ABSTRACT
Nitric oxide synthases (NOSs) generate nitric oxide (NO) and the by-product l-citrulline, via the catalytic combination of l-arginine and molecular oxygen. In mammals, there are three NOS genes: nNOS (NOS1), iNOS (NOS2) and eNOS (NOS3). The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important processes. The studies of NOS enzymes using knockout and transgenic mouse models have provided an invaluable contribution, highlighting critical roles in neuronal, renal, pulmonary, gastro-intestinal, skeletal muscle, reproductive and cardiovascular biology. This review will outline the data gleaned from complementary knockout and transgenic over-expression models in mice, and focus on the interactions between NOS enzymes and pathophysiology of the vascular system. These studies are a paradigm for the near future, which will involve the translation of an enormous amount of genomic data into physiological insights that penetrate the realms of both health care and biology.
Subject(s)
Nitric Oxide Synthase/metabolism , Animals , Arteriosclerosis/genetics , Coronary Vessels/physiology , Disease Models, Animal , Endotoxins/adverse effects , Liver Circulation/genetics , Male , Mice , Mice, Knockout , Muscle, Skeletal/blood supply , Neurons/physiology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Penis/blood supply , Pulmonary Circulation/genetics , Renal Circulation/geneticsABSTRACT
Synaptic function requires proper localization of proteins at synaptic sites. Targeting of the postsynaptic density protein 95 (PSD-95) relies on multiple signals within the protein, including twelve C-terminal amino acids. We now show that this C-terminal targeting domain of PSD-95 mediates postsynaptic localization through a short tyrosine-based motif followed by a pair of hydrophobic amino acids. Consistent with a role in cellular trafficking, the tyrosine motif resembles the canonical motif for interactions with clathrin adaptor proteins. In fact, we find that the C-terminal targeting domain of PSD-95 is sufficient to mediate clathrin-dependent endocytosis when appended to a transmembrane protein. Furthermore, systematic mutagenesis reveals that endocytosis mediated by this domain depends on both the tyrosine motif and the dihydrophobic amino acid pair. Thus, postsynaptic targeting of PSD-95 requires a tyrosine-based signal that can mediate clathrin-coated vesicle formation.
Subject(s)
Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Signal Transduction , Synapses/chemistry , Synapses/metabolism , Tyrosine/chemistry , Amino Acid Sequence , Amino Acids/metabolism , Animals , COS Cells , Cell Membrane/metabolism , Cells, Cultured , Clathrin/metabolism , Conserved Sequence , DNA, Complementary/metabolism , Disks Large Homolog 4 Protein , Endocytosis , Fluorescent Antibody Technique , Genes, Dominant , Green Fluorescent Proteins , Hippocampus/embryology , Hippocampus/metabolism , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/metabolism , Membrane Proteins , Molecular Sequence Data , Mutagenesis , Nerve Tissue Proteins/genetics , Neurons/metabolism , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Vasoactive intestinal polypeptide and peptide histidine isoleucine, two peptides with a common precursor and with strong vasodilatory actions, have been suggested to be involved in control of blood flow through the hypothalamic portal blood vessels, in this way regulating the amounts of releasing and inhibitory factors reaching the anterior pituitary. Using the indirect immunofluorescence technique, we now show that this system also contains the enzyme nitric oxide synthase, as well as acetylcholinesterase. It is therefore likely that the control of blood flow through the portal vessels is mediated via relaxation of smooth muscle cells with a high myogenic tone by neuronal release of four vasodilatory compounds, acetylcholine, vasoactive intestinal polypeptide, peptidine histidine isoleucine, and nitric oxide, i.e. a classic neurotransmitter, two neuropeptides and a gas.
Subject(s)
Cerebrovascular Circulation/physiology , Hypothalamus/blood supply , Nitric Oxide/metabolism , Acetylcholine/metabolism , Animals , Circle of Willis/physiology , Fluorescent Antibody Technique , Hypothalamus/physiology , Immunohistochemistry , Median Eminence/physiology , Muscle Relaxation/physiology , Muscle, Smooth, Vascular/physiology , Nerve Fibers/metabolism , Peptide PHI/metabolism , Peptide PHI/physiology , Rats , Vasoactive Intestinal Peptide/metabolism , Vasoactive Intestinal Peptide/physiologyABSTRACT
Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.