Development of a novel probe for measuring drug binding to the F1*S variant of human alpha 1-acid glycoprotein.

A novel probe was developed to measure drug association with the F1*S variant of the human serum protein alpha 1-acid glycoprotein (AGP). The molecule 2-hydroxy-3,5-diiodo-N-[2(diethylamino)ethyl]benzamide (DEDIC) binds to AGP, quenching its native fluorescence. This quenching was fitted to a two-site model giving apparent dissociation constants of 0.049 +/- 0.005 and 12 +/- 2 microM (mean +/- SEM). Quenching of each of the separate variants of AGP by DEDIC was itself described by a two-site model, giving for the F1*S variant K(D)(1)((F1*S)) = 0.041 +/- 0.010 microM and K(D)(2)((F1*S)) = 29 +/- 7 microM; and for the A variant K(D)(1)((A)) = 0.31 +/- 0.18 microM and K(D)(2)((A)) = 8.8 +/- 0.7 microM. The utility of DEDIC in probing drug interactions with isolated variants was demonstrated in competition experiments with the model drugs amitriptyline and bupivacaine. In addition, the selectivity of DEDIC for variant F1*S rendered it capable of probing the binding of drugs (including the variant A-selective drug amitriptyline) to F1*S in a mixture of variants, such as occurs naturally in whole AGP. DEDIC is unique as an F1*S variant-selective probe of drug binding to whole AGP that is also sufficiently soluble to serve as a probe of drug binding to the lower affinity sites on isolated A and F1*S variants.

Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils reveals interstrand contact regions between assembled beta-amyloid peptide subunits.

The assembly of the beta-amyloid peptide (Abeta) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural information about fibrillogenesis has remained elusive due to the highly insoluble, noncrystalline nature of the assembled peptide. X-ray fiber diffraction, infrared spectroscopy, and solid-state NMR studies performed on fibrils composed of Abeta peptides have led to conflicting models of the intermolecular alignment of beta-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Abeta monomers within amyloid fibrils. A photoreactive Abeta(1-40) ligand was synthesized by substituting L-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Abeta(1-40) F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS-PAGE of dissolved fibrils revealed the light-dependent formation of a covalent Abeta dimer. Enzymatic cleavage followed by mass spectrometric analysis demonstrated the presence of a dimer-specific ion at MH(+) = 1825.9, the predicted mass of a fragment composed of the N-terminal Abeta(1-5) F4Bpa tryptic peptide covalently attached to the C-terminal Abeta(29-40) tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked dimer led to the localization of the photo-cross-link between the ketone of the Bpa4 side chain and the delta-methyl group of the Met35 side chain. The Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel alignment of Abeta peptides within amyloid fibrils.

The Alzheimer's peptide a beta adopts a collapsed coil structure in water.

The self-assembly of the soluble peptide Abeta into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Abeta is of significant interest. In contrast to studies in other solvents, in water Abeta is collapsed into a compact series of loops, strands, and turns and has no alpha-helical or beta-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers approximately 25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular beta-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.

Activation barriers to structural transition determine deposition rates of Alzheimer's disease a beta amyloid.

Brain amyloid composed of the approximately 40-amino-acid human beta-amyloid peptide A beta is integral to Alzheimer's disease pathology. To probe the importance of a conformational transition in Abeta during amyloid growth, we synthesized and examined the solution conformation and amyloid deposition activity of A beta congeners designed to have similar solution structures but to vary substantially in their barriers to conformational transition. Although all these peptides adopt similar solution conformations, a covalently restricted Abeta congener designed to have a very high barrier to conformational rearrangement was inactive, while a peptide designed to have a reduced barrier to conformational transition displayed an enhanced deposition rate relative to wild-type A beta. The hyperactive peptide, which is linked to a heritable A beta amyloidosis characterized by massive amyloid deposition at an early age, displayed a reduced activation barrier to deposition consistent with a larger difference in activation entropy than in activation enthalpy relative to wild-type A beta. These results suggest that in Alzheimer's disease, as in the prion diseases, a conformational transition in the depositing peptide is essential for the conversion of soluble monomer to insoluble amyloid, and alterations in the activation barrier to this transition affect amyloidogenicity and directly contribute to human disease.

