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PubMed1998

Effect of Rev on the intranuclear localization of HIV-1 unspliced RNA.

Human immunodeficiency virus type 1 (HIV-1) Rev is a 19-kDa regulatory protein which binds to unspliced and partially spliced HIV-1 RNAs. Export, splicing, stability, and translation of HIV-1 RNAs are influenced by Rev. To further understand the effect of Rev on HIV-1 RNA splicing, the intranuclear localization of unspliced HIV-1 RNA and a cellular splicing factor was examined in the presence and absence of Rev. Splicing component-35 (SC-35) is an essential SR protein splicing factor which localizes into 20-40 nuclear granules (Fu, X. D., and Maniatis, T. Nature 343 (6257), 437-441, 1990). Laser scanning confocal microscopy was utilized to examine the colocalization of unspliced HIV-1 RNA and SC-35-containing granules. In the presence of Rev, many of the SC-35-containing granules were colocalized on their edges or completely colocalized with HIV-1 unspliced RNA speckles. In the absence of Rev, however, little colocalization of the unspliced HIV-1 RNA speckles and the SC-35-containing granules was observed. Quantitative RT-PCR was utilized to examine the effect of Rev on the level of fully spliced HIV-1 RNA. In the presence of Rev, a decrease in the level of fully spliced HIV-1 RNA was observed. Thus both the intranuclear localization and posttranscriptional processing of HIV-1 unspliced RNA are affected by Rev.

Cell Line↗

PubMed2001

Mouse hepatitis virus replicase protein complexes are translocated to sites of M protein accumulation in the ERGIC at late times of infection.

The coronavirus mouse hepatitis virus (MHV) directs the synthesis of viral RNA on discrete membranous complexes that are distributed throughout the cell cytoplasm. These putative replication complexes are composed of intimately associated but biochemically distinct membrane populations, each of which contains proteins processed from the replicase (gene 1) polyprotein. Specifically, one membrane population contains the gene 1 proteins p65 and p1a-22, while the other contains the gene 1 proteins p28 and helicase, as well as the structural nucleocapsid (N) protein and newly synthesized viral RNA. In this study, immunofluorescence confocal microscopy was used to define the relationship of the membrane populations comprising the putative replication complexes at different times of infection in MHV-A59-infected delayed brain tumor cells. At 5.5 h postinfection (p.i.) the membranes containing N and helicase colocalized with the membranes containing p1a-22/p65 at foci distinct from sites of M accumulation. By 8 to 12 h p.i., however, the membranes containing helicase and N had a predominantly perinuclear distribution and colocalized with M. In contrast, the p1a-22/p65-containing membranes retained a peripheral, punctate distribution at all times of infection and did not colocalize with M. By late times of infection, helicase, N, and M each also colocalized with ERGIC p53, a specific marker for the endoplasmic reticulum-Golgi-intermediate compartment. These data demonstrated that the putative replication complexes separated into component membranes that relocalized during the course of infection. These results suggest that the membrane populations within the MHV replication complex serve distinct functions both in RNA synthesis and in delivery of replication products to sites of virus assembly.

Animals↗

PubMed1996

Nitrergic and VIPergic neurons in the choroid and ciliary ganglion of the duck Anis carina.

