Regulation of SSAT expression by synaptic activity.

Neuronal activity is a requirement for the plasticity and normal development of the central nervous system. We have used differential cloning techniques to identify an immediate-early gene (IEG) that is rapidly induced in neurons by activity in both adult and developmental models of plasticity. Here we describe the key regulatory enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT), as a neuronal IEG. In the rat brain, kainate-induced seizures result in a 5.5-fold increase in the amount of SSAT mRNA above basal levels and the enzymatic activity is increased twofold. Expression of SSAT mRNA is rapidly and transiently upregulated in the cerebral cortex and hippocampus by seizure-induced neuronal activation. In hippocampal neurons, SSAT expression is dynamically responsive to synaptic activity in the long-term potentiation (LTP) paradigm. In developing brain, region-specific expression of SSAT mRNA is first detected at postnatal day 9 (P9) and subsequently increases through days P15, P20, before reaching maximal level in adult animals. This dynamic transcriptional and translational control suggests that SSAT may play a role in activity-dependent neuronal plasticity and development.

Heme oxygenase-2 acts to prevent neuronal death in brain cultures and following transient cerebral ischemia.

Heme oxygenase (HO) cleaves the heme ring to form biliverdin, which is rapidly reduced to bilirubin, carbon monoxide, and iron. HO1, the first form of the enzyme discovered, is an inducible protein, concentrated in tissues that are exposed to degrading red blood cells and stimulated by hemolysis and numerous other toxic perturbations to eliminate potentially toxic heme. By contrast, HO2 is constitutive and most highly concentrated in neural tissues. Carbon monoxide, formed from HO2, is a putative neurotransmitter in the brain and peripheral autonomic nervous system. HO1 regulates the efflux of potentially toxic iron from cells, as iron efflux is deficient in mice with targeted deletion of HO1 (HO1(-/-)), and transfection of HO1 facilitates iron efflux. Bilirubin appears to be a physiologic neuroprotectant. Activation of HO2 by phorbol esters, that stimulate protein kinase C to phosphorylate HO2, augments production of bilirubin which protects brain cultures from oxidative stress. Bilirubin itself in nanomolar concentrations is neuroprotective, while HO2 deletion (HO2(-/-)) leads to increased neurotoxicity in brain cultures and increased neural damage following transient cerebral ischemia in intact mice. Mechanisms whereby HO2 provides neuroprotection have not been clarified including whether protection is primarily associated with apoptotic or necrotic cell death. Moreover, the generality of neurotoxic stimuli influenced by HO2 has been unclear. We now demonstrate increased neuronal death in cerebellar granule cultures of HO2(-/-) mice with a selective augmentation of apoptotic death. We also demonstrate that HO2 transfection rescues apoptotic death. In intact mice, we show an increased incidence of apoptotic morphology in the penumbra area surrounding the infarct core in HO2(-/-) mice undergoing transient focal ischemia.

Dynamic regulation of RGS2 suggests a novel mechanism in G-protein signaling and neuronal plasticity.

Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein, and recent studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction.

The regulation of heme turnover and carbon monoxide biosynthesis in cultured primary rat olfactory receptor neurons.

Heme oxygenase (HO) converts heme to carbon monoxide (CO) and biliverdin, which is metabolized rapidly to bilirubin. CO is implicated as an intercellular messenger, whereas bilirubin could function as an antioxidant. These cellular functions differ significantly from those of HO in peripheral tissues, in which it degrades heme from senescent erythrocytes, suggesting that the regulation of HO may differ in neurons from that in other tissues. Among neurons, olfactory receptor neurons have the highest level of HO activity. Metabolic labeling with [2-14C]glycine or delta-[3H]aminolevulinic acid ([3H]ALA) was used to investigate heme metabolic turnover and CO biosynthesis in primary cultures of olfactory receptor neurons. The production rates of heme precursors and metabolites from [14C]glycine over 6 hr were (in pmol/mg protein): 100 for ALA, 8.2 for heme, and 2.9 for CO. Taking into account endogenous heme content, the amount of total CO production was determined to be 1.6 nmol/mg protein per 6 hr. Heme biosynthesis usually is subject to end-product negative feedback at the level of ALA synthase. However, metabolic control in these neurons is different. Both heme concentration (heme formation) and HO activity (heme degradation) were enhanced significantly during immature stage of neuronal differentiation in culture. Neuronal maturation, which is accelerated by transforming growth factor-beta 2 (TGF-beta 2), suppressed the activities of both heme biosynthesis and degradation. To explore the physiological importance of this endogenous production of CO, we examined the potency of CO as a soluble guanylyl cyclase activator. Exogenous CO (10-30 microM), comparable to endogenous CO production, significantly activated guanylyl cyclase, suggesting that HO activity may regulate cGMP levels in the nervous system.

Carbon monoxide: an endogenous modulator of the nitric oxide-cyclic GMP signaling system.