Alzheimer's disease amyloid propagation by a template-dependent dock-lock mechanism.

Amyloid plaques composed of the peptide Abeta are an integral part of Alzheimer's disease (AD) pathogenesis. We have modeled the process of amyloid plaque growth by monitoring the deposition of soluble Abeta onto amyloid in AD brain tissue or synthetic amyloid fibrils and show that it is mediated by two distinct kinetic processes. In the first phase, "dock", Abeta addition to the amyloid template is fully reversible (dissociation t(1/2) approximately 10 min), while in the second phase, "lock", the deposited peptide becomes irreversibly associated (dissociation t(1/2) >> 1000 min) with the template in a time-dependent manner. The most recently deposited peptide dissociates first while Abeta previously deposited becomes irreversibly "locked" onto the template. Thus, the transition from monomer to neurotoxic amyloid is mediated by interaction with the template, a mechanism that has also been proposed for the prion diseases. Interestingly, two Abeta peptides bearing primary sequence alterations implicated in heritable Abeta amyloidoses displayed faster lock-phase kinetics than wild-type Abeta. Inhibiting the initial weak docking interaction between depositing Abeta and the template is a viable therapeutic target to prevent the critical conformational transition in the conversion of Abeta((solution)) to Abeta((amyloid)) and thus prevent stable amyloid accumulation. While thermodynamics suggest that inhibiting amyloid assembly would be difficult, the present study illustrates that the protein misfolding diseases are kinetically vulnerable to intervention.

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.

Stereochemical specificity of Alzheimer's disease beta-peptide assembly.

The formation and growth of insoluble amyloid deposits composed primarily of the human beta-amyloid peptide (A beta) in brain is an essentially invariant feature of Alzheimer's disease (AD) and is widely believed to contribute to the progressive neurodegeneration of the disorder. To probe the specificity of amyloid formation and growth, we synthesized and examined the self-assembly of D- and L-stereoisomers of A beta in vitro. While both enantiomers formed insoluble aggregates at similar rates with amyloid-like fibrillar morphology, deposition of soluble A beta peptide onto preexisting A beta aggregates was stereospecific. Although the L-peptide deposited readily onto immobilized L-A beta aggregates with first-order kinetic dependence on soluble peptide concentration, essentially no association between the D-peptide and L-template was observed. Similarly, the D-peptide deposited with first-order kinetics onto a D-A beta aggregate template but did not deposit onto a similar template composed of aggregates of the L-enantiomer. Furthermore, although the L-A beta isomer deposited onto authentic AD amyloid in preparations of unfixed AD brain, no focal association between the D-peptide and brain amyloid was detected. These results establish that deposition of soluble A beta onto preexisting amyloid template is stereospecific, likely involving direct docking interactions between peptide backbone and/or side chains rather than simple hydrophobic association.

Delayed behavioral effects following intrahippocampal injection of aggregated A beta (1-42).

Beta amyloid protein (A beta) is the major extracellular component of Alzheimer's disease (AD) plaques. In the current study, A beta (1-42) was aggregated in vitro using a method which produces A beta aggregates similar to those found in the AD brain. Twelve male Sprague-Dawley rats were trained in two-lever operant chambers under an alternating lever cyclic-ratio (ALCR) schedule. When performance was stable on the ALCR schedule, six subjects were injected (bilaterally into the CA3 area of the dorsal hippocampus) with 5.0 microliters aggregated A beta in suspension, and the remaining six subjects were injected with 5.0 microliters sterile water. Behavioral testing resumed 5 days after surgery and continued for 90 days post-injection. Aggregated A beta injection did not affect the number of lever switching errors made in a daily session but did affect the number of incorrect lever response perseverations. After approximately 30 days post-injection, aggregated A beta injection detrimentally affected ability to track the changing parameters of the schedule, and decreased the efficiency by which subjects obtained reinforcers. From approximately day 50 post-injection onward, A beta-injected subjects demonstrated significantly higher numbers of incorrect lever response perseverations than did sterile water-injected subjects. These effects appeared to be central rather than peripheral, as A beta injection did not decrease running response rates under the ALCR schedule. The delayed onset of behavioral effects seen in this and other behavioral studies may be a result of a cascade of potentially harmful responses induced through glial activation following aggregated A beta injection.

