Extracts of retina and brain that excite afferent fibers innervating hair cells contain a compound related to hydroxyphenylglycine-N-carbamoyl.

To identify new neurotransmitter and modulator candidates that might be important in transmission from sensory hair cells to afferent nerves, we examined extracts of neural tissue for compounds that excite afferent fibers innervating hair cells. Here, we describe the extraction and purification from retina and brain of a potent, unstable, excitatory compound with pharmacological activity similar to glutamate on afferent fibers innervating hair cells. This compound, however, was clearly distinguished from glutamate, other common amino acids, and known endogenous glutamate-receptor agonists. After derivatization and analysis by gas chromatography-mass spectrometry, the major compound found in highly purified neuroactive chromatographic fractions had the same gas chromatographic elution time and mass spectrum as the compound formed by derivatization of L-p-hydroxyphenylglycine-N-carbamoyl. Hydroxyphenylglycine-N-carbamoyl, however, did not copurify with the neuroactive compound and was not neuroactive. We thus hypothesize that the detected compound was produced from a precursor, structurally related to L-p-hydroxyphenylglycine-N-carbamoyl, that was a major component of the neuroactive chromatographic fractions. Because several compounds related to hydroxyphenylglycine are known to act on glutamate receptors, such a compound is an interesting candidate to be an endogenous glutamate-receptor ligand in the mammalian nervous system.

Calcium and magnesium transport by isolated goldfish hair cells.

We used electron-probe analysis (EPA) to investigate the transport of the divalent cations calcium and magnesium across the plasma membranes of hair cells. Unlike ion-sensitive fluorescent dyes, EPA detects these ions regardless of the state of chemical combination inside the cell; changes in these cell ions determined by EPA indicate net transport across the cell membrane. Raising or lowering either extracellular divalent cation within 1 mM of its control level raised or lowered its cell contents, but further increases in extracellular concentration of either ion had little additional effect on the cell content of that ion. New steady-state contents could be obtained within minutes, but the net divalent cation currents required to account for the observed changes would have been smaller than most currents recorded electrophysiologically, less than 1 pA. The effects of replacing extracellular Na+ with other ions were consistent with the presence in hair cells of exchangers for divalent cations thought to occur in other tissues: electrically neutral sodium/magnesium exchange (2 Na+ per Mg2+) and electrogenic sodium/calcium exchange (at least 3 Na+ per Ca2+). The increase in cell Ca after 1 minute of potassium-depolarization was similar to that expected from electrophysiological studies of voltage-sensitive calcium currents in goldfish hair cells. After that time in elevated potassium, however, either calcium-entry pathways were inhibited or calcium-export mechanisms were enhanced.

Extracellular N-methyl-D-glucamine leads to loss of hair-cell sodium, potassium, and chloride.

The organic cation N-methyl-D-glucamine (NMDG) is often used to replace extracellular sodium in experimental studies. Replacing 100 mM of Na+ with NMDG+ in the fluid bathing isolated goldfish hair cells led to a rapid loss not only of cell sodium, but also of cell potassium and chloride. The loss of inorganic cell solutes was accompanied by acidification of the cells. Cell volume did not change significantly. These results are consistent with passage of the cationic form of NMDG, a titratable amine with a pKa of 9.6, across the hair-cell membrane. These results should have bearing in interpreting results of experiments in which this cation is used to replace extracellular sodium, particularly for periods of time longer than 3 min.

Flavin adenine dinucleotide is a major endogenous fluorophore in the inner ear.

When illuminated with visible light, hair cells can exhibit autofluorescence (Lewis et al. [1982] Science 215, 1641-1643) concentrated in the basal pole near the synapses (Sento and Furukawa [1987] J. Comp. Neurol. 258, 352-367). The autofluorescence is enhanced by formaldehyde. The level of fluorescence is high enough to interfere with fluorescence microscopy of hair cells and to suggest that the fluorescent substance might have a particular role in hair-cell function. To identify this substance, we extracted a substance with formaldehyde-enhanced fluorescence from the inner ears of goldfish and purified it chromatographically. The substance copurified with FAD and had the same fluorescence emission spectrum. Two further results supported the identity of the endogenous fluorescent substance with FAD. First, as is the case with flavins, the autofluorescence in inner ear tissue examined within a few hours after fixation was reduced by addition of dithionite. Second, as is the case with the formaldehyde-enhanced fluorophore, the fluorescence of FAD was enhanced by formaldehyde. FAD accounted for 90% of flavins in goldfish inner ears; its concentration in the sensory epithelium was estimated to be about 30 nmol/g tissue weight, one of the highest tissue concentrations known. The FAD is probably associated with an unidentified flavoprotein concentrated in the basal, synaptic region of the hair cell.