Carbon monoxide (CO) is an activator of soluble guanylyl cyclase and is implicated as a neuronal messenger. CO production, nitric oxide synthase (NOS) activity, and guanosine 3',5'-monophosphate (cGMP) levels were quantitated in cerebellar granule cell cultures. Metabolic labeling experiments enabled the direct measurement of neuronal CO production in vitro. CO production is significant, and peaked during early stages of culture. NOS activity and cGMP levels synchronously increased as cells matured. Whereas inhibition of NOS depleted cGMP in mature cultures, inhibitors of CO production potentiated the nitric oxide (NO)-mediated cGMP increase. Exogenous CO at similar concentrations to endogenous levels blocked the NO-mediated cGMP increase. These results directly demonstrate that endogenous neuronal CO production is high and indicate that while NO is the major regulator of cGMP in these neurons, CO may modulate the NO-cGMP signaling system.

Direct demonstration of a physiological role for carbon monoxide in olfactory receptor neurons.

Recent evidence suggests that, like nitric oxide (NO), carbon monoxide (CO), another activator of soluble guanylyl cyclase, may serve as an intercellular messenger in the brain. Heme oxygenase, which converts heme to biliverdin and CO, is abundantly expressed in the brain and is localized to discrete neuronal populations. However, evidence for the actual generation of CO by neurons is lacking. Heme oxygenase-2 immunoreactivity is abundantly present in olfactory receptor neurons where it essentially colocalizes with immunoreactivity to soluble guanylyl cyclase, the target of CO action. To examine the generation of CO by neurons, we measured CO production directly and determined its relationship to cyclic GMP levels in cultured rat olfactory receptor neurons. This system has the advantage of not having measurable NO production, which could confound results since NO is a more potent activator of guanylyl cyclase than CO. Metabolic labeling experiments permitted the direct measurement of 14CO production by neurons in vitro. CO release parallels endogenous cyclic GMP concentrations with its peak at the immature stage of neuronal differentiation in culture. Cyclic GMP production is inhibited by zinc protoporphyrin-9 and zinc deuteroporphyrin IX 2,4-bis glycol, inhibitors of heme oxygenase, indicating that CO is an endogenous regulator of soluble guanylyl cyclase activities in these cells. Transforming growth factor-beta 2, an olfactory neurogenic factor, specifically shows a negative effect on CO release in olfactory receptor neurons. These results indicate that CO may serve as a gaseous neuronal messenger linked to cyclic GMP production and suggests its involvement in developmental processes of the olfactory receptor neuron.

Characterization of ligand-binding properties and selectivities of three rat tachykinin receptors by transfection and functional expression of their cloned cDNAs in mammalian cells.

We investigated the ligand-binding properties and selectivities of rat substance P, substance K and neuromedin K receptors by transfection and functional expressions of the cDNAs for these receptor subtypes in monkey kidney COS cells. Selective radioligand binding analysis of both substance P and substance K receptors revealed the presence of high-affinity and low-affinity components which are governed primarily by the difference in rates of dissociation of the ligand-receptor complex. The two affinity components were interconvertible by the presence and absence of a guanine nucleotide, suggesting the involvement of a G protein in the two affinity states. The ligand bindings of the three receptors were inhibited in different potencies by naturally occurring tachykinins and a series of carboxyl-terminal fragments containing a common tachykinin sequence, and this comprehensive analysis indicated that a high and selective affinity of each of the tachykinin receptors is governed by interaction with several key amino acids under recognition of the fundamental core sequence of the tachykinin peptides. The potencies and selectivities of several synthetic agonists and an antagonist for the three receptors were also examined, and senktide was found to be a highly selective and potent agonist for neuromedin K receptor. This investigation thus indicates the detailed properties and binding selectivities characteristic of the three tachykinin receptors and also the usefulness of this receptor expression system for examination and development of a new receptor agonist or antagonist.

A set of U1 snRNA-complementary sequences involved in governing alternative RNA splicing of the kininogen genes.

The rat K and T kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the exon next to and upstream from the LMW exon (HMW exon) through alternative splicing and polyadenylation. In contrast, the T gene generates one mRNA by using selectively the LMW exon, although the T gene is extremely homologous to the K gene. In this study, we constructed a series of chimeric kininogen genes by not only exchanging equivalent restriction fragments of the two genes but also replacing nucleotides that differ between the two genes. We then examined the sequences and the mechanisms governing the different expression patterns of the two genes by transfecting the chimeric genes into heterologous COS cells. The results indicated that the different expression patterns of the K and T genes are governed by two separate internal sequences of the HMW and LMW exons. The internal HMW sequence contains a set of five repetitive sequences, and these repetitive sequences are highly complementary to the 5' portion of U1 snRNA. Furthermore, the nucleotide differences in the U1 snRNA-complementary sequences between the K and T genes have marked effects on the relative formation of the HMW and LMW mRNAs; this indicates that the repetitive sequences complementary to U1 snRNA play a crucial role in determining the relative expression of the two mRNAs. Based on these findings, we discuss a novel mechanism for alternative RNA processing, in which splicing efficiency is controlled by the interaction of U1 small nuclear ribonucleoproteins and the U1 snRNA-complementary repetitive sequences of the kininogen pre-mRNA.