Immunohistochemistry for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP), and NADPH diaphorase histochemistry, were applied to investigate neurons in the choroid and the ciliary ganglion of the muscovy duck Anis carina. Up to 1000 neurons in the choroid stained for NADPH diaphorase and showed virtually complete colocalization for nNOS immunoreactivity. Almost all of them co-stained for VIP, while about 90% of VIP immunoreactive cell bodies showed colocalization for nNOS. Two-thirds of the neurons were located, mostly singly, at nodes of a wide-meshed nerve plexus in the suprachoroid and were only rarely grouped in ganglia of up to 3 neurons. Numerous varicose nNOS/NADPH-diaphorase-positive nerve fibers were seen around large arterial blood vessels. These fibers derived mainly from paravascular cell bodies that represented about one-third of all choroidal neurons and also displayed costaining for nitrergic markers and VIP. Colocalization of nNOS/NADPH-d and VIP could be demonstrated in most of the perivascular fibers, while slightly more VIP-positive axons in the suprachoroid plexus did not costain for nNOS/NADPH-d. Small-caliber blood vessels and those localized in the choriocapillaris were not endowed with VIP/nNOS/NADPH-diaphorase-positive fibers. A few reactive neuronal cell bodies were also found in ciliary nerves, while most ciliary axons were unstained. In the ciliary ganglion a small subpopulation of neurons showed VIP/nNOS/NADPH-diaphorase colocalization. There were also nNOS/NADPH-d-positive cap-like terminals on ciliary ganglion cells. The presence of VIP/nNOS/NADPH-diaphorase positive neurons and nerve fibers in both the choroid and ciliary ganglion, and in the choroidal perivascular plexus, indicates peripheral nitrergic and VIPergic control of blood flow in the choroid of the duck.

Animals↗

PubMed1990

GABA, glycine, aspartate, glutamate and taurine in the vestibular nuclei: an immunocytochemical investigation in the cat.

The distributions of five amino acids with well-established neuroexcitatory or neuroinhibitory properties were investigated in the feline vestibular complex. Consecutive semithin sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of GABA, glycine, aspartate, glutamate and taurine. This approach allowed us to study the relative densities of the different immunoreactivities at the level of individual cell profiles. The results indicate that in the vestibular nuclei, neuronal colocalization of two or more neuroactive amino acids is the rule rather than an exception. Colocalization was found of immunoreactivities for GABA and glycine; glycine, aspartate and glutamate; glycine and aspartate, and glutamate and aspartate. GABA immunoreactive neurons were generally small and were found scattered throughout the vestibular complex. Glycine immunoreactive neurons were similarly distributed, except in the superior nucleus where the latter type of neuron could not be detected. Neuronal profiles colocalizing immunoreactivities for GABA and glycine occurred in all nuclei, but were most numerous in the lateral nucleus. The vast majority of the neurons showed noteworthy staining for glutamate and aspartate, although the level of immunoreactivities varied (e.g., the large neurons in the lateral and descending nuclei were more intensely aspartate immunoreactive than the smaller ones). Taurine-like immunoreactivity did not occur in neuronal cell bodies but appeared in Purkinje cell axons and in glial cell profiles. The functional significance of the complex pattern of amino acid colocalization remains to be clarified. In particular it needs to be distinguished between metabolic and transmitter pools of the different amino acids. The present results call for caution when attempts are made to conclude about transmitter identity on the basis of amino acid contents alone.

Amino Acids↗

PubMed2003

Proliferation-differentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes.

The topological relationships between erbB receptors and ligands of the epidermal growth factor family were characterized by immunocytochemistry in normal and psoriatic epidermis and in proliferating and differentiating human keratinocytes in culture. Spatial colocalization of receptors and ligands was assessed by dual immunostaining. Expression of epidermal growth factor receptor (EGFr), erbB2, and erbB3, but not erbB4, was detected throughout the epidermis, although labeling for erbB2 and erbB3 was accentuated in the upper spinous layers, and EGFr was more strongly labeled in basal cells. Of the tested growth factors, heparin-binding epidermal growth factor (HB-EGF) was diffusely expressed throughout normal and psoriatic epidermis and sparsely colocalized with EGFr in all viable epidermal layers, with increased colocalization in psoriatic epidermis. In contrast, betacellulin and heregulin/neu differentiation factor (NDF) alpha were largely restricted in their distribution to the upper spinous and granular layers. Betacellulin was downregulated in psoriatic keratinocytes. Although heregulin/NDF-beta was undetectable in normal epidermis, it was upregulated in psoriasis. Betacellulin and heregulin/NDF-alpha strikingly colocalized with EGFr and erbB3 receptors in the granular layer and in a declining gradient from the granular zone to the basal layer, respectively. Similar patterns were observed in cultured keratinocytes under proliferative conditions and upon differentiation in high-calcium medium. These morphological data collectively suggest divergent functions for members of the growth factor family, and in particular, we propose that betacellulin and heregulin/NDF-alpha are involved in epidermal morphogenesis and/or in maintenance of the differentiated phenotype.

