Direct evidence for the interaction of neurokinin A with the tachykinin NK(1) receptor in tissue.

Neurokinin A (NKA) is a tachykinin peptide that binds with high affinity to the tachykinin NK(2) receptor. Recent homologous binding studies, however, have shown that neurokinin A is also a high-affinity ligand for the tachykinin NK(1) receptor. In this report, we demonstrate that a photoreactive neurokinin A analogue specifically labels the NK(1) receptor in rat submandibular gland membranes and show via bioassay that neurokinin A is a potent stimulator of salivary secretion. Through the use of specific non-peptide antagonists in both photolabeling and functional assays, we unequivocally demonstrate that neurokinin A can specifically interact with the NK(1) receptor in vivo and elicit NK(1) receptor-mediated physiological responses.

Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex.

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.

Evidence for spatial proximity of two distinct receptor regions in the substance P (SP)*neurokinin-1 receptor (NK-1R) complex obtained by photolabeling the NK-1R with p-benzoylphenylalanine3-SP.

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide.NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.

Molecular characterization of the substance P*neurokinin-1 receptor complex: development of an experimentally based model.

Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed. The ligand.receptor complex is based on experimental data from a series of photoaffinity labeling experiments and spectroscopic structural studies of extracellular domains of the NK-1R. Using the ligand/receptor contact points derived from incorporation of photolabile probes (p-benzoylphenylalanine (Bpa)) into SP at positions 3, 4, and 8 and molecular dynamics simulations, the topological arrangement of SP within the NK-1R is explored. The model incorporates the structural features, determined by high resolution NMR studies, of the second extracellular loop (EC2), containing contact points Met(174) and Met(181), providing important experimentally based conformational preferences for the simulations. Extensive molecular dynamics simulations were carried out to probe the nature of the two contact points identified for the Bpa(3)SP analogue (Bremer, A. A., Leeman, S. E., and Boyd, N. D. (2001) J. Biol. Chem. 276, 22857-22861), examining modes of ligand binding in which the contact points are fulfilled sequentially or simultaneously. The resulting ligand.receptor complex has the N terminus of SP projecting toward transmembrane helix (TM) 1 and TM2, exposed to the solvent. The C terminus of SP is located in proximity to TM5 and TM6, deeper into the central core of the receptor. The central portion of the ligand, adopting a helical loop conformation, is found to align with the helices of the central regions EC2 and EC3, forming important interactions with both of these extracellular domains. The model developed here allows for atomic insight into the biochemical data currently available and guides targeting of future experiments to probe specific ligand/receptor interactions and thereby furthers our understanding of the functioning of this important neuropeptide system.

Further definition of the substance P (SP)/neurokinin-1 receptor complex. MET-174 is the site of photoinsertion p-benzoylphenylalanine4 SP.

The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in position 8 of the C-terminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor. In this study, a second photoreactive SP analogue, Bpa(4)-SP, in which the Bpa residue is located in the N-terminal portion of the peptide, was used to define further the peptide-receptor interface. The NK-1 receptor expressed in Chinese hamster ovary cells was specifically and efficiently photolabeled with a radioiodinated derivative of Bpa(4)-SP. Fragmentation analysis of the photolabeled receptor restricted the site of photoincorporation of Bpa(4)-SP to an amino acid within the sequence Thr-173 to Arg-177 located on the N-terminal side of the E2 loop. To identify the specific amino acid in this sequence that serves as the covalent attachment site for Bpa(4)-SP, a small photolabeled receptor fragment was generated by chemical cleavage with cyanogen bromide. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the purified fragment identified a single protonated molecular ion with a molecular mass of 1801.3 +/- 1.8, indicating that upon irradiation, the bound photoligand covalently attaches to the terminal methyl group of a methionine residue. This result, taken together with the results of the peptide mapping studies, establishes that the site of Bpa(4)-SP covalent attachment to the NK-1 receptor is Met-174.

The common C-terminal sequences of substance P and neurokinin A contact the same region of the NK-1 receptor.

Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK-1 receptor (NK-1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high-affinity interaction with this receptor. To document the site of interaction between NKA and the NK-1R, we have used a photoreactive analogue of NKA containing p-benzoyl-L-phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by (125)I-iodohistidyl(1)-Bpa(7)NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that (125)I-BH-Bpa(8)SP, a photoreactive analogue of SP, covalently attaches to M(181) within this same receptor sequence. Importantly, both of these peptides ((125)I-iodohistidyl(1)-Bpa(7)NKA and (125)I-BH-Bpa(8)SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl-terminal tail. In this report, we also show that site-directed mutagenesis of M(181) to A(181) in the NK-1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK-1R, contact the same residue.

