A major receptor for nitric oxide (NO) is the cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC), but it is not known how this enzyme behaves in cells. In cerebellar cells, NO (from diethylamine NONOate) increased astrocytic cGMP with a potency (EC 50 < 20 nM) higher than that reported for purified sGC. Deactivation of NO-stimulated sGC activity, studied by trapping free NO with hemoglobin, took place within seconds (or less) rather than the minute time scale reported for the purified enzyme. Measurement of the rates of accumulation and degradation of cGMP were used to follow the activity of sGC over time. The peak activity, occurring within seconds of adding NO, was swiftly followed by desensitization to a steady-state level 8-fold lower. The same desensitizing profile was observed when the net sGC activity was increased or decreased or when cGMP breakdown was inhibited. Recovery from desensitization was relatively slow (half-time ‫؍‬ 1.5 min). When the cells were lysed, sGC desensitization was lost. Analysis of the transient cGMP response to NO in human platelets showed that sGC underwent a similar desensitization. The results indicate that, in its natural environment, sGC behaves much more like a neurotransmitter receptor than had been expected from previous enzymological studies, and that hitherto unknown sGC regulatory factors exist. Rapid sGC desensitization, in concert with variations in the rate of cGMP breakdown, provides a fundamental mechanism for shaping cellular cGMP responses and is likely to be important in decoding NO signals under physiological and pathophysiological conditions. N itric oxide (NO) performs numerous physiological functions, including relaxation of smooth muscle, inhibition of platelet aggregation, and neural communication in the brain (1, 2). A major receptor for NO is the enzyme, soluble guanylyl cyclase (sGC), which catalyzes the production of the effector molecule, cGMP from GTP (3, 4).Compared with neurotransmitter receptors or related adenylyl and guanylyl cyclases (5, 6), the NO receptor enzyme appears rather unremarkable. It is composed of two different subunits (␣ and ␤), but only two isoforms have been shown to exist at the protein level: the ␣1␤1 isoform, which is expressed widely, and the ␣2␤1 isoform present in human placenta (7-9). Also, sGC appears to lack the functional complexity exhibited by related enzymes or receptors. For example, there is no established mechanism for regulation of the enzyme (e.g., by phosphorylation) and, on activation by NO, purified sGC generates cGMP at a constant rate for long periods of time (10, 11). Furthermore, the two naturally occurring sGC isoforms possess very similar functional and pharmacological properties (9).How sGC responds to NO in living cells, however, has not been investigated, nor is it understood why different cells display very different patterns of NO-stimulated cGMP accumulation ranging from a transient spike-like response (12) to a more slowly developing plateau (13). Here we have analyzed the kinetics of NO...

Nitric oxide (NO) signal transduction may involve at least two targets: the guanylyl cyclase-coupled NO receptor (NO GC R), which catalyzes cGMP formation, and cytochrome c oxidase, which is responsible for mitochondrial O 2 consumption and which is inhibited by NO in competition with O 2 . 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 NO GC R was about 100-fold more sensitive to NO than reported previously, 50% of maximal activity requiring only 4 nM NO. Conversely, at physiological O 2 concentrations (20 -30 M), 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 NO GC R (20 nM) inhibited respiration only when the O 2 concentration was pathologically low (50% inhibition at 5 M O 2 ). 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 NO GC R without inhibiting mitochondrial respiration. Nitric oxide (NO)1 is a diffusible biological messenger that subserves cell-to-cell signaling functions in most tissues. NO can also be cytotoxic and has been incriminated in many different pathologies, including atherosclerosis, septic shock, cancer, and neurodegenerative disorders (1). Although much has been learned about the mechanism of NO synthesis (2), the transduction pathways engaged by physiological NO signals to modify cell and tissue function remain to be clearly defined.The established target is the guanylyl cyclase-coupled receptor, or NO GC R, 2 which exists in at least two different heterodimeric isoforms (␣1␤1 and ␣2␤1). This is a metabotropic type of receptor equipped with a heme prosthetic group to which NO binds, triggering the formation of cGMP from GTP in the cyclase domain of the protein. Through this route, NO elicits many effects such as smooth muscle relaxation, inhibition of platelet aggregation, and synaptic plasticity (3, 4). Knowledge of the NO concentrations that engage the NO GC R is important for understanding the receptor kinetics, for informing on the physiological NO concentrations likely to exist in tissues, and for developing realistic models of NO signaling. Currently, however, the information on this issue is incoherent. Studies on the purified ␣1␤1 receptor protein have suggested that the NO concentration giving half-maximal activation (the EC 50 ) is 250 nM (5). More recently, an EC 50 of 1.6 M has been obtained for the enzyme in an extract of rat aorta (6). The validity of this range appears to be supported by several studies that have used the NONOate, diethylamine/NO adduct (DEA/NO), which degrades to release NO with ...

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.

Soluble guanylyl cyclase (sGC) catalyzes cGMP synthesis and serves as a physiological receptor for nitric oxide (NO). Recent evidence indicates that key properties of sGC within cells differ from those of purified sGC. We have devised a technique for resolving NO-stimulated sGC activity in cells on a sub-second time scale, enabling the first quantitative description of the kinetics of the enzyme within its natural environment. Upon release of NO from a caged derivative, sGC became activated without any lag observable at a 20-ms sampling time. Deactivation of sGC on removal of NO occurred with a rate constant of 3.7 s ؊1 , which is 25-fold faster than the fastest estimate for purified sGC. Desensitization of sGC occurred with a time constant of 6.9 s at an estimated 70 nM NO and became faster at a higher concentration, indicating that NO accelerates desensitization. The concentration-response curve for NO consequently became increasingly bell-shaped with time, a phenomenon that causes the apparent potency of NO to increase with time. The results indicate that sGC within cells behaves in a highly dynamic fashion, allowing the NO-cGMP pathway to operate within a kinetic framework more resembling that of neurotransmission than the properties of purified sGC suggest. Nitric oxide (NO)1 performs diverse biological functions, ranging from relaxation of smooth muscle and inhibition of platelet aggregation to neural signaling in the peripheral and central nervous systems (1, 2). The principal receptor mediating the physiological actions of NO is the cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC). Exposure to NO increases the sGC catalytic activity up to several hundred-fold (3-5), and the resulting cellular accumulation of cGMP can engage a number of downstream targets, including cGMP-dependent protein kinase (6), cGMP-regulated phosphodiesterases (7), and cyclic nucleotide-gated ion channels (8) to bring about the various biological effects.Since the purification of sGC in the late 1970s, much knowledge of the structure and the mechanism of activation of the enzyme by NO has accrued (9). The enzyme is an ␣␤-heterodimer with a prosthetic heme group, the NO binding site, attached to the ␤-subunit. Somewhat unexpectedly by comparison with analogous receptors, sGC exhibits limited molecular heterogeneity; only 2 ␣-and 2 ␤-subunits have been identified, and only 2 isoforms have so far been found to exist at the protein level as follows: ␣ 1 ␤ 1 , which is widespread, and ␣ 2 ␤ 1 , which is found in human placenta (10). The two isoforms appear to have similar functional and pharmacological properties (10).As with any other signaling molecule, knowledge of the kinetic properties of the receptor is a prerequisite for understanding how the signals are decoded. When studied in tissue homogenates or in its purified form, sGC exhibits simple Michaelis-Menten-type kinetics. Consequently, in the presence of NO, excess substrate (GTP), and cofactor (Mg 2ϩ ), sGC synthesizes cGMP at a constant rate over long periods, more in ...