Betacellulin↗

PubMed2000

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina.

The histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunoreactivity of neuronal nitric oxide synthase (nNOS-IR) can be demonstrated in various cell types of the vertebrate retina. In this study, we have focused on characterizing the different NADPH-d-positive amacrine cell types in turtle retina. Cryostat sections were examined by confocal laser scanning microscopy for double immunofluorescence with antibodies against nNOS and either GABA or glycine, or by combining histochemistry with immunocytochemistry to obtain triple labeling with NADPH-d, GABA, and glycine. Forty-eight percent of the NADPH-d-labeled amacrine cells colocalized GABA, 52% glycine. Here we show that two morphologically different types of amacrine cell are nNOS/glycine-IR and three types are nNOS/GABA-IR. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize with nNOS-IR or NADPH-d-labeled amacrine cells, but 15% of the NOS-labeled amacrine cells showed immunoreactivity against calbindin. Only GABA has been seen to colocalize with NADPH-d in amacrine cells in previous reports in other species. The finding here of glycine colocalizing with NO-containing cells is novel. We suggest that NO, apart from its well known function in gap junction regulation, can also modulate the release of both GABA and glycine in the turtle retina.

Animals↗

PubMed1995

Nitrinergic and peptidergic innervations and their inter-relationships in human colon.

The distribution and colocalization of nitrinergic and peptidergic nerves were examined in six human colons. The tissues were fixed, cryosectioned, and standard immunohistochemistry was performed for several known neuropeptides. The same sections were stained for NADPH-diaphorase to denote nitric oxide synthase. NADPH-diaphorase-positive myenteric neurons were counted and colocalization noted for each peptide, as well as for peptide terminations. Galanin was the only neuropeptide that colocalized to a significant extent (23.0 +/- 7.21%) with NADPH-diaphorase-positive myenteric neurons. Many neuropeptide-containing nerve fibers had extensive terminations onto NADPH-diaphorase-positive neurons. Vasoactive intestinal peptide was the only neuropeptide that colocalized with NADPH-diaphorase to any extent in nerve fibers within circular muscle (59.5 +/- 9.3%). Fiber distribution in the longitudinal muscles showed a similar, but less dense pattern. These observations provide morphological evidence for the presence of nitric oxide, a candidate nonadrenergic noncholinergic neurotransmitter in the human colon.

Aged↗

PubMed1995

GAP-43 and its relation to autonomic and sensory neurons in sciatic nerve and gastrocnemius muscle in the rat.

The presence of the growth-associated protein, GAP-43, in rat sciatic nerve and gastrocnemius muscle was studied, using indirect immunofluorescence, in lumbar sympathectomized and sham-sympathectomized rats. To study fast axonal transport and accumulation of immunogenic GAP-43, the sciatic nerves were crush operated, 6 h before perfusion fixation. In sections of normal, crushed sciatic nerve GAP-43-like immunoreactivity (LI) rapidly accumulated, on both sides of the crushes, in medium and thin sized axons. In double immunostaining experiments, GAP-43-LI was mainly colocalized with tyrosine hydroxylase (TH)-LI, or neuropeptide Y (NPY)-LI, markers of sympathetic nerves. In some axons, GAP-43-LI was colocalized with Substance P (SP)-LI. In perivascular nerve terminals in the gastrocnemic muscle, strong GAP-43-immunofluorescence was observed, in most cases colocalized with TH-LI, but in some terminals with SP-LI. Three days after lumbar sympathectomy (removal of the L1-L4 sympathetic ganglia bilaterally), TH-LI and NPY-LI positive axons in the sciatic nerve were reduced in number by more than 90%. GAP-43-LI positive axons were reduced by about 50%. In the gastrocnemic muscle, some GAP-43-LI positive terminals, but very few TH-LI positive nerve fibres, were found around blood vessels. No further changes were seen 8 days after sympathectomy. SP-LI in axons in the sciatic nerve and in perivascular nerve terminals of the gastrocnemic muscle, did not change after sympathectomy; most of these axons also contained GAP-43-LI. S-100-LI was present periaxonally in the sciatic nerves, but it did not colocalize with GAP-43, indicating that Schwann cells contained no GAP-43-LI in these experiments. The results show that, in normal adult rats, GAP-43-LI is mainly present in sympathetic and sensory nerve fibers in sciatic nerve and in perivascular nerve terminals. The peptide is axonally transported, mainly in sensory and adrenergic axons.