Rapid resting ion fluxes in goldfish hair cells are balanced by (Na+,K+)-ATPase.

Inhibition of sodium/potassium pumping by isolated goldfish hair cells led to a rapid gain of sodium and loss of potassium. Half-times for turnover were about 10 min, among the fastest of any cell type examined by electron-probe analysis. Pumping was inhibited by removal of extracellular potassium or by treatment with 1 mM ouabain, as expected of a classical (Na+,K+)-ATPase. The initial rate of entry of sodium after inhibition, about 4 mM/min, provided an estimate of resting sodium-entry and sodium-pumping rates. After return to control medium, cells loaded with sodium by removal of extracellular potassium could recover their normal high-potassium/low-sodium status. The initial rate of recovery (an estimate of the cells' maximum sodium-pumping rate) was sufficient to lower cell sodium by 10 mM/min. This functional estimate of hair-cell (Na+,K+)-ATPase activity was of the same order of magnitude as the biochemical activity of (Na+,K+)-ATPase previously reported for sensory epithelia of other species. The balance between sodium entry and sodium pumping determines hair-cell ionic composition, and thus the resting potential and the driving forces for sodium-coupled transport processes. Imbalance due to excess sodium entry or loss of pump capacity could have significant consequences for hair-cell function and integrity.

Electron-probe analysis of isolated goldfish hair cells: implications for preparing healthy cells.

Electron-probe analysis provides an objective criterion for the physiological status of cells: whether they show the high potassium and low sodium that are expected of healthy animal cells. Preparing isolated goldfish hair cells that were healthy by this criterion required several precautions, including: limited exposure to enzymes and to simple salt solutions, a rest period between enzyme treatment and mechanical disruption of the tissue, and presence of bovine albumin in the medium both during the rest period and during mechanical dispersion and plating. Cells prepared with these precautions from the saccule and lagena and kept in an enriched medium had the following elemental composition (mole percentages with respect to phosphorus): K, 103; Na, 18; Cl, 23; S, 13; Mg, 8; Ca, 1.5. These mole percentages were close to these elements' total millimolar concentrations in the cells. If the precautions were not taken, cells with intact surface membranes (as assessed by exclusion and retention of dyes) could be obtained, but the cells had elevated cell sodium and low cell potassium.

Purification of a low-molecular-weight excitatory substance from the inner ears of goldfish.

The neurotransmitter released by the hair cell has not been identified; little is known about other neuroactive substances that may be important in hair-cell organ function. To identify neuroactive substances in hair cell tissue, we have examined substances in extracts of the inner ears of goldfish that can excite afferent fibers innervating hair cells. The extracts contain an unidentified low-molecular-weight (LMW) excitatory substance that is a candidate to be the hair-cell neurotransmitter. The LMW excitatory substance has been highly purified by a sequential combination of (1) treatment with cation-exchange resin; (2) gel-permeation chromatography; (3) gradient-elution cation-exchange chromatography; (4) isocratic-elution cation-exchange chromatography; and (5) high-performance anion-exchange chromatography. Based upon its separation behavior in these purification steps, the LMW excitatory substance may be a small, zwitterionic compound with titratable anionic and cationic groups.

Pharmacological alterations of the activity of afferent fibers innervating hair cells.

To determine whether some of the substances that may be present in hair-cell sensory organs could affect neural activity in afferent fibers, we examined 56 compounds for the ability to alter the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis, the African clawed frog. These compounds included amino acids, glutamyl dipeptides, standard neurotransmitter candidates, and other constituents of tissues and body fluids. Substances found to be excitatory included some neutral amino acids (alanine, serine, threonine, asparagine, glutamine, and proline), ATP, carnosine, histidine, and barium chloride. Compounds that suppressed discharge included the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), serotonin, and gamma-glutamyl dipeptides. GABA and acidic amino acids (glutamate, aspartate, and cysteine sulfinate) produced a brief excitation followed by a suppression of discharge rate. Several of these substances were active at sufficiently low concentrations that their presence in body fluids may affect afferent fiber discharge rate under normal or pathological conditions.

