Steven R. Vincent

Nitric oxide: A radical neurotransmitter in the central nervous system

Abbreviations

Histochemical characterization of neuronal NADPH-diaphorase.

We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons and provided a Golgi-like image of the neurons exhibiting this activity. The NADPH-diaphorase reaction appeared to be enzyme catalyzed, since it was abolished by pre-treatment with proteases, heat, and acid or alkaline denaturation. Under anaerobic conditions, any tetrazolium salt with a redox potential more positive than NADPH could be reduced by the enzyme. NADPH-diaphorase activity was sensitive to inhibition by sulfhydryl reagents but was unaffected by metal chelators, superoxide dismutase, and catalase. Therefore, the enzyme is unlikely to be a metalloenzyme or to reduce tetrazoliums by producing superoxide anions or hydrogen peroxide. Various analogues of beta-NADPH could be used by the enzyme; however, beta-NADH, which can be used by DT-diaphorase, was ineffective. The enzyme was also resistant to dicumarol, an inhibitor of DT-diaphorase activity. Electron microscopy indicated that the NADPH-diaphorase reaction resulted in staining of various membranous organelles. We conclude that neuronal NADPH-diaphorase is a membrane-bound enzyme distinct from DT-diaphorase and other known enzymes with diaphorase activity. The histochemical characteristics presented here should now enable meaningful biochemical studies of neuronal NADPH-diaphorase to be undertaken.

Neuropeptides and NADPH-diaphorase activity in the ascending cholinergic reticular system of the rat.

A major group of cholinergic neurons is present in the midbrain and pontine tegmentum. These cells could be selectively stained using either monoclonal antibodies to choline acetyltransferase, the pharmacohistochemical acetylcholinesterase procedure, or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Using these three techniques, the precise distribution of this cell group was determined. By combining these techniques with immunohistochemical staining for various neuropeptides, examples of peptide-cholinergic coexistence could be demonstrated in this cell group. Approximately 30% of these cholinergic neurons displayed substance P immunoreactivity. Most of these cells also showed corticotropin-releasing factor immunoreactivity and bombesin/gastrin-releasing peptide immunoreactivity. These results therefore provide evidence for the coexistence of various neuropeptides together with NADPH-diaphorase activity in the ascending cholinergic reticular system.

Cytokines, nitric oxide, and cGMP modulate the permeability of an in vitro model of the human blood-brain barrier

The endothelial cells (EC) of the microvasculature in the brain form the anatomical basis of the blood-brain barrier (BBB). In the present study, the effects of agents that modify the permeability of a well-established in vitro model of the human BBB were studied. The monolayers formed by confluent human brain microvessel endothelial cell (HBMEC) cultures are impermeable to the macromolecule tracer horseradish peroxidase (HRP) and have high electrical resistance. Exposure of HBMEC to various cytokines including TNF-alpha, IL-1beta, interferon gamma (IFN-gamma), or lipopolysaccharide (LPS) decreased transendothelial electrical resistance (TEER) mainly by increasing the permeability of the tight junctions. Primary cultures of HBMEC express endothelial nitric oxide synthase (eNOS) and produce low levels of NO. Treatment with the NO donors sodium nitroprusside (SNP) and DETA NONOate or the cGMP agonist 8-Br-cGMP significantly increased monolayer resistance. Conversely, inhibition of soluble guanylyl cyclase with ODQ rapidly decreased the resistance, and pretreatment of HBMEC with Rp-8-CPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, partially prevented the 8-Br-cGMP-induced increase in resistance. Furthermore, NO donors and 8-Br-cGMP could also reverse the increased permeability of the monolayers induced by IL-1beta, IFN-gamma, and LPS. These results indicate that NO can decrease the permeability of the human BBB through a mechanism at least partly dependent on cGMP production and cGMP-dependent protein kinase activation.

The immunohistochemical localization of choline acetyltransferase in the cat brain

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the feline brain using a monoclonal antibody. Groups of ChAT-immunoreactive neurons were detected that have not been identified previously in the cat or in any other species. These included small, weakly stained cells found in the lateral hypothalamus, distinct from the magnocellular rostral column cholinergic neurons. Other small, lightly stained cells were also detected in the parabrachial nuclei, distinct from the caudal cholinergic column. Many small ChAT-positive cells were also found in the superficial layers of the superior colliculus. Other ChAT-immunoreactive neurons previously detected in rodent and primate, but not in cat, were observed in the present study. These included a dense cluster of cells in the medial habenula, together with outlying cells in the lateral habenula. Essentially all of the cells in the parabigeminal nucleus were found to be ChAT-positive. Additional ChAT-positive neurons were detected in the periolivary portion of the superior olivary complex, and scattered in the medullary reticular formation. In addition to these new observations, many of the cholinergic cell groups that have been previously identified in the cat as well as in rodent and primate brain such as motoneurons, striatal interneurons, the magnocellular rostral cholinergic column in the basal forebrain and the caudal cholinergic column in the midbrain and pontine tegmentum were confirmed. Together, these observations suggest that the feline central cholinergic system may be much more extensive than previous studies have indicated.

Metalloporphyrins inhibit nitric oxide-dependent cGMP formation in vivo.