Fibrillar beta-amyloid induces microglial phagocytosis, expression of inducible nitric oxide synthase, and loss of a select population of neurons in the rat CNS in vivo.

To determine the stability of beta-amyloid peptide (Abeta) and the glial and neuronal changes induced by Abeta in the CNS in vivo, we made single injections of fibrillar Abeta (fAbeta), soluble Abeta (sAbeta), or vehicle into the rat striatum. Injected fAbeta is stable in vivo for at least 30 d after injection, whereas sAbeta is primarily cleared within 1 d. After injection of fAbeta, microglia phagocytize fAbeta aggregates, whereas nearby astrocytes form a virtual wall between fAbeta-containing microglia and the surrounding neuropil. Similar glial changes are not observed after sAbeta injection. Microglia and astrocytes near the injected fAbeta show a significant increase in inducible nitric oxide synthase (iNOS) expression compared with that seen with sAbeta or vehicle injection. Injection of fAbeta but not sAbeta or vehicle induces a significant loss of parvalbumin- and neuronal nitric oxide synthase-immunoreactive neurons, whereas the number of calbindin-immunoreactive neurons remains unchanged. These data demonstrate that fAbeta is remarkably stable in the CNS in vivo and suggest that fAbeta neurotoxicity is mediated in large part by factors released from activated microglia and astrocytes, as opposed to direct interaction between Abeta fibrils and neurons.

Oligomerization of endogenous and synthetic amyloid beta-protein at nanomolar levels in cell culture and stabilization of monomer by Congo red.

Amyloid beta-proteins (A beta) are proteolytic fragments of the beta-amyloid precursor protein (beta APP) that are secreted by mammalian cells throughout life but also accumulate progressively as insoluble cerebral aggregates in Alzheimer's disease (AD). Because mounting evidence indicates that A beta aggregation and deposition are early, critical features of AD leading to neurotoxicity, many studies of A beta aggregation have been conducted using synthetic peptides under generally nonphysiological conditions and concentrations. We recently described the oligomerization of A beta peptides secreted by beta APP-expressing cells at low nanomolar (20-30 ng/mL) levels into sodium dodecyl sulfate- (SDS-) stable oligomers of 6-16 kDa. Here, we extensively characterize this in vitro system and show that the amyloid binding dye, Congo red, acts to markedly decrease oligomer/monomer ratios by stabilizing the 4 kDa A beta monomers (ID50 approximately equal to 3.4 microM). Addition of radioiodinated synthetic A beta 1-40 to the cultures or to their conditioned media at physiological concentrations (0.25-2.5 nM) reveals that it undergoes progressive aggregation into SDS-stable oligomers of 6-25 kDa during brief (approximately 4 h) incubation at 37 degrees C, and this is inhibitable by Congo red. The level of A beta oligomers can be quantitated in the Chinese hamster ovary (CHO) conditioned medium by size-exclusion chromatography as well as by SDS-polyacrylamide gel electrophoresis (PAGE), and comparison of these two methods suggests that aggregation of A beta into higher molecular weight polymers that are not detectable by SDS-PAGE occurs in the cultures. We conclude that both endogenous and synthetic A beta can assemble into stable oligomers at physiological concentrations in cell culture, providing a manipulable system for studying the mechanism of early A beta aggregation and identifying inhibitors thereof under biologically relevant conditions.

New insights into the neuropathology and cell biology of Alzheimer's disease.