A possible neurotransmitter role for CGRP in a hair-cell sensory organ.

We report that calcitonin gene-related peptide (CGRP) increases the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis. We have localized CGRP-like immunoreactivity in small, presumably efferent, fibers innervating the lateral line organ. In addition to providing evidence for a neurotransmitter role for CGRP in a sensory system, these results may help explain the non-cholinergic excitatory effect seen with efferent stimulation in this and other hair cell organs such as the inner ear.

Neuroactive substances in inner ear extracts.

To identify the neurotransmitter released by sensory hair cells, as well as to find other substances that might influence neural function of the inner ear, we have prepared extracts from inner ears of fishes (which have large numbers of hair cells), fractionated the extracts, and studied the effects of the fractionated extracts on the discharge rate of afferent fibers innervating hair cells in the lateral line organ of the African clawed frog Xenopus laevis. The extracts contain active substances that do not bind to a cation-exchange resin at neutral pH. Gel-permeation chromatography suggests that at least 2 unidentified excitatory substances are present in the extracts: one of low molecular weight (Mr about 200) and one of high molecular weight (Mr less than or equal to 5000). Some extracts also contain a high-molecular-weight inhibitory substance (Mr greater than 5000). The low-molecular-weight active substance is detected in extracts of inner ear, but not in brain or muscle. The high-molecular-weight excitatory substance is present both in brain and in inner ear.

Isolation and culture of auditory cells from the goldfish (Carassius auratus).

Large numbers of hair cells and VIIIth nerve ganglion cells are obtained from the inner ears of adult goldfish by a combination of enzymatic and mechanical dissociation. Centrifugation of the dissociated tissue through a discontinuous density gradient produces one fraction enriched in hair cells and another enriched in nerve cells. The fraction of cells enriched in neurons can be put into cell culture and kept for a period of weeks. During that period, the neurons send out processes that can extend for millimeters. The morphology of these cultured neurons is similar to that of the goldfish auditory neurons in histological material.

Pancreatic zymogen granules differ markedly in protein composition.

The activities of both chymotrypsin and amylase in individual zymogen granules of rat pancreas were measured by means of micromanipulation and microfluorometric methods. The enzyme content and the ratio of amylase to chymotrypsin varied widely among granules taken from the same animal. These results are compatible with short-term nonparallel bulk secretion of the two enzymes through exocytosis. The distribution of each enzyme activity in a population of granules suggests quantal packaging of amylase and chymotrypsinogen into the granules.

Nucleotides in a single mammalian ovum or preimplantation embryo.

ATP, ADP, and AMP have been measured jointly on a single mouse ovum or preimplantation embryo using an ultramicrofluorescence technique. The method uses the traditional approach of enzymatic analysis based on changes in the concentrations of nucleotide cofactors, but eliminates the need for enzymatic recycling. It permits the measurement of as little as 10 fmol, and may be adapted for numerous metabolites.

An NADH-coupled assay for femtogram or nanogram quantities of chymotrypsin.

Chymotrypsin can be determined with an NADH-coupled assay. Hydrolysis of the substrate benzoyltyrosine ethyl ester is monitored by coupling the liberation of ethanol to the production of NADH and determining the NADH spectrophotometrically or fluorometrically. Nanogram quantities of chymotrypsin can be determined in milliliter volumes. With these microfluorescence methods this assay can be performed in a final volume of less than a nanoliter, allowing determination of femtogram quantities of chymotrypsin, the amount present in an individual zymogen granule.

On the origin of substance P and glutamic acid decarboxylase (GAD) in the substantia nigra.

Knife cuts in the frontal plane separating the anterior part of the caudate-putamen from the globus pallidus resulted in marked decreases in substances P levels in the reticular part of the substantia nigra. More caudal knife cuts were required in order to effect maximal decreases in nigral glutamic acid decarboxylase levels. Thus, there is a clear anatomical dissociation between the striatal neurons which project to the reticular part of the substantia nigra and which contain SP, and the more caudally located GAD-containing striatal and pallidal neurons, all of which travel through the globus pallidus on their way to the substantia nigra.