Biomedical subjects

Nathan G Hatcher

Publications and source records attributed to Nathan G Hatcher.

7 recordsLinked to original sources

PubMed2006

Serotonin catabolism and the formation and fate of 5-hydroxyindole thiazolidine carboxylic acid.

Serotonin (5-HT) functions as a neurotransmitter and neuromodulator in both the central and enteric nervous systems of mammals. The dynamic degradation of 5-HT metabolites in 5-HT-containing nervous system tissues is monitored by capillary electrophoresis with wavelength-resolved laser-induced native fluorescence detection in an effort to investigate known and novel 5-HT catabolic pathways. Tissue samples from wild type mice, genetically altered mice, Long Evans rats, and cultured differentiated rat pheochromocytoma PC-12 cells, are analyzed before and after incubation with excess 5-HT. From these experiments, several new compounds are detected. One metabolite, identified as 5-hydroxyindole thiazoladine carboxylic acid (5-HITCA), has been selected for further study. In 5-HT-incubated central and enteric nervous system tissue samples and differentiated PC-12 cells, 5-HITCA forms at levels equivalent to 5-hydroxyindole acetic acid, via a condensation reaction between L-cysteine and 5-hydroxyindole acetaldehyde. In the enteric nervous system, 5-HITCA is detected without the addition of 5-HT. The levels of L-cysteine and homocysteine in rat brain mitochondria are measured between 80 and 140 microm and 1.9 and 3.4 microm, respectively, demonstrating that 5-HITCA can be formed using available, free L-cysteine in these tissues. The lack of significant accumulation of 5-HITCA in the central and enteric nervous systems, along with data showing the degradation of 5-HITCA into 5-hydroxyindole acetaldehyde, suggests that an equilibrium coupled to the enzyme, aldehyde dehydrogenase type 2, prevents the accumulation of 5-HITCA. Even so, the formation of 5-HITCA represents a catabolic pathway of 5-HT that can affect the levels of 5-HT-derived compounds in the body.

Animals↗

PubMed2006

The first gamma-carboxyglutamate-containing neuropeptide.

A key factor in the characterization of peptide transmitters used in neuronal signaling is the correct elucidation of post-translational modifications, especially as they are often required to confer biological activity. A rare carboxylation modification is described on the D-peptide from the insulin prohormone in the sea slug, Aplysia californica. Using liquid chromatography purification coupled with electrospray ionization and nanoelectrospray ionization-ion trap-mass spectrometry (ESI- and nanoESI-MS), the presence of this D-peptide within Aplysia insulin (AI)-producing neurons is confirmed. Further detailed mass spectrometric analyses demonstrate that the Aplysia insulin D-peptide is carboxylated on the single glutamate residue within the sequence. This gamma-carboxy D-peptide, along with other identified AI-related peptides, is secreted from the central nervous system in response to ionophore stimulation, thus suggesting a signaling role within the nervous system. Although carboxylated peptides have been described previously, the Aplysia gamma-carboxy D-peptide appears to be the first reported carboxylated neuropeptide.

1-Carboxyglutamic Acid↗

PubMed2006

Nitric oxide potentiates cAMP-gated cation current in feeding neurons of Pleurobranchaea californica independent of cAMP and cGMP signaling pathways.

Critical roles for nitric oxide (NO) in regulating cell and tissue physiology are broadly appreciated, but aspects remain to be explored. In the mollusk Pleurobranchaea, NO synthase activity is high in CNS ganglia containing motor networks for feeding and locomotion, where a cAMP-gated cation current (I(Na,cAMP)) is also prominent in many neurons. We examined effects of NO on I(Na,cAMP) using voltage-clamp methods developed to analyze cAMP signaling in the live neuron, focusing on the identified metacerebral giant neuron of the feeding network. NO donors enhanced the I(Na,cAMP) response to injected cAMP by an averaged 85%. In dose-response measures, NO increased the current stimulated by cAMP injection without altering either apparent cAMP binding affinity or cooperativity of current activation. NO did not detectably alter levels of native cAMP or synthesis or degradation rates as observable in both current saturation and decay rate of I(Na,cAMP) responses to cAMP injection. NO actions were not exerted by cGMP signaling, as they were not mimicked by cGMP analogue nor blocked by inhibitors of guanylate cyclase and protein kinase G. NO potentiation of I(Na,cAMP) was broadly distributed among many other neurons of the feeding motor network in the buccal ganglion. However, NO did not affect a second type of I(Na,cAMP) found in locomotor neurons of the pedal ganglia. These results suggest that NO acts through a novel mechanism to regulate the gain of cAMP-dependent neuromodulatory pathways that activate I(Na,cAMP) and may thereby affect the set points of feeding network excitability and reactivity to exogenous input.