Conformationally constrained substance P analogues: the total synthesis of a constrained peptidomimetic for the Phe7-Phe8 region.

A lactam-based peptidomimetic for the Phe7-Phe8 region of substance P has been synthesized. The synthesis used an anodic amide oxidation to selectively functionalize the C5-position of a 3-phenylproline derivative. The resulting proline derivative was coupled to a Cbz-protected phenylalanine, and an intramolecular reductive amination strategy used to convert the coupled material into a bicyclic piperazinone ring skeleton. The net result was a dipeptide building block that imbedded one of two proposed receptor bound conformations for the Phe7-Phe8 region of substance P into a bicyclic ring skeleton. The building block was then converted into a constrained substance P analogue with the use of solid-phase peptide synthesis. A similar intramolecular reductive amination strategy was used to synthesize a substance P analogue having only Phe7 constrained, and the original 3-phenylproline was converted into a substance P analogue having only Phe8 constrained. All of the analogues were examined for their ability to displace substance P from its NK-1 receptor.

Conformational probes for elucidating the nature of substance P binding to the NK1 receptor: initial efforts to map the Phe7-Phe8 region.

Three substance P analogs with conformation constraints in the Phe7-Phe8 region have been prepared in connection with an effort to differentiate two families of potential conformations for the binding of substance P to its NK1 receptor. While the analogs did not bind the NK1 receptor with high affinity, the synthesis of the analogs demonstrated the utility of a general method for constructing piperazinone based peptidomimetics.

A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization.

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by approximately 8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar Kd ( approximately 0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 x 10(5) and 0.3 x 10(5) for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca2+]i), which consisted of the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC50 values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.

Identification of methionine as the site of covalent attachment of a p-benzoyl-phenylalanine-containing analogue of substance P on the substance P (NK-1) receptor.

Previously we have been able to restrict the site of covalent attachment of a photolabile and radiolabeled derivative of substance P (SP), p-benzoylphenylalanine8-SP (Bpa8-SP), to residues 178-183 located on the second extracellular loop (E2) of the SP (NK-1) receptor (Boyd, N. D., Kage, R., Dumas, J. J., Krause, J. E., and Leeman, S. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 433-437). To ascertain the specific amino acid in this sequence that serves as the site of covalent attachment for 125I-Bolton-Hunter reagent (BH)-Bpa8-SP, we have employed here a novel solid-phase approach to cyanogen bromide cleavage of the photolabeled receptor followed by mass spectrometric analysis of a purified labeled fragment. SP receptors on transfected Chinese hamster ovary cells were photolabeled with isotopically diluted 125I-BH-Bpa8-SP. A membrane preparation of the photolabeled receptors was adsorbed onto C-18-derivatized silica gel and cleaved with cyanogen bromide. A single radiolabeled fragment containing 63% of the photoincorporated radioactivity was generated and purified by high performance liquid chromatography. Mass spectrometric analysis identified a single molecular ion with a molecular mass of 1751.4 +/- 2, establishing that upon irradiation the bound photoligand forms a covalent link with the methyl group of a methionine residue at the peptide binding site. In view of our previous findings, this methionine is Met-181 on the primary sequence of the SP receptor.

Photoaffinity labeling of the human substance P (neurokinin-1) receptor with [3H2]azido-CP-96,345, a photoreactive derivative of a nonpeptide antagonist.

An azido derivative of [3H2](2S, 3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl) methyl)-1-azabicyclo[2.2.2]octon-3-amine (CP-96,345), a potent nonpeptide antagonist of the substance P (SP) (neurokinin-1) receptor, was synthesized and shown to have an affinity for the human SP receptor similar to that of the parent compound, CP-96,345. When Chinese hamster ovary cells expressing the human SP receptor were photolabeled with this compound and analyzed with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, several radioactive bands were observed, including a major band centered at molecular mass 80 kDa, the expected value for the SP receptor expressed in Chinese hamster ovary cells. Only the labeling of the 80-kDa protein was specific: nonradiolabeled CP-96,345 but not its optical enantiomer, CP-96,344 was a potent inhibitor of photoincorporation. SP prevented photolabeling only at concentrations higher than expected from its binding affinity but similar to those shown in a competition binding assay to displace radioiodinated analogue of CP-96,345. Antiserum generated against a synthetic peptide corresponding to the carboxyl terminus of the human SP receptor immunoprecipitated only the 80-kDa photoaffinity labeled protein, confirming that it is the human SP receptor. Interestingly, a second antiserum that was generated against the third extracellular loop of the G protein-coupled receptor no longer immunoprecipitated the receptor when covalently labeled with [3H2]azido-CP-96,345. This result indicates either that attachment of the antagonist modified the antigenic region directly, suggesting involvement of this domain in the binding of CP-96,345, or that the loss of recognition by the antiserum is secondary to a change in conformation induced by the covalent attachment of the antagonist at a different site.