Several lines of evidence, including newly discovered genetic mutations, suggest that beta-amyloid (A beta) is directly involved in the neuropathology observed in familial and sporadic forms of Alzheimer's disease (AD). Rather than exerting its neurotoxicity directly, results from our laboratory suggest that fibrillar A beta (fA beta) activates microglia and astrocytes upon injection into the rat brain. The microglia and astrocytes, in turn, form a functional barrier between A beta and surrounding neurons. An increase in inducible nitric oxide synthase (iNOS) immunoreactivity is observed in activated microglia and astrocytes, and specific subpopulations of neurons are lost in fA beta injection areas versus controls. These data, coupled with recent discoveries of the A beta association with the receptor for advanced glycation end products (RAGE) and the class A scavenger receptors (SR), support the hypothesized role of inflammatory mechanisms in AD neurotoxicity.

Expression of endothelin-B receptors by glia in vivo is increased after CNS injury in rats, rabbits, and humans.

Previous studies have demonstrated that neonatal cultures of astrocytes express functional endothelin (ET) receptors. To determine if similar ET receptors are expressed by adult glia we used 125I-ET-1 to examine the expression of ET receptors both in vivo in the normal and transected optic nerves of the rabbit and rat and in vitro in cultures of astrocytes, microglia, or oligodendrocytes. Additionally, we examined the expression of ET receptors in the human optic nerve. Moderate levels of ET(B) receptors were identified in the rabbit and rat forebrain, whereas in the normal rabbit, rat, and human optic nerves a low density of ET(B) receptors was observed, mainly in association with glial fibrillary acidic protein + (GFAP+) astrocytes. After unilateral optic nerve transection, or damage to the retina, the density of glial ET(B) receptors in the optic nerve is significantly increased in all species examined. Thus, at 7 days posttransection there is a significant increase in ET(B) receptors, and by 90 days posttransection the density of ET(B) receptors in the rabbit or rat optic nerve was among the highest of any area in the central nervous system (CNS). Primary cultures of astrocytes or microglia, but not oligodendrocytes, express 125I-ET-1 binding sites. These data demonstrate that in the normal CNS, astrocytes express low but detectable levels of ET(B) receptors, and, after CNS injury, both astrocytes and microglia express high levels of ET(B) receptors. ET(B) receptors provide a therapeutic target for regulating glial proliferation and the release of neurotrophic factors from glia that occur in response to neuronal injury.

p-(4-Hydroxybenzoyl)phenylalanine: a photoreactive amino acid analog amenable to radioiodination for elucidation of peptide-protein interaction. Application to substance P receptor.

Benzoylphenylalanine, a photoreactive phenylalanine analog that can be incorporated into a peptide during solid-phase synthesis, is a useful probe for investigating the interactions of bioactive peptides with their receptors. This probe, however, lacks versatility because it is not detectable by Edman sequencing and because it cannot be labeled with radioiodine, requiring radiolabeling of the peptide ligand at a site distal to the photoreactive amino acid. The separation of the radioisotope and photoaffinity labels along the primary sequence limits identification of the photoinsertion site to a peptide fragment rather than a specific amino acid of the receptor protein. We have now synthesized p-(4-hydroxybenzoyl)phenylalanine by a synthetic route involving reaction of 4-(chloromethyl)benzoic anhydride with phenol in polyphosphoric acid to give the 4-(chloromethyl)benzoyl ester of 4-(chloromethyl)-4'-hydroxybenzophenone followed by reaction of the benzophenone derivative with ethyl acetamidocyanoacetate and subsequent hydrolysis of the product to give p-(4-hydroxybenzoyl)phenylalanine. The novel photolabile amino acid was incorporated into substance P (replacing Phe8 or Lys3) to give 11-mer peptides that bind with high (nM) affinity and specificity to the substance P receptor. Radioiodination of the substance P analogs resulted in the incorporation of 125I at the photoreactive amino acid residue, yielding probes of high (approximately 2000 Ci/mmol) specific activity. Subsequent photolysis of the radiolabeled peptides in the presence of substance P receptor caused covalent attachment of the peptide to the receptor with high photoinsertion yield (approximately 30%); photolabeling was abolished in the presence of excess unlabeled SP. p-(4-Hydroxybenzoyl)phenylalanine retains p-benzoylphenylalanine's high insertion yield and low reactivity with water, but in contrast allows placement of radioiodine and the photoactive moieties within the same residue, providing the ability to identify the specific site(s) of interaction, and identification of the residue by Edman sequencing. This novel amino acid may be useful in the elucidation of the interaction of a variety of peptides with their receptors.

