Despite the widespread use of nitric oxide as a signalling molecule in the central nervous system, the molecular makeup of its receptor, soluble guanylyl cyclase (sGC), therein is poorly understood. Accordingly, RT-PCR and in situ hybridization were used to identify sGC subunits expressed in rat brain. In addition to the expected mRNA for alpha 1 and beta1 subunits, message for the beta 2 subunit was detected in the cerebellum at all developmental stages investigated (1--150 days postnatum). The use of degenerate primers allowed the identification of mRNA coding for the rat alpha 2 subunit, which was also expressed at every age studied. All but beta 2 were detected by in situ hybridization in the brains of both 8-day-old and adult rats. The distribution patterns indicated that in some areas, e.g. caudate-putamen and nucleus accumbens, sGC probably exists mainly as the alpha 1 beta 1 heterodimer. In others, e.g. hippocampus and olfactory bulb, alpha 2 beta 1 is likely to be dominant. In the cerebellum, alpha 1 and beta 1 message was strong in the Purkinje cell layer but was not confined to Purkinje cells: smaller cells, presumed to be the Bergmann glia, were also labelled. In contrast, alpha 2 mRNA was concentrated in cerebellar granule cells. Western blotting indicated an excess of alpha 1 over beta 1 protein in the cerebellum, the reverse of what was found in the lung. It is concluded that, in molecular terms, sGC is likely to be more complex and exhibit more regional variation in the brain than previously thought. The functional consequences of this heterogeneity require investigation.

Protein phosphorylation is well established as a regulatory mechanism in higher plants, but only a handful of plant enzymes are known to be regulated in this manner, and relatively few plant protein kinases have been characterized. AMP-activated protein kinase regulates key enzymes of mammalian fatty acid, sterol and isoprenoid metabolism, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. We now show that there is an activity in higher plants which, by functional criteria, is a homologue of the AMP-activated protein kinasc, although it is not regulated by AMP. The plant kinase inactivates mammalian HMG-CoA reductase and acetyl-CoA carboxylase, and peptide mapping suggests that it phosphorylates the same sites on these proteins as the mammalian kinase. However, with the target enzymes purified from plant sources, it inactivates HMG-CoA reductase but not acetyl-CoA carboxylasc. The kinasc is located in the solublc, and not the chloroplast, fraction of leaf cells, consistent with the idea that it regulates HMG-CoA reductase, and hence isoprenoid biosynthesis, in vivo. The plant kinase also appears to be part of a protcin kinase cascade which has been highly conserved during evolution, since the kinase is inactivated and reactivated by mammalian protein phosphatases (2A or 2 C) and mammalian kinase kinase, respectively. This contrasts with thc situation for many othcr mammalian protein kinascs involved in signal transduction, which appear to have no close homologue in higher plants. 'To our knowledge, this represents the first direct evidence for a protein kinase cascade in higher plants.

In order to investigate the effect on tau of manipulating glycogen synthase kinase (GSK)-3beta activity in the brain, we created transgenic mice harbouring wild-type GSK-3beta genes or a mutant GSK-3beta that is predicted to be more active. Transgene-derived mRNAs were detected in the brains of a number of the transgenic mouse lines and several of these transgenic lines displayed transgenic GSK-3beta activity. Western blot analyses of the two lines with the highest levels of transgenic GSK-3beta activity revealed that the phosphorylation status of tau was elevated at the AT8 epitope. These observations strongly suggest that GSK-3beta is an in vivo tau kinase in the brain. Only low levels of expression of GSK-3beta were obtained and it is possible that high levels of GSK-3beta activity are lethal.

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.

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