Animals↗

PubMed1985

Anatomy and physiology of the neuroendocrine arcuate nucleus.

The arcuate nucleus surrounds the ventral part of the third ventricle and contains densely packed small neurons with 1-3 dendrites. At least fifteen transmitters and neuropeptides have been found in perikarya of arcuate neurons. Each transmitter and neuropeptide have a characteristic distribution. In many cases distributions overlap (for example, dopamine and somatostatin, dopamine and neurotensin, neuropeptide Y and somatostatin) and alpha-MSH and beta-endorphin seem to have identical distributions but there are also distinctive neuronal populations containing only one of the described transmitters or neuropeptides (neuropeptide Y and alpha-MSH). Studies show extensive colocalization of dopamine and neurotensin and sparse colocalization of dopamine and GABA, neuropeptide Y and FMRF-NH2 and neuropeptide Y and somatostatin. Colocalization does not seem to be the rule in the arcuate, however, it is possible that colocalization may vary with the physiological state or sex of the animal. It also should be noted that our techniques may not be sensitive enough. To study efferent projections as a possible organizing principle within the arcuate, retrograde fluorescent tracing was combined with transmitter and neuropeptide immunohistochemistry. Mainly NPY and alpha-MSH neurons were studied and both peptides are present in projections to the preoptic area as well as to the midbrain periaqueductal gray. Some arcuate neurons were found to have collateral axons to both these areas. The arcuate communicates primarily with the pituitary gland, hypothalamus, limbic system, midbrain periaqueductal gray and autonomic nuclei of the brain stem. In this way, the arcuate may be involved in integrating emotional, sensory, vegetative homeostatic and autonomic functions with endocrine functions.

Animals↗

PubMed1987

Coexistence of putative neuroactive substances in lateral olivocochlear neurons of rat and guinea pig.

We have used the retrograde axonal transport of Fast Blue, injected intra-cochlearly, to identify in the rat lateral superior olive (LSO) neurons which belong to the lateral olivocochlear system (LOCS). Using immunohistofluorescence technique, we have localized within Fast Blue-labeled neurons immunostainings for enkephalins (Met-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8), dynorphins (alpha-neo-endorphin, dynorphin 1-17) or choline acetyltransferase (ChAT). Many Fast Blue-labeled neurons did not show any immunostaining, but all the immunostained neurons found in the LSO were Fast Blue-labeled. In immunohistofluorescence colocalization experiments of two antigens, we could colocalize within the same neurons of the rat LSO immunostainings for ChAT and enkephalins and for ChAT and dynorphins. In each case, neurons only immunostained for ChAT, enkephalins or dynorphins could also be observed. A colocalization of the immunostainings for Met-enkephalin and dynorphins within neurons of the guinea pig and rat LSO was also found. However, in this case, neurons which did not show colocalization were only Met-enkephalin-immunoreactive, thus suggesting that all the dynorphins immunoreactive LSO neurons also contain enkephalins. These findings support the idea that the neurons of the LSO which contain ChAT-, enkephalin- or dynorphin-immunostainings project to the cochlea and belong to the LOCS. It can also be concluded that acetylcholine, enkephalins and dynorphins coexist within a same population of neurons of the LOCS, although other patterns of co-containment of neuroactive substances within LOCS neurons may also exist.