A beta deposition inhibitor screen using synthetic amyloid.

The formation, growth, and maturation of brain amyloid "senile" plaques are essential pathological processes in Alzheimer's disease (AD) and key targets for therapeutic intervention. The process of in vitro deposition of A beta at physiological concentrations onto plaques in AD brain preparations has been well characterized, but is cumbersome for drug discovery. We describe here a high-through put screen for inhibitors of A beta deposition onto a synthetic template (synthaloid) of fibrillar A beta immobilized in a polymer matrix. Synthaloid is indistinguishable from plaques in AD brain (the natural template) in deposition kinetics, pH profile, and structure-activity relationships for both A beta analogs and inhibitors. Synthaloid, in contrast to current A beta aggregation screens, accurately predicted inhibitor potency for A beta deposition onto AD cortex preparations, validating its use in searching for agents that can slow the progression of AD and exposing a previously inaccessible target for drug discovery.

Absorption and excretion of undegradable peptides: role of lipid solubility and net charge.

Absorption and excretion of undegradable peptides were investigated with use of octapeptides synthesized from D-amino acids. D-Tyrosine was included in each peptide to permit labeling with 125I, D-glutamic acid or D-lysine were included to vary net electric charge and D-serine or D-leucine were included to vary lipid solubility. Peptides were administered parenterally or orally to normal rats drinking 5% glucose or maltose. Forty-five percent of a lipid-insoluble, negatively charged octapeptide added to the drinking fluid in milligram quantities was absorbed from the intestine and excreted intact in urine; 90% of this peptide was recovered in urine after parenteral injection. In contrast, lipophilic D-octapeptides were largely excreted in feces, even after subcutaneous injection; the amounts excreted in feces were correlated with oil/aqueous partition coefficients. Evidence is presented that lipophilic peptides entering liver cells combine with bile salts to form hydrophilic complexes that are secreted rapidly at high concentration in bile. At physiological concentrations of bile salts (5-40 mM) and nanomolar concentrations of peptide the binding is so complete that these undegradable peptides are rapidly cleared from liver to duodenal fluid in association with the bile salts. After reaching the ileum the bile salts are reabsorbed to blood, leaving the original lipophilic peptides to be excreted in the feces from which they can be extracted, purified and identified by high-pressure liquid chromatography. These mechanisms are discussed in relation to a) the paracellular absorption of peptides and other solutes by solvent drag and b) the delivery and fate of biologically active peptides.

Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma.

The amyloid beta-peptide (Abeta) is the major constituent of neuritic plaques in Alzheimer's disease and occurs as a soluble 40-42-residue peptide in cerebrospinal fluid and blood of both normal and AD subjects. It is unclear whether Abeta, once it is secreted by cells, remains free in biological fluids or is associated with other proteins and thus transported and metabolized with them. Such knowledge of the normal fate of Abeta is a prerequisite for understanding the changes that may lead to the pathological aggregation of soluble Abeta in vivo, the possible influence of certain extracellular proteins, particularly apolipoprotein E, on plaque formation, and the pharmacology of putative Abeta-lowering drugs. To address the question of Abeta distribution in human biological fluids, we incubated fresh human plasma from 38 subjects with physiological concentrations (0.5-0.7 nM) of radioiodinated Abeta1-40 and seven plasma samples with Abeta1-42. Lipoproteins and lipid-free proteins were separated and analyzed for bound iodinated Abeta1-40. We found that up to 5% of Abeta added to plasma is bound to selected lipoproteins: very low density, low density, and high density, but not lipoprotein(a). The large majority ( approximately 89%), however, is bound to albumin, and very little Abeta is free. Abeta distribution in plasma was not significantly influenced by apolipoprotein E genotype. We conclude that Abeta is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions.

