THE JOURNAL OF EXPERIMENTAL ZOOLOGY 261:355-358 (1992)
Changes in Monoamine Transmitter Concentration in Freshwater Mussel Tissues THOMAS H. DIETZ, JOHN M. WILSON, AND HAROLD SILVERMAN Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803 ABSTRACT Freshwater mussels were analyzed for biogenic amine transmitter substances in gill tissue, suprabranchial nerve and blood. Gill tissue from normal pondwater-acclimated mussels contained significant amounts of monoamine neurotransmitter substances. In comparison with the suprabranchial nerve the gill tissue contained 42% of the dopamine, 7% of the serotonin and 490% of norepinephrine. Exposing the animals to deionized water (salt-depleted) resulted in a loss of transmitter substances from gill tissue, but serotonin reduction was modest. The mussel gill tissue content of serotonin and the precursor tryptophan was regulated a t nearly constant levels. Serotonin is a n important transmitter substance in mussels and the many functions it controls, including sodium transport regulation, would depend on its continued presence.
Freshwater mussels are hyperosmotic regulators and must obtain ions from their environment by transport processes. In vitro studies have demonstrated that the gills are a principal site of ion transport in the freshwater mussels, and sodium is a major cation that must be transported to maintain the electrolyte balance (Dietz and Graves, '81). We have demonstrated that sodium transport in freshwater bivalves is under neural control (Dietz et al., '82). Application of exogenous serotonin (5-HT) to isolated gills stimulates Na influx and this effect can be mimicked by exogenous cyclic AMP or theophylline. Application of other biogenic amines (dihydroxyphenylalanine,DOPA;dopamine, DA; norepinephrine, NE) had no effect on sodium transport (Dietz et al., '82). We have demonstrated previously a serotonin-stimulated adenylate cyclase being present in the gills of mussels (Scheide and Dietz, '83; '84;'86). Salt depletion or selective ion depletion also will cause an increase in ion transport rates when the mussels are returned to freshwater, however the mechanism of action of salt depletion has not been studied (Murphy and Dietz, '76; Scheide and Dietz, '82). The gills of bivalves are innervated with a large number of serotonergic and dopaminergic neurons (Hiripi '68; '72; Dietz et al., '85). Serotonin exerts control over several functions in the gill including stimulation of Na influx, ciliary activity and causes gill musculature to relax (Gardiner et al., '91a). Exogenous DA increases ciliary movement i n isolated mussel gills but does not alter Na influx or muscular activity (Dietz et al., '85; Gardiner et al., '91a). 0 1992 WILEY-LISS, INC.
Here we report the major monoamines detected in freshwater mussels using a high-performance liquid chromatography (HPLC) separation system that provides good quantitation of catecholamines, serotonin, their amino acid precursors, and their associated metabolites. Qrosine is converted into DOPA that in most tissues is not accumulated but converted into dopamine or norepinephrine and stored in synaptic vesicles. These two transmitters are subject to reaccumulation by neurons following their release or are, respectively, converted into dihydroxyphenylacetic acid (DOPAC)or dihydroxyphenylglycol (DOPEG) by monoamine oxidase activity. Tryptophan is converted into 5-hydroxytryptophan (5-HTP) and then into serotonin (5-hydroxytryptamine, 5-HT) and stored in transmitter vesicles. Serotonin released from neurons also is subject to reaccumulation or is metabolized into 5-hydroxyindoleacetic acid (5-HIAA).
MATERIALS AND METHODS Mussels were collected from ponds near Baton Rouge, Louisiana. All animals were maintained at 22-25°C in artificial pondwater (in mM; 0.5 NaC1, 0.4 CaC12,0.2 NaHC03, and 0.05 (KC1) under laboratory photoperiod. Representatives of the family Lampsilinae that we examined were Ligumia subrostrata, and Toxolasma (Carunculina) texasensis.
Received December 17,1990; revision accepted August 22,1991
356
T.H. DIETZ ET AL.
