Solomon H. Snyder

Targeted disruption of the neuronal nitric oxide synthase gene

By homologous recombination, we have generated mice that lack the neuronal nitric oxide synthase (NOS) gene. Neuronal NOS expression and NADPH-diaphorase (NDP) staining are absent in the mutant mice. Very low level residual catalytic activity suggests that other enzymes in the brain may generate nitric oxide. The neurons normally expressing NOS appear intact, and the mutant NOS mice are viable, fertile, and without evident histopathological abnormalities in the central nervous system. The most evident effect of disrupting the neuronal NOS gene is the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles the human disorder infantile pyloric stenosis, in which gastric outlet obstruction is associated with the lack of NDP neurons in the pylorus.

Nitric Oxide: A Physiologic Messenger

A noxious, unstable gas is an unlikely candidate to act as a biological messenger. However, in the last 7 years, nitric oxide, a byproduct of automobile exhaust, electric power stations, and lightning, was discovered in the body, where it participates in various functions, including suppression of pathogens, vasodilation, and neurotransmission [1-8]. We describe what is known about nitric oxide, focusing on its clinical relevance. Unusual Properties of Nitric Oxide Metabolism Nitric oxide is an unusual messenger. It is a small molecule composed of one atom each of nitrogen and oxygen, not to be confused with nitrous oxide, N2O, an inhalational anesthetic commonly referred to as laughing gas. Nitric oxide is an uncharged molecule with an unpaired electron. These characteristics of nitric oxide make it an ideal messenger molecule: Uncharged, nitric oxide can diffuse freely across membranes. With an unpaired electron, it is called a radical molecule, which is highly reactive (having a half-life of 2 to 30 seconds); after transmitting a signal spontaneously, it decays into nitrite. Nitric oxide is made by nitric oxide synthase in an unusual reaction that converts arginine and oxygen into citrulline and nitric oxide. The mechanism of nitric oxide synthesis is not completely understood, but it involves the transfer of electrons between various cofactors, including flavin adenine dinucleotide, flavin mononucleotide, nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin, and heme. Finally, one atom of oxygen from oxygen binds with the terminal guanidine nitrogen from arginine to form nitric oxide [9, 10]. Although several nitric oxide synthase isoforms have been isolated [11-17], all are homologous and divided into two categories with different regulation and activities (Figure 1). The constitutive isoforms in neuronal or endothelial cells are always present [11, 18]. These nitric oxide synthase isoforms are inactive until intracellular calcium levels increase, the calcium-binding protein calmodulin binds to calcium, and the calcium-calmodulin complex binds to and activates nitric oxide synthase [19-22]. The constitutive nitric oxide synthase isoforms then synthesize small amounts of nitric oxide until calcium levels decrease. This intermittent production of small amounts of nitric oxide transmits signals. In contrast, the inducible nitric oxide synthase isoform is normally absent from macrophages and hepatocytes, but when these cells are activated by specific cytokines, an inducible nitric oxide synthase enzyme is produced; once produced, it always synthesizes large amounts of nitric oxide. Induced nitric oxide synthase is transcriptionally regulated [16, 23]. The continuous production of large amounts of nitric oxide kills or inhibits pathogens. Figure 1. Diagrammatic representation of the structure of cloned forms of nitric oxide synthase with sites for cofactor binding. P Other mechanisms of regulating nitric oxide synthase enzymes recently were discovered. Neuronal nitric oxide synthase can be phosphorylated by protein kinases to decrease its activity [24]. The subcellular location of nitric oxide synthase in endothelial cells can also be changed by its phosphorylation [25]. Although the constitutive isoforms are regulated by calcium, the inducible isoforms also appear to bind calmodulin, although calcium has little effect on their activity [26]. The role of calmodulin in inducible nitric oxide synthase function is unknown. Most molecules that transmit signals between cells, such as hormones, neurotransmitters, and growth factors, act through specific protein receptors that are often associated with the plasma membrane. In contrast, nitric oxide diffuses out of the cell that generates it and into target cells, where it interacts with specific molecular targets (Table 1). The best-characterized receptor of nitric oxide is iron, contained in certain proteins as a heme group or as an iron-sulfur complex. Nitric oxide exerts some of its effects by binding to iron-containing enzymes and either activating or inactivating the enzymes. When nitric oxide binds to the iron in the heme group of guanylate cyclase, the enzyme is activated. Guanylate cyclase then produces cyclic guanosine monophosphate (cGMP), and the increase in cGMP activates other cellular processes (Figure 2). By changing the activity of guanylate cyclase, nitric oxide dilates arteries, signals neurons, and kills cells. Table 1. Molecular Targets of Nitric Oxide* Figure 2. Nitric oxide arterial smooth