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D S Bredt

Publications and source records attributed to D S Bredt.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein.

Nitric oxide (NO) formation in brain is regulated by the calcium/calmodulin dependence of neuronal NO synthase (nNOS). Calcium influx through NMDA-type glutamate receptors is efficiently coupled to nNOS activity, whereas many other intracellular calcium pathways are poorly coupled. To elucidate possible mechanisms responsible for this coupling, we performed yeast two-hybrid screening to identify proteins that interact with nNOS. Two nNOS interacting proteins were identified: the postsynaptic density proteins PSD-93 and PSD-95. Here, we report the cloning and characterization of PSD-93. PSD-93 is expressed in discrete neuronal populations as well as in specific non-neuronal cells, and it exhibits complex molecular diversity attributable to tissue-specific alternative splicing. PSD-93, like PSD-95, binds to nNOS and to the NMDA receptor 2B. PSD-93, however, is unique among PSD-95/SAP-90 family members in its expression in Purkinje neuron cell bodies and dendrites. We also demonstrate that the PDZ domain at the N terminus of nNOS is required, but it is not sufficient for interaction with PSD-93/95. Given that PSD-93 and PSD-95 each contain multiple potential binding sites for nNOS and the NMDA receptor, complexes involving oligomers of PSD-93/95 may help account for the functional as well as the physical coupling of nNOS to NMDA receptors.

Alternative Splicing↗
PubMed1996

Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Animals↗
PubMed1996

cGMP-dependent protein kinase in dorsal root ganglion: relationship with nitric oxide synthase and nociceptive neurons.

Nitric oxide and cGMP influence plasticity of nociceptive processing in spinal cord. However, effectors for cGMP have not been identified in sensory pathways. We now demonstrate that cGMP-dependent protein kinase I (cGKl) occurs in the DRGs at levels comparable to that in cerebellum, the richest source of cGKl in the body. Immunohistochemical studies reveal that cGKl is concentrated in a subpopulation of small- and medium-diameter DRG neurons that partially overlap with substance P and calcitonin gene-related polypeptide containing cells. During development, cGKl expression throughout the embryo is essentially restricted to sensory neurons and to the spinal floor and roof plates. Neuronal nitric oxide synthase (nNOS) is coexpressed with cGKl in sensory neurons during embryonic development and after peripheral nerve axotomy. The primary target for cGKl in cerebellum, G-substrate, is not present in developing, mature, or regenerating sensory neurons, indicating that other proteins serve as effectors for cGKl in sensory processing. These data establish sensory neurons as a primary locus for cGMP actions during development and suggest a role for cGKl in plasticity of nociception.

Animals↗
PubMed1996

Neuronal nitric-oxide synthase-mu, an alternatively spliced isoform expressed in differentiated skeletal muscle.

Nitric oxide (NO) functions as a molecular mediator in numerous processes in cellular development and physiology. Differential expression and regulation of a family of three NO synthase (NOS) gene products help achieve this diversity of action. Previous studies identify post-translational modification and interaction of NOS with specific protein targets as tissue-specific modes of regulation. Here, we show that alternative splicing specifically regulates neuronal NOS (nNOS, type I) in striated muscle. nNOS in skeletal muscle is slightly more massive than nNOS from brain owing to a 102-base pair (34-amino acid) alternatively spliced segment between exons 16 and 17. Following purification, this novel nNOS mu isoform has similar catalytic activity to that of nNOS expressed in cerebellum. nNOS mu appears to function exclusively in differentiated muscle as its expression occurs coincidentally with myotube fusion in culture. An isoform-specific antibody detects nNOS mu protein only in skeletal muscle and heart. This study identifies alternative splicing as a means for tissue-specific regulation of nNOS and reports the first additional protein sequence for a mammalian NOS since the original cloning of the gene family.

Alternative Splicing↗
PubMed1996

Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains.

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

Amino Acid Sequence↗
PubMed1996

Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: regulation by NGF.