Intra-arterial infusion of [125I]A beta 1-40 labels amyloid deposits in the aged primate brain in vivo.

Alzheimer's disease is characterized by extracellular amyloid deposits in the brain at both vascular sites (cerebrovascular amyloid, CVA) and within the neuropil (plaques). In the present study we demonstrated that brain amyloid of aged non-human primates is efficiently detected by [125I]A beta in vitro, and assessed the detection of that amyloid in vivo by intravascular infusion of [125I]A beta. Aged squirrel monkeys (Saimiri sciureus) were anesthetized and infused intra-arterially with [125I]A beta, and sacrificed 2 h later. Analysis of the anterior frontal and temporal cortices by autoradiography demonstrated that [125I]A beta was deposited on CVA and that essentially every amyloid deposit which could be detected with thioflavin S or anti-A beta antibodies was also labeled by [125I]A beta. These experiments suggest that intravascular infusion of radiolabeled A beta can be used to detect and image amyloid deposits in the human AD brain.

Point substitution in the central hydrophobic cluster of a human beta-amyloid congener disrupts peptide folding and abolishes plaque competence.

Alzheimer's disease (AD) is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain composed primarily of a 40-43 amino acid peptide, the human beta-amyloid peptide (A beta). The process of A beta deposition can be modeled in vitro by deposition of physiological concentrations of radiolabeled A beta onto preexisting amyloid in preparations of unfixed AD cerebral cortex. Using this model system, it has been shown that A beta deposition is biochemically distinct from A beta aggregation and occurs readily at physiological A beta concentrations, but which regions and conformations of A beta are essential to A beta deposition is poorly understood. We report here that an active congener, A beta (10-35)-NH2, displays time dependence, pH-activity profile, and kinetic order of deposition similar to A beta (1-40), and is sufficiently soluble for NMR spectroscopy in water under conditions where it actively deposits. To examine the importance of the central hydrophobic cluster of A beta (LVFFA, residues 17-21) for in vitro A beta deposition, an A beta (10-35)-NH2 analog with a single point substitution (F19T) in this region was synthesized and examined. Unlike A beta (10-35)-NH2, the F19T analog was plaque growth incompetent, and NMR analysis indicated that the mutant peptide was significantly less folded than wild-type A beta. These results support previous studies suggesting that the plaque competence of A beta correlates with peptide folding. Since compounds that alter A beta folding may reduce amyloid deposition, the central hydrophobic cluster of A beta will be a tempting target for structure-based drug design when high-resolution structural information becomes available.

Differential expression of two isoforms of the neurokinin-1 (substance P) receptor in vivo.

Recent pharmacological and biochemical studies have suggested that there may be more than one molecular form of the neurokinin-1 receptor (NK-1), a long and short isoform differing in the length of their cytoplasmic carboxyl-terminal tails, but no definitive evidence of the existence of such NK-1 receptor isoforms in tissue has been presented. To examine whether these different isoforms are expressed in vivo we have compared the distribution of high affinity substance P (SP) binding sites (visualized by autoradiography with [125I]SP), with the distribution of the C-terminal epitope of the full length receptor (visualized with a specific antibody against the extreme C-terminal sequence). The former method labels both long and short forms of the NK-1 receptor, while the latter labels only the long form of the protein. In the rat there is a close correspondence of [125I]SP binding and NK-1 immunoreactivity in the striatum, suggesting that the long isoform predominates in this tissue. In the parotid and submaxillary gland, there are very high levels of [125I]SP binding but only low levels of NK-1 immunoreactivity, suggesting that expression of the short form predominates in these tissues. These results imply that different tissues express different ratios of the two isoforms of the NK-1 receptor. This differential expression provides the theoretical basis for tissue specific pharmacological targeting of NK-1 receptors.