Animals↗

PubMed1991

Neuropeptide-Y innervation of the rat spleen: another potential immunomodulatory neuropeptide.

Neuropeptide-Y (NPY) is a 36 amino acid peptide that acts as a chemical messenger in the central and peripheral nervous systems. NPY often is found colocalized with the classical neurotransmitter norepinephrine (NE) and can potentiate the effects of this neurotransmitter postsynaptically in many systems. Using immunocytochemistry for NPY and specific lymphoid cell markers, we mapped the distribution of NPY-positive nerve fibers in the rat spleen. NPY-positive nerve fibers were present along the vasculature, trabeculae, and capsule, and also were found associated with specific lymphoid parenchymal compartments of the spleen, in close contact with lymphocytes and macrophages. These contacts were investigated further at the electron microscopic level. NPY-positive nerve terminals were found in close apposition with lymphocytes in the periarteriolar lymphatic sheath, and with lymphocytes and macrophages in the marginal zone. Previous studies have reported that postganglionic noradrenergic nerve fibers innervate specific lymphoid compartments of the rat spleen, with nerve terminals forming direct appositions with cells of the immune system. The possible colocalization of NPY and NE in these nerve fibers was investigated by chemical sympathectomy with 6-hydroxydopamine, followed by immunocytochemical labeling of NPY and tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine synthesis. Colocalization also was investigated by labeling for NPY with a fluorescent label, eluting the NPY, and staining for TH with diaminobenzidine as the label. These studies demonstrate that norepinephrine and NPY are colocalized in the postganglionic sympathetic nerve fibers of the rat spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Cytoskeletal interactions of synapsin I in non-neuronal cells.

Synapsin I is a neuronal phosphoprotein involved in the localization and stabilization of synaptic vesicles. Recently, synapsin I has been detected in several non-neuronal cell lines, but its function in these cells is unclear. To determine the localization of synapsin I in non-neuronal cells, it was transiently expressed in HeLa and NIH/3T3 cells as an enhanced green fluorescent protein fusion protein. Synapsin I-enhanced green fluorescent protein colocalized with F-actin in both cell lines, particularly with microspikes and membrane ruffles. It did not colocalize with microtubules or vimentin and it did not cause major alterations in cytoskeletal organization. Synapsin Ia-enhanced green fluorescent protein colocalized with microtubule bundles in taxol-treated HeLa cells and with F-actin spots at the plasma membrane in cells treated with cytochalasin B. It did not noticeably affect F-actin reassembly following drug removal. Synapsin Ia-enhanced green fluorescent protein remained colocalized with F-actin in cells treated with nocodazole, and it did not affect reassembly of microtubules following drug removal. These results demonstrate that synapsin I interacts with F-actin in non-neuronal cells and suggest that synapsin I may have a role in regions where actin is highly dynamic.

Actins↗

PubMed2006

P2X5 receptors are expressed on neurons containing arginine vasopressin and nitric oxide synthase in the rat hypothalamus.

In this study, the P2X(5) receptor was found to be distributed widely in the rat hypothalamus using single and double labeling immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. The regions of the hypothalamus with the highest expression of P2X(5) receptors in neurons are the paraventricular and supraoptic nuclei. The intensity of P2X(5) immunofluorescence in neurons of the ventromedial nucleus was low. 70-90% of the neurons in the paraventricular nucleus and 46-58% of neurons in the supraoptic and accessory neurosecretory nuclei show colocalization of P2X(5) receptors and arginine vasopressin (AVP). None of the neurons expressing P2X(5) receptors shows colocalization with AVP in the suprachiasmatic and ventromedial nuclei. 87-90% of the neurons in the lateral and ventral paraventricular nucleus and 42-56% of the neurons in the accessory neurosecretory, supraoptic and ventromedial nuclei show colocalization of P2X(5) receptors with neuronal nitric oxide synthase (nNOS). None of the neurons expressing P2X(5) receptors in the suprachiasmatic nucleus shows colocalization with nNOS. These findings provide a morphological basis for possible functional interactions between the purinergic and nitrergic or vasopressinergic neurotransmitter systems.

