Proteins and lipids define the diffusional field of nitric oxide.

Nitric oxide (NO) fluxes released from the surface of individual activated macrophages or cells localized in small aggregates were measured with a novel polarographic self-referencing microsensor. NO fluxes could be detected at distances from the cells of 100-500 microm. The initial flux and the distance from the cells at which NO could be detected were directly related to the number of cells in the immediate vicinity of the probe releasing NO. Thus, whereas NO fluxes of approximately 1 pmol. cm(-2). s(-1) were measured from individual macrophages, aggregates composed of groups of cells varying in number from 18 to 48 cells produced NO fluxes of between approximately 4 and 10 pmol. cm(-2). s(-1). NO fluxes required the presence of L-arginine. Signals were significantly reduced by the addition of hemoglobin and by N-nitro-L-arginine methyl ester. NO fluxes were greatest when the sensor was placed immediately adjacent to cell membranes and declined as the distance from the cell increased. The NO signal was markedly reduced in the presence of the protein albumin but not by either oxidized or reduced glutathione. A reduction in the NO signal was also noted after the addition of lipid micelles to the culture medium. These results demonstrate that NO can be detected at significant distances from the cell of origin. In addition, both proteins and lipids strongly influence the net movement of free NO from macrophages. This suggests that these tissue components play an important role in regulating the biological activity of NO.

Minimal amidine structure for inhibition of nitric oxide biosynthesis.

Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on gamma-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K(i) formamidine approximately 800 microM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.

Isolation and characterization of a skate retinal GABA transporter cDNA.

PURPOSE: The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is believed to play a crucial role in the processing of information within the vertebrate retina. Extracellular concentrations of GABA are thought to be tightly regulated by carrier-mediated transport proteins in neurons and glial cells. The purpose of this work was to isolate the gene that encodes one of these transport proteins in the skate retina. METHODS: cDNA clones were isolated from a skate retinal cDNA library using a mouse retinal GABA transporter (GAT1) cDNA as a probe. The PCR technique was used to fill sequence gaps, and 5' and 3' RACE were employed to amplify the 5' and 3' untranslated regions. The amplified fragments were subcloned into a T-vector. Blots containing RNA from 10 different tissues were probed to determine the size of the transcript and the tissue distribution. RESULTS: Sequence analysis revealed that the skate retinal GABA transporter cDNA shared 72% identity with the mouse GABA transporter-1 at the DNA level and 80% identity at the amino acid level. Multiple sequence alignments showed that our sequence is closest to the Torpedo GABA transporter-1. Two transcripts, 4.5 and 7 kb, were detected in retina and possibly brain by RNA blot analysis. Fourteen introns were detected in the skate GABA transporter gene. CONCLUSIONS: We successfully isolated a full length GABA transporter cDNA from the retina of the skate. The size of the full length sequence of the skate retinal GABA transporter is in agreement with the size of the smaller transcript detected on RNA blots. The larger transcript observed on the RNA blot may be the result of either alternative splicing or utilization of a downstream poly A signal.