The Anodontidae representative was Anodonta grand is. Gill tissue or suprabranchial nerve was homogenized in 10 vol of cold mobile phase (with an internal standard, 5-hydroxymethyltryptamine, 5-HMT). The homogenate was centrifuged (75,000 g - min) and 500 pl of supernatant filtered using an Amincon micropartition filtration system (MPS-1).Blood samples were prepared using HC104precipitation. Perchlorate precipitated blood was centrifuged 150,000 g * min and the supernatant neutralized with Tris followed by alumina extraction. The alumina was collected by centrifugation and washed with deionized water; the catecholamines were then collected with HC104. The extracted blood sample was neutralized and filtered with the MPS-1 filter. Samples (20-60 pl) were analyzed with a Beckman HPLC model 342 dual pump gradient system equipped with a 250-p1 loop and analytical C-18 reverse-phase ODS column. An electrochemical detector (Bioanalytic Systems, LC-4A) with a carbon paste electrode, and the signal output was recorded with a Hewlett Packard 3390 integrating recorder. The mobile phase was 84:16 viv mixture of 0.1 M NaH2P04(deionized distilled water) and methanol (HPLC grade) with 3.6 mM sodium heptanesulfonate, 0.1 mM ethylenediamine tetraacetic acid, and 0.24 mM triethylamine at a flow rate of 1ml/min adapted from Wagner et al. ('82). The pH was ordinarily 3.00; slight pH changes had minimal effect on most of the biogenic amines or metabolites, but 5-hydroxytryptophan and dihydroxyphenylalanine retention times were inversely related to pH. The mobile phase was filtered (0.22 pm) and degassed under vacuum prior to use. The monoamines from tissue were measured with the detector set at 900 mV potential relative to the reference electrode allowing us to detect tyrosine (precursor of catecholamine synthesis) and the serotonin precursor, tryptophan. We were able to identify the major transmitter substances (norepinephrine, dopamine, and serotonin)and metabolites (3,4-dihydroxyphenylaceticacid, and 5-hydroxyindoleacetic acid) exceeding 5 pg. The detection of monoamines from blood samples were obscured by a substantial amount of unidentified material. Using an electrode potential of 750 mV improved the detection of blood monoamines, but the precursor amino acids could not be measured. All samples were analyzed at two different volumes injected into the HPLC. The unknowns were
identified by retention times and the peak heights were automatically printed out by the integrating recorder. The specific sample peaks were quantitated by comparison with a mixture of standards that were analyzed at about 4 to 5 hr intervals (every 5th sample).The detector sensitivity declined with time, and the quantity of each metabolite was calculated from standard curves for each specific metabolite peak height using the internal standard 5-HMT as the point of reference. The recovery of the sample through the preparation procedure was determined by the quantity of internal standard measured in the sample. The tissue data were converted t o ng (g wet tissue) and blood samples were expressed as ng ml All replicates were averaged for each animal and the data are reported as mean 2 1 SEM, with the number of animals in parentheses. Differences between means were determined by Student's t-test and considered significant i f P
Changes in Monoamine Transmitter Concentration in Freshwater Mussel Tissues THOMAS H. DIETZ, JOHN M. WILSON, AND HAROLD SILVERMAN Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803 ABSTRACT Freshwater mussels were analyzed for biogenic amine transmitter substances in gill tissue, suprabranchial nerve and blood. Gill tissue from normal pondwater-acclimated mussels contained significant amounts of monoamine neurotransmitter substances. In comparison with the suprabranchial nerve the gill tissue contained 42% of the dopamine, 7% of the serotonin and 490% of norepinephrine. Exposing the animals to deionized water (salt-depleted) resulted in a loss of transmitter substances from gill tissue, but serotonin reduction was modest. The mussel gill tissue content of serotonin and the precursor tryptophan was regulated a t nearly constant levels. Serotonin is a n important transmitter substance in mussels and the many functions it controls, including sodium transport regulation, would depend on its continued presence.
Freshwater mussels are hyperosmotic regulators and must obtain ions from their environment by transport processes. In vitro studies have demonstrated that the gills are a principal site of ion transport in the freshwater mussels, and sodium is a major cation that must be transported to maintain the electrolyte balance (Dietz and Graves, '81). We have demonstrated that sodium transport in freshwater bivalves is under neural control (Dietz et al., '82). Application of exogenous serotonin (5-HT) to isolated gills stimulates Na influx and this effect can be mimicked by exogenous cyclic AMP or theophylline. Application of other biogenic amines (dihydroxyphenylalanine,DOPA;dopamine, DA; norepinephrine, NE) had no effect on sodium transport (Dietz et al., '82). We have demonstrated previously a serotonin-stimulated adenylate cyclase being present in the gills of mussels (Scheide and Dietz, '83; '84;'86). Salt depletion or selective ion depletion also will cause an increase in ion transport rates when the mussels are returned to freshwater, however the mechanism of action of salt depletion has not been studied (Murphy and Dietz, '76; Scheide and Dietz, '82). The gills of bivalves are innervated with a large number of serotonergic and dopaminergic neurons (Hiripi '68; '72; Dietz et al., '85). Serotonin exerts control over several functions in the gill including stimulation of Na influx, ciliary activity and causes gill musculature to relax (Gardiner et al., '91a). Exogenous DA increases ciliary movement i n isolated mussel gills but does not alter Na influx or muscular activity (Dietz et al., '85; Gardiner et al., '91a). 0 1992 WILEY-LISS, INC.