muscle. Another unusual way in which nitric oxide affects cells is by facilitating transfer of an ADP-ribose group to an accepting molecule (a process called ADP ribosylation). Normally an ADP-ribose group is attached to a protein target by an enzyme, for example, when cholera toxin ADP-ribosylates a guanosine triphosphate-binding protein. However, when nitric oxide diffuses into a cell, it can cause auto-ADP ribosylation, that is, ADP ribosylation of a target without enzyme catalysis. For example, nitric oxide inactivates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase by attaching an ADP-ribose group to it, thereby blocking the production of adenosine triphosphate from glycolysis [27-32]. Nitric Oxide in the Cardiovascular System Although the effect of exogenously administered nitrates on the vasculature has been studied for decades, the first clue that endothelial cells released a substance that could cause vasodilation was found only recently. In 1980, Furchgott and Zawadski [33] discovered that sections of the aorta would relax in response to agonists only if the inner linings of endothelial cells were intact. However, aortic rings with no endothelial cells could not relax. Endothelial cells thus released an agent that relaxed vascular smooth muscle; within several years, it was discovered that this endothelial-derived relaxation factor was nitric oxide [34]. The Role of Nitric Oxide in Mediating an Active State of Vasodilation The vasculature is in a constant state of active dilation mediated by nitric oxide. Endothelial cells continuously release small amounts of nitric oxide, producing a basal level of vascular smooth muscle relaxation. When inhibitors of nitric oxide synthase, such as nitro-arginine methyl ester or n-mono-methyl-arginine, are infused into animals [35-44] or humans [45], nitric oxide production is inhibited, vascular smooth muscle contracts, and blood pressure increases. Nitric oxide dilates blood vessels by directly relaxing vascular smooth muscle cells (see Figure 2). An agonist such as acetylcholine binds to its receptor on endothelial cells, causing a transient increase in intracellular calcium. Calcium binds to calmodulin, and the calcium-calmodulin complex activates endothelial nitric oxide synthase, which makes nitric oxide. Nitric oxide then diffuses out of the endothelial cell and into adjacent smooth muscle cells, where it binds to the heme group of guanylate cyclase. Guanylate cyclase is activated to produce cGMP, which, through a cascade of protein kinases, induces smooth muscle relaxation. Because the half-life of nitric oxide in biological fluids is between 2 and 30 seconds, the effect of nitric oxide spontaneously diminishes and the vessel constricts unless more nitric oxide is produced. Nitric Oxide: An Autoregulator and Neural Regulator of Blood Flow Nitric oxide automatically regulates blood flow in response to local changes in some regions of the vasculature. Ischemia and reperfusion cause vasodilation only in the affected tissue, and this response is mediated by nitric oxide. For example, a study of ischemia in patients arms and legs showed an increase in nitric oxide production [46]. However, whereas some investigators found that ischemia and reperfusion increase nitric oxide production in animals [47, 48], others observed a decrease in nitric oxide [49-51]. Shear-stress, an increase in blood flow through a vessel, is another physical stimulus to which endothelial cells respond by increasing nitric oxide production [52, 53]. In animal studies, increases in blood flow but not pressure caused an increase in nitric oxide production and dilation in excised hearts and vessels [54-59]. Factors locally released by adjacent tissue, such as bradykinin or acetylcholine, can also induce nitric oxide release in some vessels but not in others. A basal level of nitric oxide regulates blood flow in the brain [60-62], heart [59, 63-67], lung [68], gastrointestinal tract [69], and kidney [38, 42, 43, 70]. Thus, nitric oxide is an endogenous autoregulator of blood flow. The release of nitric oxide into the vasculature is also controlled by the autonomic nervous system. Parasympathetic nerves containing nitric oxide synthase terminate in the adventitia of certain large vessels, such as the cerebral and retinal arteries [71]. Nitric oxide is released from the nerves and diffuses into the muscular media from the outside of the vessel, causing vasorelaxation. The Role of Nitric Oxide in Hypertension and Vasospasm Because nitric oxide plays a central role in regulating blood pressure, defects in the regulation of nitric oxide synthase could lead to vasospasm or hypertension, although this has not been proved. Clearly the endothelium is abnormal in persons with hypertension of unknown cause (essential hypertension), because acetylcholine causes less vasodilation in hypertensive than in healthy persons [72-74]. In contrast, the vascular smooth muscle is normal in persons with hypertension because intravenous nitroprusside, which releases nitric oxide, causes equal amounts of vasodilation in both hypertensive and healthy persons [74, 75]. Abnormal endothelial nitric oxide synthase could cause this defective response to acetylcholine in persons with hypertension. In fact, the blood pressure of healthy persons increases when nitric oxide synthase inhibitors are infused, but the blood pressure of persons wi