Animals↗

PubMed2006

Protein expression and mRNA cellular distribution of the NKCC1 cotransporter in the dorsal root and trigeminal ganglia of the rat.

Primary afferent neurons maintain depolarizing responses to GABA into adulthood. The molecular basis for this GABAergic response appears to be the Na+K+2Cl- cotransporter NKCC1 that contributes to the maintenance of a high intracellular chloride concentration. Recently, a role for NKCC1 has been proposed in nociceptive processing which makes it timely to gain a better understanding of the distribution of NKCC1 in sensory ganglia. Here, we describe that, in the rat, NKCC1 mRNA is predominately expressed by small and medium diameter dorsal root (DRG) and trigeminal (TG) ganglion neurons. The colocalization of NKCC1 mRNA with sensory neuron population markers was assessed. In the DRG, many NKCC1 mRNA-expressing neurons colocalized peripherin (57.0+/-2.5%), calcitonin-gene-related peptide (CGRP, 39.2+/-4.4%) or TRPV1 immunoreactivity (50.0+/-1.9%) whereas only 8.7+/-1.2% were co-labeled with a marker for large diameter afferents (N52). Similarly, in the TG, NKCC1 mRNA-expressing neurons frequently colocalized peripherin (50.0+/-3.0%), CGRP (35.4+/-2.6%) or TRPV1 immunoreactivity (44.7+/-1.2%) while 14.8+/-1.3% were co-labeled with the N52 antibody. NKCC1 mRNA was also detected in satellite glial (SGCs) in both the DRG and TG. Colocalization of NKCC1 protein with the SGC marker NG2 confirmed the phenotype of these NKCC1-expressing glial cells. In contrast to in situ hybridization experiments, we did not observe NKCC1 immunoreactivity in primary afferent somata. These findings suggest that NKCC1 is expressed in anatomically appropriate cells in order to modulate GABAergic responses in nociceptive neurons. Moreover, these results suggest the possibility of a functional role of NKCC1 in the glial cells closely apposed to primary sensory afferents.

Animals↗

PubMed2005

Cellular and synaptic distribution of NR2A and NR2B in macaque monkey and rat hippocampus as visualized with subunit-specific monoclonal antibodies.

The functional and pharmacological attributes of the N-methyl-D-aspartate (NMDA) receptor are related to its subunit composition, thus resolving the subunit composition of NMDA receptors in specific classes of synapses is an important step in characterizing excitatory circuits. Toward this end, mouse monoclonal antibodies were raised against fusion protein antigens corresponding to the putative amino acid sequences of human NMDA receptor subunits NR2A and NR2B. The subunit specificity of these monoclonal antibodies was demonstrated with transfected human and rat NMDA receptor cDNAs, and their immunoreactivity was established in rat, macaque monkey, and human brain tissue. At the light microscopic level, both NR2A and NR2B exhibit a distribution in monkey and rat hippocampus very similar to NMDA receptor subunit NR1, and both are highly colocalized with NR1. Electron microscopic immunogold studies demonstrated that both NR2A and NR2B are often present in asymmetric synapses in CA1, commonly colocalized with NR1, and often colocalized with each other in the same asymmetric synapses. Both assembly and synthetic pools are present within spines and spine necks, respectively, particularly for NR2A. The confocal and ultrastructural data suggest that whereas NR1, NR2A, and NR2B are essentially uniformly colocalized in hippocampal projection neurons, there is extensive heterogeneity at the synaptic level that would lead to multiple functional classes of NMDA receptor-mediated synapses, and extensive capacity for plasticity at the synapse. Thus, the subunit profile of a given synapse may be dynamic, with regulation of local synthesis and insertion of different subunits into the synapse leading to a complex, heterogeneous, and shifting set of functional attributes of the NMDA receptor.

Animals↗

PubMed2026

Cross-Ancestry Proteogenomic Analyses Identified New Therapeutic Insights for Ischemic Heart Disease.

