This study suggests that the natural history of this subgroup of dysraphic patients may be more benign than hitherto considered. Conservative management with adoption of a novel surveillance policy and timely intervention only in the presence of symptomatic deterioration resulted in 71% of this series remaining clinically asymptomatic at mean follow up period of 5.9 years (range, 1.0-19.3 years). At 10 years, the cumulative risk of deterioration determined by the Kaplan-Meier method was 40%. Children aged<2 years, female, with presence of a transitional type of LSL and associated syrinx were independently associated with a higher risk of deterioration.

The signaling molecule nitric oxide (NO) could engage multiple pathways to influence cellular function. Unraveling their relative biological importance has been difficult because it has not been possible to administer NO under the steady-state conditions that are normally axiomatic for analyzing ligand-receptor interactions and downstream signal transduction. To address this problem, we devised a chemical method for generating constant NO concentrations, derived from balancing NO release from a NONOate donor with NO consumption by a sink. On theoretical grounds, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) was selected as the sink. The mixture additionally contained urate to convert an unwanted product of the reaction (NO 2 ) into nitrite ions. The method enabled NO concentrations covering the physiological range (0 -100 nM) to be formed within approximately 1 s. Moreover, the concentrations were sufficiently stable over at least several minutes to be useful for biological purposes. When applied to the activation of guanylyl cyclase-coupled NO receptors, the method gave an EC 50 of 1.7 nM NO for the protein purified from bovine lung, which is lower than estimated previously using a biological NO sink (red blood cells). The corresponding values for the ␣1␤1 and ␣2␤1 isoforms were 0.9 nM and 0.5 nM, respectively. The slopes of the concentrationresponse curves were more shallow than before (Hill coefficient of 1 rather than 2), questioning the need to consider the binding of more than one NO molecule for receptor activation. The discrepancies are ascribable to limitations of the earlier method. Other biological problems can readily be addressed by adaptations of the new method.

1 Physiological nitric oxide (NO) signal transduction occurs through activation of guanylyl cyclase (GC)-coupled receptors, resulting in cGMP accumulation. There are five possible receptors: four heterodimers (a1b1, a2b1, a1b2, a2b2) and a presumed homodimer (nb2). The present study investigated the kinetic and pharmacological properties of all these putative receptors expressed in COS-7 (or HeLa) cells. 2 All exhibited NO-activated GC activity, that of a1b1 and a2b1 being much higher than that of the b2-containing heterodimers or nb2. All were highly sensitive NO detectors. Using clamped NO concentrations, EC 50 values were 1 nm for a1b1 and 2 nm for a2b1. With a1b2, a2b2 and nb2, the EC 50 was estimated to be lower, about 8 nm.3 All the GCs displayed a marked desensitising profile of activity. Consistent with this property, the concentration -response curves were bell-shaped, particularly those of the b2 heterodimers and nb2. 4 Confocal microscopy of cells transfected with the fluorescently tagged b2 subunit suggested targeting to the endoplasmic reticulum through its isoprenylation sequence, but no associated particulate GC activity was detected. 5 The NO-stimulated GC activity of all heterodimers and nb2 was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and, except for nb2, was enhanced by the allosteric activator YC-1. 6 It is concluded that all the four possible heterodimers, as well as the putative nb2 homodimer, can function as high-affinity GC-coupled NO receptors when expressed in cells. They exhibit differences in NO potency, maximal GC activity, desensitisation kinetics and possibly subcellular location but, except for nb2, cannot be differentiated using existing pharmacological agents.

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 of cGMP 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.