Messenger molecules in the cerebellum

As data accumulate, the mammalian brain reveals its complex and subtle synaptic mechanisms. In the simplest system, a neurotransmitter binds to the receptor portion of a molecular complex incorporating an ion channel and thus alters the membrane potential, leading to excitatory or inhibitory effects. In more complex systems, receptors are coupled to second messenger systems to generate signals of longer duration and to modulate more diverse molecular mechanisms. The cerebellar cortex has a relatively simple wiring diagram with the primary neurotransmitter of most inhibitory and excitatory synapses well established. The second messenger signalling systems are more complex and those of the cerebellar output, the Purkinje cells, are the best characterized. More recently, molecules that might act as neuromodulators, carrying messages between neurons and between neurons and glial cells, have been identified, such as endothelin and nitric oxide. The classic neurotransmitters and novel neuromodulators, together with second messenger-activated trophic factors, can interact in complex ways; in this review Christopher Ross, David Bredt and Solomon Snyder discuss how studies of cerebellar circuitry and biochemistry are revealing such interrelations.

Nitric oxide and neurons

In the past 2 years powerful evidence has emerged to suggest that nitric oxide functions as a neurotransmitter in both the central and peripheral nervous systems. Recent evidence suggests that it may play a role in mediating forms of synaptic plasticity such as long-term potentiation in the CA1 region of the hippocampus, and long-term depression in the cerebellum. Abnormal secretion of nitric oxide may be responsible for the neurotoxicity mediated by NMDA receptors that results in the pathophysiology of strokes and neurodegenerative diseases.

Inhibition of neuronal nitric oxide synthase increases aggressive behavior in mice.

BackgroundMice with targeted disruption of the gene for the neuronal isoform of nitric oxide synthase (nNOS) display exaggerated aggression. Behavioral studies of mice with targeted gene deletions suffer from the criticism that the gene product is missing not only during the assessment period but also throughout development when critical processes, including activation of compensatory mechanisms, may be affected. To address this criticism, we have assessed aggressive behavior in mice treated with a specific pharmacological inhibitor of nNOS.Materials and MethodsAggressive behavior, as well as brain citrulline levels, were monitored in adult male mice after treatment with a specific nNOS inhibitor, 7-nitroindazole (7-NI) (50 mg/kg ip), which is known to reduce NOS activity in brain homogenates by >90%. As controls, animals were treated with a related indazole. 3-indazolinone (3–1) (50 mg/kg ip) that does not affect nNOS, or with an oil vehicle.ResultsMice treated with 7-NI displayed substantially increased aggression as compared with oil- or 3-I-injected animals when tested in two different models of aggression. Drug treatment did not affect nonspecific locomotor activities or body temperature. Immunohistochemical staining for citrulline in the brain revealed a dramatic reduction in 7-NI-treated animals.Conclusions7-NI augmented aggression in WT mice to levels displayed by nNOS- mice, strongly implying that nNOS is a major mediator of aggression. NOS inhibitors may have therapeutic roles in inflammatory, cardiovascular, and neurologic diseases. The substantial aggressive behavior soon after administration of an nNOS inhibitor raises concerns about adverse behavioral sequelae of such pharmacological agents.