Here we report the major monoamines detected in freshwater mussels using a high-performance liquid chromatography (HPLC) separation system that provides good quantitation of catecholamines, serotonin, their amino acid precursors, and their associated metabolites. Qrosine is converted into DOPA that in most tissues is not accumulated but converted into dopamine or norepinephrine and stored in synaptic vesicles. These two transmitters are subject to reaccumulation by neurons following their release or are, respectively, converted into dihydroxyphenylacetic acid (DOPAC)or dihydroxyphenylglycol (DOPEG) by monoamine oxidase activity. Tryptophan is converted into 5-hydroxytryptophan (5-HTP) and then into serotonin (5-hydroxytryptamine, 5-HT) and stored in transmitter vesicles. Serotonin released from neurons also is subject to reaccumulation or is metabolized into 5-hydroxyindoleacetic acid (5-HIAA).
MATERIALS AND METHODS Mussels were collected from ponds near Baton Rouge, Louisiana. All animals were maintained at 22-25°C in artificial pondwater (in mM; 0.5 NaC1, 0.4 CaC12,0.2 NaHC03, and 0.05 (KC1) under laboratory photoperiod. Representatives of the family Lampsilinae that we examined were Ligumia subrostrata, and Toxolasma (Carunculina) texasensis.
Received December 17,1990; revision accepted August 22,1991
356
T.H. DIETZ ET AL.
The Anodontidae representative was Anodonta grand is. Gill tissue or suprabranchial nerve was homogenized in 10 vol of cold mobile phase (with an internal standard, 5-hydroxymethyltryptamine, 5-HMT). The homogenate was centrifuged (75,000 g - min) and 500 pl of supernatant filtered using an Amincon micropartition filtration system (MPS-1).Blood samples were prepared using HC104precipitation. Perchlorate precipitated blood was centrifuged 150,000 g * min and the supernatant neutralized with Tris followed by alumina extraction. The alumina was collected by centrifugation and washed with deionized water; the catecholamines were then collected with HC104. The extracted blood sample was neutralized and filtered with the MPS-1 filter. Samples (20-60 pl) were analyzed with a Beckman HPLC model 342 dual pump gradient system equipped with a 250-p1 loop and analytical C-18 reverse-phase ODS column. An electrochemical detector (Bioanalytic Systems, LC-4A) with a carbon paste electrode, and the signal output was recorded with a Hewlett Packard 3390 integrating recorder. The mobile phase was 84:16 viv mixture of 0.1 M NaH2P04(deionized distilled water) and methanol (HPLC grade) with 3.6 mM sodium heptanesulfonate, 0.1 mM ethylenediamine tetraacetic acid, and 0.24 mM triethylamine at a flow rate of 1ml/min adapted from Wagner et al. ('82). The pH was ordinarily 3.00; slight pH changes had minimal effect on most of the biogenic amines or metabolites, but 5-hydroxytryptophan and dihydroxyphenylalanine retention times were inversely related to pH. The mobile phase was filtered (0.22 pm) and degassed under vacuum prior to use. The monoamines from tissue were measured with the detector set at 900 mV potential relative to the reference electrode allowing us to detect tyrosine (precursor of catecholamine synthesis) and the serotonin precursor, tryptophan. We were able to identify the major transmitter substances (norepinephrine, dopamine, and serotonin)and metabolites (3,4-dihydroxyphenylaceticacid, and 5-hydroxyindoleacetic acid) exceeding 5 pg. The detection of monoamines from blood samples were obscured by a substantial amount of unidentified material. Using an electrode potential of 750 mV improved the detection of blood monoamines, but the precursor amino acids could not be measured. All samples were analyzed at two different volumes injected into the HPLC. The unknowns were
identified by retention times and the peak heights were automatically printed out by the integrating recorder. The specific sample peaks were quantitated by comparison with a mixture of standards that were analyzed at about 4 to 5 hr intervals (every 5th sample).The detector sensitivity declined with time, and the quantity of each metabolite was calculated from standard curves for each specific metabolite peak height using the internal standard 5-HMT as the point of reference. The recovery of the sample through the preparation procedure was determined by the quantity of internal standard measured in the sample. The tissue data were converted t o ng (g wet tissue) and blood samples were expressed as ng ml All replicates were averaged for each animal and the data are reported as mean 2 1 SEM, with the number of animals in parentheses. Differences between means were determined by Student's t-test and considered significant i f P