Chemical cross-linking of the substance P (NK-1) receptor to the alpha subunits of the G proteins Gq and G11.

We have previously shown that the high-affinity binding of substance P (SP) to its receptor is dependent on an interaction with a PTX-insensitive G protein. This G protein couples SP receptor activation to stimulation of its effector, phospholipase C. In this study, we combined photoaffinity labeling, chemical cross-linking techniques, and immunological characterization using sequence-specific antibody probes to identify G proteins that couple to the SP receptor. First we covalently labeled the SP receptor present on rat submaxillary gland membranes with a radioiodinated photoreactive derivative of SP, p-benzoyl-L-phenylalanine(8)-substance P (125I-[Bpa8]SP). Photoincorporation of this SP derivative was susceptible to guanine nucleotide inhibition, indicating that the receptor was coupled to its G protein during labeling. We then used a chemical cross-linking agent to covalently link the photoaffinity labeled SP receptor and its associated G protein. Cross-linking generated a 96 kDa product, formation of which was prevented by the addition of a guanine nucleotide, but not an adenine nucleotide, following photolabeling, but prior to cross-linking. Furthermore, the 96 kDa cross-linked complex was absent in membranes which had been depleted of G proteins by treatment with alkaline buffer prior to addition of the cross-linking agent. Reductive cleavage of the cross-link in the isolated 96 kDa complex yields two products: the 53 kDa SP receptor and a 42 kDa protein identified by immunoblot analysis as either G alpha q or G alpha 11. Antisera against a common sequence within G alpha s, G alpha i, and G alpha o showed no immunoreactivity to the complex or its cleavage products. These results provide the first direct evidence of specific interaction between photoaffinity labeled SP receptor and the alpha subunits of Gq and G11, members of a family of G proteins known to be associated with pertussis toxin-insensitive phospholipase C activation.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Identification of the site in the substance P (NK-1) receptor for modulation of peptide binding by sulfhydryl reagents.

Substance P (SP) is a peptide neurotransmitter that is involved in multiple responses in both the central and the peripheral nervous systems through a G-protein-coupled contains a number of conserved cysteine residues. To localize and identify the cysteine residues that participate in receptor binding, intact Chinese hamster ovary cells expressing the SP receptor were treated with various sulfhydryl reagents and the effect of these reagents on radioiodinated SP binding affinity and dissociation rate was determined. We used a series of amphiphilic maleimide derivatives in which the reactive maleimide group penetrates to different depths within the plane of membrane. Only the maleimide derivatives with intermediate chain lengths modified receptor binding properties, indicating that the reactive sulfhydryl group is located within a transmembrane domain of the receptor close (within 1.7 nm) to the extracellular border. Since peptide binding to a mutant receptor C199S, in which Cys-199 was replaced by a serine, was found to be insensitive to modulation by sulfhydryl reagents, this reactive sulfhydryl group is on Cys-199 of the receptor. Receptor occupancy by SP protects Cys-199 from modification and thus this residue is either located at or conformationally linked to the SP binding site.

Characterization of the substance P (NK-1) receptor in tunicamycin-treated transfected cells using a photoaffinity analogue of substance P.

Chinese hamster ovary cells expressing the N-glycosylated substance P (NK-1) receptor were treated with the glycosylation inhibitor tunicamycin and photolabeled with 125I-Bolton-Hunter-p-benzoyl-L-phenylalanine8-substance P. Two radioactive proteins of M(r) 80,000 and 46,000, representing the glycosylated and nonglycosylated substance P (NK-1) receptor, respectively, were observed. The IC50 for the inhibition of photolabeling of both receptor forms was 0.3 +/- 0.1 nM for substance P and 30 +/- 5 nM for neurokinin A (substance K). Thus, glycosylation of the substance P (NK-1) receptor has no detectable effect on the affinity of the substance P (NK-1) receptor for substance P or neurokinin A (substance K).

Measurement of total mercury in biological specimens by cold vapor atomic fluorescence spectrometry.