BACKGROUND: Most drugs target proteins, and proteome-wide genetic analyses in diverse populations could discover potential novel and repurposed targets for improved prevention and treatment of ischemic heart disease (IHD) beyond statin therapy. OBJECTIVES: The purposes of this study were to use cis-acting single nucleotide polymorphisms (cis-pQTLs) identified for plasma proteins in East Asians and Europeans to discover and validate potential drug targets for IHD. METHODS: We measured plasma levels of 9,520 (Olink/SomaScan: 2,923/7,297) proteins in a case-cohort study of IHD (1,976 incident cases and 2,001 subcohort controls) in statin-free individuals in the prospective China Kadoorie Biobank (CKB). Genome-wide association studies identified 2,895 (Olink/SomaScan: 1,301/1,594) cis-pQTLs for these proteins in CKB. Two-sample Mendelian randomization (MR) and colocalization analyses assessed associations of all available cis-pQTLs for these proteins with IHD in East Asians (n = 29,319 cases), with further replication in Europeans (n = 181,522 cases) and comparison with findings in previous MR studies. RESULTS: In CKB observational analyses, a total of 959 (Olink/SomaScan: 426/533) proteins were associated at false discovery rate-corrected P < 0.05 with IHD after adjusting for major IHD risk factors. Two-sample MR analyses provided genetic support for 54 unique (Olink/SomaScan: 36/28) proteins in IHD etiology. Colocalization analyses confirmed shared gene-protein-IHD associations (posterior probability of hypothesis 4 [PPH4] ≥0.8) for 15 unique (Olink/SomaScan: 10/10) proteins, including 8 lipid-related, 3 inflammation-related, 1 blood pressure-related, and 3 alcohol-related proteins in East Asians. In Europeans, MR analyses of 12 non-alcohol-related proteins showed directionally concordant results for 8 proteins, with 5 having strong colocalization evidence of shared gene-protein-IHD associations (PPH4 ≥0.8), including 4 lipid-related (proprotein convertase subtilisin/kexin type 9, LPA, APOE, cadherin-1) and 1 systolic blood pressure-related (fibroblast growth factor 5) protein. However, 4 proteins showed directionally discordant MR results, including 2 lipid-related (APOA5, SORT1) and 1 inflammation-related (transforming growth factor beta 1) proteins with strong colocalization evidence of shared gene-protein-IHD associations (PPH4 ≥0.8). Comparison with previous MR studies revealed little consistency across studies in the number and identity of target proteins for IHD beyond well-established lipid-related (low-density lipoprotein cholesterol, lipoprotein(a), and triglycerides) or inflammation-related (interleukin-6) protein targets. CONCLUSIONS: The findings support a role for lipid-driven chronic inflammation in IHD etiology, and treatment strategies simultaneously targeting multiple lipid and inflammation pathways should be prioritized for further research to improve drug treatment of IHD beyond statin therapy.

Aged↗

PubMed2005

Nitroxidergic neurons in rat nucleus tractus solitarii express vesicular glutamate transporter 3.

Earlier we reported that glutamate transporter (VGLUT) 2 and neuronal nitric oxide synthase (nNOS) are colocalized in some fibers and are present in apposing fibers in the nucleus tractus solitarii (NTS). Those findings provided anatomical support for a hypothesized physiological link between glutamate and nitric oxide (NO.) in the NTS. Recently a third class of VGLUT, VGLUT3, was identified, but its distribution in NTS and its anatomical relationship with nNOS have not been shown. In this study we tested the hypothesis that neurons and fibers containing VGLUT3 lie in close proximity to those containing nNOS and that both proteins colocalize in some neurons and fibers in the NTS. We perfused rats and obtained brain stem sections and nodose ganglion sections for immunofluorescent staining analyzed by confocal microscopy. The NTS contained moderate VGLUT3-immunoreactivity (IR), with the intermediate, medial and interstitial subnuclei containing higher VGLUT3-IR than other subnuclei. Although all three forms of VGLUT were present in the NTS, VGLUT3-IR was not colocalized with either VGLUT1-IR or VGLUT2-IR in either processes or cells in the brain stem. Cells and processes containing both VGLUT3-IR and nNOS-IR were noted in all NTS subnuclei and in the nodose ganglion. Triple immunofluorescent staining revealed that cells double-labeled for nNOS-IR and VGLUT3-IR were all additionally labeled for neuronal nuclear antigen (NeuN), a neuronal marker. These findings support our hypothesis that neurons and fibers containing VGLUT3 lie in close proximity to those containing nNOS and that both proteins colocalize in some neurons and fibers in the NTS.