Sodium nitroprusside produced a dose-dependent increase in extracellular levels of cGMP in the cerebellar cortex in vivo. This was independent of nitric oxide synthase activity. The metalloporphyrins zinc-protoporphyrin-IX, tin-protoporphyrin-IX and zinc-deuteroporphyrin-IX,2,4-bis glycol prevented the increase in cGMP in the cerebellar cortex produced by sodium nitroprusside. At high doses, tin-protoporphyrin-IX also decreased the basal extracellular levels of cGMP. These drugs had no effect on nitric oxide synthase activity. We conclude that the neuropharmacological effects of metalloporphyrins may result from their direct inhibition of soluble guanylyl cyclase, rather than from an effect on carbon monoxide synthesis.

Molecular characterization of a type II cyclic GMP-dependent protein kinase expressed in the rat brain

Abstract: We applied reverse transcription‐PCR to examine the gene expression of cyclic GMP (cGMP)‐dependent protein kinase in the rat brain. A PCR product with the size predicted from the type II cGMP‐dependent protein kinase (cGK II) cDNA was detected in various regions of the brain, with highest expression in the thalamus. The amplified product of this cDNA was subcloned, sequenced, and consequently shown to be cGK II. Northern analysis confirmed that this kinase was highly expressed in the thalamus. In situ hybridization with riboprobes derived from this cDNA indicated that cGK II mRNA was highly expressed in the outer layers of the cortex, the septum, amygdala, and olfactory bulb with highest levels in the thalamus. High amounts of cGK II mRNA were also found in specific brainstem loci, including the medial habenula, the subthalamic nucleus, the locus ceruleus, the pontine nucleus, the inferior olivary nuclei, and the nucleus of the solitary tract. Only low levels of cGK II mRNA were detected in the striatum, cerebellum, and hippocampus. These data suggest that the effects of guanylyl cyclase activators, such as nitric oxide and the atriopeptides, in various regions of the CNS may be mediated through cGK II.

Histochemical demonstration of separate populations of somatostatin and cholinergic neurons in the rat striatum

Striatal neurons containing acetylcholine and somatostatin were examined using a pharmacohistochemical procedure for acetylcholinesterase and NADPH-diaphorase histochemistry respectively. The use of these two histochemical procedures allowed both cholinergic and somatostatin cells to be visualized simultaneously in single sections of the striatum. The results indicate that somatostatin and acetylcholine are contained in separate populations of striatal neurons and illustrate the utility of simple histochemical procedures to visualize biochemically defined neurons.

NMDA AND D1 RECEPTORS REGULATE THE PHOSPHORYLATION OF CREB AND THE INDUCTION OF C-FOS IN STRIATAL NEURONS IN PRIMARY CULTURE

Numerous in vivo studies have demonstrated that psychostimulant drugs such as amphetamine and cocaine can induce the expression of the immediate early gene c‐fos in striatal neurons via the activation of D1 dopamine receptors. NMDA receptor activation is also known to induce c‐fos in the striatum. In the present study we have used a primary striatal neuronal culture preparation to examine the mechanisms whereby these stimuli lead to changes in gene expression. Direct application of NMDA to striatal cells in culture caused a rapid increase in the expression of c‐fos as well as an increase in the phosphorylation of the transcription factor CRE binding protein (CREB). This was prevented by NMDA receptor antagonists, and required extracellular calcium, but did not involve L‐type calcium channels. The induction of c‐fos and CREB phosphorylation following NMDA were unaffected by inhibition of protein kinase C, tyrosine kinases or nitric oxide synthase. However, the response to NMDA was blocked by KN62, a selective inhibitor of calcium/calmodulin‐dependent protein kinase. Application of the D1 agonist SKF 38393, or direct stimulation of adenylyl cyclase with forskolin, also resulted in the phosphorylation of CREB and the induction of c‐fos in striatal neurons. These effects were blocked by the protein kinase A inhibitor H89. These observations are consistent with the hypothesis that calcium/calmodulin‐dependent phosphorylation of CREB induced by NMDA, or cAMP‐dependent phosphorylation of CREB induced by D1 agonists, underlie the induction of c‐fos seen following activation of these receptors in striatal neurons. Synapse 25:227–233, 1997.

Nitric oxide neurons and neurotransmission

Nitric oxide was identified as a biological intercellular messenger just over 20 years ago, and its presence and potential importance in the nervous system was immediately noted. With the cloning of NO synthase and the physiological NO receptor soluble guanylyl cyclase, a variety of histochemical methods quickly led to a rather complete picture of where NO is produced and acts in the nervous system. However, the details regarding the subcellular localization of NO synthase and the identity of its molecular binding partners require further clarification. Although the hypothesis that calcium influx via activation of NMDA receptors is a key trigger for NO production has proven very popular and led to suggested roles for NO in synaptic plasticity, there is little direct evidence to support this notion. Instead, studies from the peripheral nervous system indicate a key role for voltage-sensitive calcium channels in regulating NO synthase activity. A similar mechanism may also be important in central neurons, and it remains an important task to identify the precise sources of calcium regulating NO production in specific NO neurons. Also, although cGMP production appears to mediate the physiological signaling by NO, the specific roles of cGMP-dependent ion channels, protein kinases and phosphodiesterases in mediating NO action remain to be determined.