Nerve growth factor (NGF) acts through the receptor tyrosine kinase trkA to serve as a trophic factor for cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band. We have previously shown that the neuronal isoform of nitric oxide synthase (NOS) is selectively expressed in a large fraction of trkA-expressing cholinergic neurons in these brain regions in the adult rat, and that NGF induces the expression of neuronal-NOS in these cells. Herein, we show that: 1) neuronal-NOS is also localized to these neurons in the developing septum; 2) the expression of neuronal-NOS is regulated in the developing medial septal nucleus and vertical limb of the diagonal band; 3) neuronal-NOS regulation parallels that for other markers of basal forebrain cholinergic neuron differentiation, such as cholineacetyltransferase; and 4) NGF infusion in the postnatal period induces robust increases in neuronal-NOS mRNA and in NOS activity in the basal forebrain. Taken together with earlier findings, our results suggest that neuronal-NOS has a role in the differentiation and mature function of septal cholinergic neurons. Through enhancing neuronal-NOS synthesis, endogenous NGF is likely to regulate NO functions in vivo.

Acetylcholinesterase↗
PubMed1996

Binding of the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase A phosphorylation.

Dynamic regulation of ion channel interactions with the cytoskeleton mediates aspects of synaptic plasticity, yet mechanisms for this process are largely unknown. Here, we report that two inwardly rectifying K+ channels, Kir 2.1 and 2.3, bind to PSD-95, a cytoskeletal protein of postsynaptic densities that clusters NMDA receptors and voltage-dependent K+ channels. Kir 2.3 colocalizes with PSD-95 in neuronal populations in forebrain, and a PSD-95/Kir 2.3 complex occurs in hippocampus. Within the C-terminal tail of Kir 2.3, a serine residue critical for interaction with PSD-95, is also a substrate for phosphorylation by protein kinase A (PKA). Stimulation of PKA in intact cells causes rapid dissociation of the channel from PSD-95. This work identifies a physiological mechanism for regulating ion channel interactions with the postsynaptic density.

Amino Acid Sequence↗
PubMed1996

Targeting nitric oxide to its targets.

Nitric oxide (NO) is now recognized as a major messenger molecule in the nervous system. In specific neuronal pathways, NO functions alternatively as a neurotransmitter and as a second messenger. Neuronal-type nitric oxide synthase is a widely distributed (nNOS or Type I) calmodulin-regulated enzyme and is coupled to a variety of neurotransmitter systems in brain and peripheral tissues. NO formation is linked in cerebellum to NMDA receptor activity, in myenteric neurons to neuronal nicotinic receptor activity, and in skeletal muscle to sarcolemmal depolarization. Coupling of nNOS activity to alternative calcium sources appears to be mediated, in part, by tethering nNOS to specific membrane proteins. In skeletal muscle, nNOS physically associates with the sarcolemmal dystrophin complex through a GLGF protein-association motif present near the N terminus of nNOS. This GLGF motif is likely also involved in anchoring nNOS to synaptic membrane complexes in brain. Subcellular targeting of nNOS represents a crucial mechanism for regulation of NO actions in the nervous system.

Amino Acid Sequence↗
PubMed1995

Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.

Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

Amino Acid Oxidoreductases↗
PubMed1995

Cloning and expression of two brain-specific inwardly rectifying potassium channels.

We have cloned two inwardly rectifying K+ channels that occur selectively in neurons in the brain and are designated BIRK (brain inwardly rectifying K+) channels. BIRK1 mRNA is extremely abundant and is enriched in specific brainstem nuclei, BIRK1 displays a consensus phosphate-binding loop, and expression in Xenopus oocytes has shown that its conductance is inhibited by ATP and adenosine 5'-[gamma-thio]triphosphate. BIRK2 is far less abundant and is selectively localized in telencephalic neurons. BIRK2 has a consensus sequence for cAMP-dependent phosphorylation.

Adenosine Triphosphate↗
PubMed1995

Expression of nitric oxide synthase in human central nervous system tumors.

