Protein inhibitor of nitric oxide synthase (NOS) and the N-methyl-D-aspartate receptor are expressed in the rat and mouse penile nerves and colocalize with penile neuronal NOS.

Nitrergic neurotransmission triggering penile erection is mediated by nitric oxide (NO) synthesized in the cavernosal nerves of the penis by penile neuronal NO synthase (PnNOS). In the central nervous system, nNOS is activated by the N-methyl-D-aspartate receptor (NMDAR) and, presumably, is inhibited by the protein inhibitor of NOS (PIN). The PnNOS and NMDAR are expressed in the penis, and PnNOS has been localized in penile nerves. Both proteins colocalize with PIN in the hypothalamus and the spinal cord involved in the control of erection. The present study aimed to elucidate the relationship between PnNOS, PIN, and NMDAR in the penis. It was found that in the rat, PIN was expressed in the pelvic ganglion and the cavernosal nerve, and penile PIN cDNA was cloned, sequenced, and expressed. Immunohistochemistry localized PIN to the cavernosal and dorsal nerve of the penis, whereas NMDAR was not detected in the latter. Dual-fluorescence labeling showed that PnNOS colocalized with PIN in both nerves but with NMDAR only in the cavernosal nerve. Aging did not affect the mRNA levels of PnNOS, nNOS, NMDAR, and PIN. Both PIN and NMDAR were detected in penile nerves of the wild-type and nNOS(-/-) mouse. The PIN protein did not inhibit or bind NOS in penile extracts, and in vivo, PIN cDNA reduced the erectile response to electrical field stimulation. In conclusion, PIN and NMDAR colocalize with PnNOS in penile nerves, but the functional significance of these protein interactions for penile erection remains to be elucidated.

Expression of penile neuronal nitric oxide synthase variants in the rat and mouse penile nerves.

Penile erection is mediated by nitric oxide (NO) synthesized by the neuronal nitric oxide synthase (nNOS). In the rat penis, the main nNOS mRNA variant, PnNOS, differs from cerebellar nNOS (CnNOS) by a 102 base pair insert encoding a 34-amino acid sequence. In the mouse, two nNOS mRNAs have been identified: nNOSalpha, encoding a 155-kDa protein, and an exon 2-deletion variant, nNOSbeta, encoding a 135-kDa protein that lacks a domain where a protein inhibitor of nNOS (PIN) binds. We wished to determine whether PnNOSalpha and beta are expressed in the rat penis and are located in the nerves and whether the beta form persists in the potent nNOS knock-out mouse (nNOS( big up tri, open big up tri, open)). A PnNOS antibody against the insert common to both PnNOSalpha and beta detected the expected 155-kDa protein in PnNOSalpha-transfected cells. This antibody, and the one common to PnNOS/CnNOS, showed (on Western blots) the 155- and 135-kDa nNOS variants in rat penile tissue during development and aging. PnNOSalpha mRNA and its subvariants were found as the main nNOS in the penile corpora, the cavernosal nerve, and the pelvic ganglia, with lower levels of PnNOSbeta mRNA. In tissue sections, PnNOS protein was immunodetected in the penile nerve endings in the rat and in the nNOS wild-type and nNOS( big up tri, open big up tri, open) mice. An antibody against the sequence encoded by exon 2 did not react (on Western blots) with the 135-kDa band, which confirms that this protein is the beta form. In conclusion, both PnNOSalpha and beta are expressed in the rat penis at all ages and are located in the nerves. The beta form may allow nitric oxide synthesis during erection to be partially insensitive to PIN. The residual expression of PnNOS, and possibly CnNOS, in the penis of the nNOS( big up tri, open big up tri, open) mouse occurs through transcription of the beta mRNA, and this may explain the retention of erectile function when the expression of nNOSalpha is disrupted.

Linkage analysis narrows the critical region for oculodentodigital dysplasia to chromosome 6q22-q23.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant condition with high penetrance and variable expressivity. The anomalies of the craniofacial region, eyes, teeth, and limbs indicate abnormal morphogenesis during early fetal development. Neurologic abnormalities occur later in life and appear to be secondary to white matter degeneration and basal ganglia changes. In familial cases, the dysmorphic and/or neurodegenerative components of the phenotype can be more severe and/or present at a younger age in subsequent generations, suggesting genetic anticipation. These clinical features suggest that the ODDD gene is pleiotropic with important functions throughout pre- and postnatal development. We have performed two-point linkage analysis with seven ODDD families and 19 microsatellite markers on chromosome 6q spanning a genetic distance of approximately 11 cM in males and 20 cM in females. We have refined the location of the ODDD gene between DNA markers D6S266/D6S261 (centromeric) and D6S1639 (telomeric), an interval of 1.01 (male) to 2.87 (female) cM. The strongest linkage was to DNA marker D6S433 (Zmax = 8.96, thetamax = 0.001). Families show significant linkage to chromosome 6q22-q23 and no evidence for genetic heterogeneity.

Detection and isolation of lectin-transfected COS cells based on cell adhesion to immobilized glycosphingolipids.

Two methods are described, one for detection and one for isolation of COS cells transiently expressing vertebrate lectins. The methods are based on specific cell adhesion to polystyrene microwells or magnetic beads adsorbed with glycosphingolipids. In the first method, glycolipids were adsorbed to wells of 96-well polystyrene plates. A suspension of lectin-transfected COS cells was added and the plate was incubated to allow cell adhesion to occur. The plate was then immersed in buffer, inverted (while immersed), and placed in a fluid-filled Plexiglas centrifugation chamber which was sealed to avoid introducing an air-liquid interface. The chamber, with the inverted plate enclosed, was centrifuged to remove nonadherent cells. The plate was then removed from the carrier (while immersed) and righted, and adherent cells were quantitated enzymatically or immunochemically using a 96-well plate reader. COS cells transfected with an expression plasmid carrying the gene for the rat Kupffer cell lectin (fucose and N-acetylgalactosamine specific) adhered specifically to globotetraosylceramide. Glycolipid- and lectin-specific cell adhesion was readily detected even when COS cells were transfected with a plasmid mixture containing 0.5% lectin-carrying plasmid and 99.5% irrelevant plasmid. This sensitivity will facilitate screening of plasmid pools to detect and isolate plasmids expressing mammalian lectin genes. To isolate COS cells transiently expressing lectin, glycosphingolipids were adsorbed to carboxylated magnetic polystyrene microspheres, which were mixed with the lectin-transfected COS cells. Adherent cells were collected on a fixed magnet and plasmid recovered for subsequent amplification.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Gangliosides as modulators of cell function.

Gangliosides, sialic acid-containing glycosphingolipids, are found in the outer leaflet of the plasma membrane of all vertebrate tissues and species. This report presents a brief introduction to the gangliosides and reviews the chemistry and topography of their biosynthesis. It also presents an overview of the present evidence supporting a physiological significance for the gangliosides in a variety of experimental systems. This includes consideration of their potential roles in development and cell adhesion. In addition, experimental examples in which gangliosides appear to influence signal transduction processes through their interactions with plasma membrane proteins are discussed.