An ultrasensitive method for determining total mercury concentrations in biological specimens is a prerequisite for monitoring exposure to chronic low-dose levels of Hg vapor such as those from dental silver amalgam fillings. The clinical consequences of such doses are currently in question. We describe an adaptation of a two-stage gold amalgamation preconcentration step combined with cold vapor atomic fluorescence spectrometric detection for Hg. At Hg concentrations of 40 and 350 nmol/L, the within-day assay CVs were 5% and 3%, respectively; between-day assay CVs were 8% and 5%, respectively. Accuracy, as demonstrated by analytical recovery, ranged from 98% to 105%. The detection limit for the assay is 50 pmol/L, which is suitable for measuring total Hg concentrations in specimens of human urine, blood, and breast milk, and in monkey kidney cortex and feces, obtained from subjects with and without amalgam fillings.

A porcine model of Staphylococcus epidermidis catheter-associated infection.

The suitability of using catheterized, partially nephrectomized (uremic) pigs to study catheter-associated infection in peritoneal dialysis was investigated. In some pigs, an inoculum of 10(9) cfu of Staphylococcus epidermidis ATCC 35984 was deposited around the catheter exit site and the organism was allowed to colonize over 21 days. The strain was recoverable from tissues and catheter samples at various locations along the catheter tract from inoculated pigs at postmortem examination. Uninoculated control catheters were colonized to a significantly lesser degree and by various other staphylococcal species. Immune response by inoculated pigs toward catheter-associated bacteria was indicated by a significant increase in serum antistaphylococcal IgG concentration and an increased percentage of peripheral polymorphonuclear leukocytes. Uremia had no significant effect on serologic response. Immunoblotting against S. epidermidis lysostaphin-extracted proteins showed that although incubation with inoculated group antisera produced more intense banding and reacted to a wider range of protein than did antisera from uninoculated controls, common antigenic proteins among the groups were found.

Biochemical characterization of two different forms of the substance P receptor in rat submaxillary gland.

Studies were designed to examine the basis for the difference in molecular weights of the two proteins detected in membrane preparations of rat submaxillary glands after photolabeling with a radioactive analogue of substance P, 125I-p-benzoyl-L-phenylalanine8-substance P. When the two proteins were separated and individually digested with endoglycosidase F, the relative molecular weight of each protein was reduced by approximately 10,000, indicating that the extent of glycosylation of both proteins is the same. To test whether the difference in their molecular weights can be attributed to a difference in the lengths of the two proteins, photolabeled membranes were treated with carboxypeptidase Y before solubilization to remove from each photolabeled protein the carboxy-terminal portion that extends beyond the membrane. Only one, albeit diffuse, band was now observed that on subsequent deglycosylation with endoglycosidase F was more clearly seen to be a single band, indicating that differing lengths of peptide chains were cleaved from the two proteins. These results permit the interpretation that the difference in the two forms of the substance P receptor present in rat submaxillary glands is due to differences in the length of their carboxy termini.

Mercury from dental "silver" tooth fillings impairs sheep kidney function.

In humans Hg vapor is released from "silver" amalgam fillings that contain 50% Hg by weight. Previous studies show that when 12 such fillings are placed in sheep teeth, the kidneys will concentrate amalgam Hg at levels ranging from 5 to 10 micrograms Hg/g renal tissue 4-20 wk after placement. In the present study 12 occlusal fillings were placed in each of six adult female sheep under general anesthesia, using standard dental procedures. Glass ionomer occlusal fillings (12) were inserted in two control sheep. At several days before dental surgery, and at 30 and 60 days after placement of fillings, renal function was evaluated by plasma clearance of inulin and by plasma and urine electrolytes, urea, and proteins. An average plasma inulin clearance rate of 69.5 +/- 7.2 ml/min before amalgam placement was reduced to 32.3 +/- 8.1 ml/min by 30 days and remained low at 27.9 +/- 8.7 ml/min after 60 days. Inulin clearance did not change in controls. After amalgam placement urine concentration of albumin decreased from 93.0 +/- 20.5 to 30.1 +/- 15.3 mg/l and urine Na+ concentration increased steadily from 24.8 +/- 7.7 to 82.2 +/- 20.3 mmol/l at 60 days. Concentrations of K+, urea, gamma-glutamyl transpeptidase, alkaline phosphatase, and total protein did not change significantly from 0 to 60 days in urine. Plasma levels of Na+, K+, urea, and albumin remained unchanged from 0 to 60 days after amalgam. Renal histology remained normal in amalgam-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)