Animals↗

PubMed2005

monitoring activity-dependent peptide release from the CNS using single-bead solid-phase extraction and MALDI TOF MS detection.

To investigate dynamic peptidergic cell-cell communication, single micrometer-sized solid-phase extraction (SPE) beads were used to collect peptides from specific locations of well-characterized neurosecretory structures and even individual neuronal processes for off-line MALDI MS analyses. Peptide binding parameters of single SPE beads, including limits of collection, detection, and saturation capacity, were tested with 14C-labeled cytochrome c as well as with mixtures of multiple neuropeptides (bradykinin, Aplysia acidic peptide 1-20, and insulin). MALDI MS detection of secreted peptides was demonstrated in two well-characterized neurosecretory structures, the rat pituitary gland and single cultured Aplysia bag cell neurons. With cultured cells, precise placement of SPE beads allowed peptide collection from distinct neurites with spatial localization on the order of 200 microm, and SPE beads could be replaced within time frames that allowed analyte collection before and after cell stimulation paradigms. Comparison between pre- and poststimulation peptide profiles in both model systems allowed a directed strategy to determine which compounds were released with neuronal activity. Single SPE bead MALDI MS offers a novel approach to investigate peptide signaling that allows the detection and discovery of unknown intercellular signals secreted from a large variety of biological tissues.

Animals↗

PubMed2005

Online microdialysis-dynamic nanoelectrospray ionization-mass spectrometry for monitoring neuropeptide secretion.

Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.

Animals↗

PubMed2004

Spurious serotonin dimer formation using electrokinetic injection in capillary electrophoresis from small volume biological samples.

One normally assumes that the analytical measurement process does not introduce spurious compounds. Capillary electrophoresis is a separation method frequently used for small-volume biological measurements. We demonstrate the potential for creating new peaks in a capillary electropherogram when using electrokinetic injections and illustrate the potential deleterious effects with biological samples involving serotonin and nitric oxide measurements. Specifically, when measuring the serotonin content from individual neurons using electrokinetic injections from 360 nL stainless steel vials, we detect a new peak that we identify as a serotonin dimer. We do not observe this peak when using hydrodynamic injections.

Animals↗

PubMed2004

Systemic serotonin sulfate in opisthobranch mollusks.

Serotonin (5-hydroxytryptamine, 5-HT) is a ubiquitous modulatory neurotransmitter with roles as a neurohormone and neurotransmitter. However, few studies have been performed characterizing this molecule and its related metabolites in circulating fluids. Here, we demonstrate native 5-HT sulfate, but much lower levels of 5-HT, in hemolymph of the marine mollusk Pleurobranchaea californica. The metabolite 5-HT sulfate forms from 5-HT uptake and metabolism in central ganglia of Aplysia californica and in the visceral nerve and eye of Pleurobranchaea, but not in hemolymph itself. In addition, 5-hydroxyindole acetic acid (5-HIAA), while not detected in hemolymph, forms in higher quantities than does 5-HT sulfate in the eye and visceral nerve, and gamma-glu-5-HT is also observed in this area but never in hemolymph. As systemic 5-HT sulfate appears not to originate from the optic region or from systemic 5-HT, 5-HT sulfate likely derives from the nervous system. Circulating 5-HT sulfate is at least 10-fold higher during the light portion of a 12 : 12-h light/dark cycle than during the dark portion (p < 0.0007), but there is no obvious trend for free systemic tryptophan (Trp) (p > 0.3) in Pleurobranchaea. 5-HT in mollusks is associated with general arousal state; thus, diurnal systemic changes in a 5-HT catabolite may reflect a regulatory role for indole catabolism in behavioral rhythms.

5-Hydroxytryptophan↗