The nitric oxide synthases (NOS) are a family of related enzymes which regulate the production of NO, a free radical gas implicated in a wide variety of biological processes. Vasodilation and increased tumor blood flow, increased vascular permeability, modulation of host tumoricidal activity, and free radical injury to tumor cells and adjacent normal tissues are pathophysiological features of malignant tumors that may be mediated by NO. We examined human brain tumors for three NOS isoforms and NADPH diaphrase, a histochemical marker of NOS activity in the brain. We detected increased expression of the brain and endothelial forms of NOS [NOS I and NOS II, respectively (C. Nathan and Q. Xie. Cell, 78: 915-919, 1994)] in astrocytic tumors, and the highest levels of expression was found in higher grade tumors. Each of these two isoforms was found in tumor cells and tumor endothelial cells. The macrophage isoform of NOS (NOS III) was less frequently detected and expressed at a lower level, predominantly in tumor endothelial cells. NADPH diaphorase staining for NOS activity paralleled this pattern of NOS expression. Western blot analysis of tumor tissues for these NOS isoforms confirmed these observations. Our data indicate that malignant central nervous system neoplasms express unexpectedly high levels of NOS and suggest that NO production may be associated with pathophysiological processes important to these tumors.

Amino Acid Oxidoreductases↗
PubMed1995

Possible regulation of capacitative Ca2+ entry into colonic epithelial cells by NO and cGMP.

A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.

Amino Acid Oxidoreductases↗
PubMed1995

Localization of nitric oxide synthase in the reproductive organs of the male rat.

Nitric oxide synthase (NOS), which catalyzes the production of nitric oxide (NO), was characterized within the reproductive tract of adult male Sprague-Dawley rats by means of biochemical and immunohistochemical techniques. Tissues examined included the testis, epididymis (caput, corpus, and cauda regions), vas deferens, ejaculatory duct, seminal vesicle, and coagulating gland. NOS activity was measured by use of an assay based on the stoichiometric conversion of [3H]-L-arginine to [3H]-L-citrulline and NO, catalyzed by NOS. Low levels of NOS activity were detected in the testis and seminal vesicle (< 0.5 fmol [3H]-L-citrulline formed/min/mg protein in each tissue). The highest levels of NOS activity were present in the cauda segment of the epididymis and in the vas deferens, each having a sevenfold greater amount of NOS activity than the testis (p < 0.05). Intermediate levels of NOS activity were detected in the coagulating gland (0.863 +/- 0.248 fmol [3H]-L-citrulline formed/min/mg protein), caput epididymidis (0.457 +/- 0.180 fmol [3H]-L-citrulline formed/min/mg protein), and corpus epididymidis (0.631 +/- 0.215 fmol [3H]-L-citrulline formed/min/mg protein). NADPH diaphorase histochemistry and NOS immunohistochemistry localized NOS to neuronal fibers coursing throughout the smooth musculature and subepithelial regions of the epididymis, vas deferens, and ejaculatory duct. Endothelial cells and nerve plexuses within the adventitia of blood vessels supplying reproductive tissues were also positive for NOS. Additional localizations of NOS were within epithelial cells of the epididymis and coagulating gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

Nitric oxide in skeletal muscle.

Reactive oxygen intermediates modulate skeletal muscle contraction, but little is known about the role of nitric oxide (NO). Here we show that rat skeletal muscle expresses neuronal-type NO synthase and that activity varies among several respiratory and limb muscles. Immunohistochemistry showed prominent staining of type II (fast) fibre cell membranes with antibodies against neuronal-type NO synthase. NO synthase activity in muscles correlated with type II fibre density. Resting diaphragm muscle produced detectable NO chi, but no reactive oxygen intermediates. In contrast, actively contracting muscle generated increased levels of reactive oxygen intermediates. Contractile function was augmented by blockers of NO synthase, extracellular NO chelation, and guanylyl cyclase inhibition; it was depressed by NO donors and by increased levels of cyclic GMP. Force-frequency plots of different muscles showed an inverse correlation between NO synthase activity and force development. Our results support two physiological functions of NO in skeletal muscle. The first is to promote relaxation through the cGMP pathway. The second is to modulate increases in contraction that are dependent on reactive oxygen intermediates and which are thought to occur through reactions with regulatory thiols on the sarcoplasmic reticulum.

Amino Acid Oxidoreductases↗