Afferent Pathways↗

PubMed2006

The role of phosphodiesterase isoforms 2, 5, and 9 in the regulation of NO-dependent and NO-independent cGMP production in the rat cervical spinal cord.

NO-responsive, cGMP-producing structures are abundantly present in the cervical spinal cord. NO-mediated cGMP synthesis has been implicated in nociceptive signaling and it has been demonstrated that cGMP has a role establishing synaptic connections in the spinal cord during development. As cGMP levels are controlled by the activity of soluble guanylyl cyclase (synthesis) and the phosphodiesterase (PDE) activity (breakdown), we studied the influence of PDE activity on NO-stimulated cGMP levels in the rat cervical spinal cord. cGMP-immunoreactivity (cGMP-IR) was localized in sections prepared from slices incubated in vitro. A number of reported PDE isoform-selective PDE inhibitors was studied in combination with diethylamineNONOate (DEANO) as a NO-donor including isobutyl-methylxanthine (IBMX) as a non-selective PDE inhibitor. We studied 8-methoxy-IBMX as a selective PDE1 inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitor, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. cGMP-IR structures (nerve fibers, axons, and terminals) were characterized using the following neurochemical markers: vesicular transporter molecules for acetylcholine, GABA, and glutamate (type 1 and type 2), parvalbumin, glutamate transporter molecule EAAT3, synaptophysin, substance P, calcitonin gene-related peptide, and isolectin B4. Most intense cGMP-IR was observed in the dorsal lamina. Ventral motor neurons were devoid of cGMP-IR. cGMP-IR was observed in GABAergic, and glutamatergic terminals in all gray matter laminae. cGMP-IR was abundantly colocalized with anti-vesicular glutamate transporter 2 (vGLUT2), however not with the anti-vesicular glutamate transporter 1 (vGLUT1), suggesting a functional difference between structures expressing vGLUT1 or vGLUT2. cGMP-IR did not colocalize with substance P- or calcitonin-gene related peptide-IR structures, however did partially colocalize with isolectin B4 in the dorsal horn. cGMP-IR in cholinergic structures was observed in dorsal root fibers entering the spinal cord, occasionally in laminae 1-3, in laminae 8 and 9 in isolated boutons and in the C-type terminals, and in small cells and varicosities in lamina 10. This latter observation suggests that the proprioceptive interneurons arising in lamina 10 are also NO-responsive. No region-specific nor a constant co-expression of cGMP-IR with various neuronal markers was observed after incubation of the slices with one of the selected PDE inhibitors. Expression of the mRNA of PDE2, 5, and 9 was observed in all lamina. The ventral motor neurons and the ependymal cells lining the central canal expressed all three PDE isoforms. Incubation of the slices in the presence of IBMX, DEANO in combination with BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase, provided evidence for endogenous NO synthesis in the slice preparations and enhanced cGMP-IR in all lamina. Under these conditions cGMP-IR colocalized with substance P in a subpopulation of substance P-IR fibers. It is concluded that NO functions as a retrograde neurotransmitter in the spinal cord but that also postsynaptic structures are NO-responsive by producing cGMP. cGMP-IR in a subpopulation of isolectin B4 positive fibers and boutons is indicative for a role of NO-cGMP signaling in nociceptive processing. cGMP levels in the spinal cord are controlled by the concerted action of a number of PDE isoforms, which can be present in the same cell.

3',5'-Cyclic-AMP Phosphodiesterases↗