neuronal and vascular networks. There is increasing evidence that astrocytes are able to discriminate between different Ca2+-linked stimuli, as the efficiency of some Ca2+ dependent processes – notably release of gliotransmitters – depends on the stimulus that initiates the Ca2+ signal. The spatiotemporal complexity of Ca2+ signals is substantial, and we here tested the hypothesis that variation in the kinetics of Ca2+ responses could offer a means of selectively engaging downstream targets, if agonists exhibited a “signature shape” in evoked Ca2+ response. To test this, astrocytes were exposed to three different receptor agonists (ATP, glutamate and histamine) and the resultant Ca2+ signals were analysed for systematic differences in kinetics that depended on the initiating stimulus. We found substantial heterogeneity between cells in the time course of Ca2+ responses, but the variation did not correlate with the type or concentration of the stimulus. Using a simple metric to quantify the extent of difference between populations, it was found that the variation between agonists was insufficient to allow signal discrimination. We conclude that the time course of global intracellular Ca2+ signals does not offer the cells a means for distinguishing between different neurotransmitters.