Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes

A procedure is described for the synthetic incorporation into membrane proteins of the non-natural amino acid TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid), which is coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by electron paramagnetic resonance (EPR). Also included is a protocol for the functional reconstitution of the spin-labeled protein in lipid vesicles. This protocol can be completed in 17 d.     

TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better ¹³C and ¹⁵N signal NMR enhancements and ¹H spin-lattice T₁ relaxation than T-MacW.     

Characterization of a monoclonal antibody against the lymphoblast substance P receptor

A murine monoclonal antibody to the IM-9 lymphoblast substance P (SP) receptor has been produced which recognizes the membrane-associated proteins of the SP receptor as demonstrated by immunoprecipitation of [125I]SP affinity-labeled and [35S]methionine biosynthetically labeled IM-9 soluble membranes. SP and anti-SP receptor binding to [35S]methionine-labeled IM-9 cell proteins were directly compared by attachment of each to affinity supports. Eluants from these affinity columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed an equivalent 33-kDa protein in both cases. This protein corresponds to one of the previously described [125I]SP specifically affinity-labeled membrane-associated proteins. In addition, two-color fluorescence-activated cell sorter analysis with human peripheral blood T lymphocytes with fluorescein-SP and rhodamine-labeled antireceptor antibody revealed a distinct population of cells (20 to 30%) that were equally labeled by both the fluorescent peptide and antibody. This result indicates that the anti-SP receptor antibody recognizes an epitope of the receptor that is common to both human peripheral blood T lymphocytes and IM-9 lymphoblast cells.     

Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist

To probe the molecular nature of the binding pocket of a G protein-coupled receptor and the events immediately following the binding and activation, we have modified the substance P peptide, a potent agonist for the neurokinin-1 receptor, with a nitroxide spin probe specifically attached at Lys-3. The agonist properties and binding affinity of the spin-labeled substance P are similar to the native peptide. Using electron paramagnetic resonance (EPR) spectroscopy, the substance P analogue is capable of reporting the microenvironment found in the binding pocket of the receptor. The EPR spectrum of bound peptide indicates that the Lys-3 portion of the agonist is highly flexible. In addition, we detect a slight increase in the mobility of the bound peptide in the presence of a non-hydrolyzable analogue of GTP, indicative of the alternate conformational states described for this class of receptor. The down-regulation of neurokinin-tachykinin receptors is accomplished by a rapid internalization of the activated protein. Thus, it was also of interest to establish whether spin-labeled substance P could serve as a real time reporter for endocytosis. Our findings show the receptor agonist is efficiently endocytosed and the loss of EPR signal upon internalization provides a real time monitor of endocytosis. The rapid loss of signal suggests that endosomal trafficking vesicles maintain a reductive environment. Whereas the reductive capacity of the lysosome has been established, our findings indicate this capacity in early endosomes as well.     

Characterization of octapeptin-membrane interactions using spin-labeled octapeptin

Octapeptin is a membrane-active peptide antibiotic that contains a C10 fatty acid covalently attached to the peptide through an amide bond. Interactions of octapeptin with bacterial membranes and phospholipids were characterized by using spin-labeling techniques and octapeptin derivatives containing fatty acids of varying chain length. Acyl modification of octapeptin demonstrated that the fatty acid of the antibiotic contributed to the antimicrobial activity of octapeptin and its affinity for membranes. The influence of octapeptin and C2 acyloctapeptin on the rates of ascorbate reduction of several membrane-bound doxyl stearates was also examined. These studies demonstrated that octapeptin increaed the rate of diffusion of ascorbate into the lipid bilayer and suggested that the acyl chain contributed to this activity. In addition, an acyl spin-labeled analogue of octapeptin was prepared and shown to retain biological activity. Spectral analysis showed that octapeptin does not aggregate in solution over a wide concentration range. However, the isotropic splitting constant indicated that the acyl chain of octapeptin is not completely exposed to water. It is proposed that the acyl chain of octapeptin in solution interacts with hydrophobic amino acids in the peptide, which partially shields the acyl chain from water. Spectral features of the spin-labeled antibiotic bound to phospholipid dispersions were consistent with directional binding of octapeptin to lipid bilayers with insertion of the fatty acid into the hydrocarbon domain.     

Solid-phase synthesis of peptides containing the spin-labeled 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC).

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino4-carboxylic acid (TOAC) is a nitroxide spin-labeled, achiral Calpha-tetrasubstituted amino acid recently shown to be not only an effective beta-turn and 3(10)/alpha-helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid-phase synthesis in an automated apparatus.     

Characterization of a conformationally sensitive murine monoclonal antibody directed to the metal ion-dependent adhesion site face of integrin CD11b.

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the alpha subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.     

Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle.

The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic localization of substance P receptors using I substance P

This paper describes a method for localization of substance P receptors in the rat central nervous system using ¹²⁵I labeled substance P in an autoradiographic procedure. Particularly high densities of substance P receptors were observed in the olfactory bulb, dentate gyrus, amygdala, superior colliculus, and locus coeruleus. Surprisingly low densities of substance P receptors were found in the substantia nigra pars reticulata, a region which contains high concentrations of substance P.     

A topographically and conformationally constrained, spin-labeled, alpha-amino acid: crystallographic characterization in peptides.

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) is a topographically and conformationally restricted, nitroxide containing, C(alpha)-tetrasubstituted alpha-amino acid. Here, we describe the molecular and crystal structures, as determined by X-ray diffraction analyses, of a TOAC terminally protected derivative, the cyclic dipeptide c(TOAC)(2).1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solvate, and five TOAC-containing, terminally protected, linear peptides ranging in length from tetra- to hepta-peptides. Incipient and fully developed, regular or distorted 3(10)-helical structures are formed by the linear peptides. A detailed discussion on the average geometry and preferred conformation for the TOAC piperidine ring is also reported. The X-ray diffraction structure of an intramolecularly cyclized side product resulting from a C-activated TOAC residue has also been determined.     

The dog common carotid artery: a sensitive bioassay for studying vasodilator effects of substance P and of kinins

In order to develop a sensitive pharmacological preparation which would allow the measurement of the inhibitory effects of kinins and substance P (SP) in vascular smooth muscles, several large arteries of the dog were studied in vitro. The common carotid artery was found to be one of the most sensitive preparations to SP and kinins. When contracted with low concentrations of noradrenaline (between 3.0 x 10(-8) and 3.0 x 10(-7) M), this artery responds to SP (6.5 x 10(-11)-6.5 x 10(-9) M) and bradykinin (BK) (8.1 x 10(-11)-9.1 x 10(-8) M) with relaxations that are proportional to the concentrations of the two peptides. SP and BK appear to exert their relaxant effects through the activation of specific receptors as the exposure of the common carotid artery to concentrations of [Leu8]-angiotensin II, propranolol, methysergide, cimetidine, or atropine sufficient to inhibit the effects of the corresponding agonists do not affect the relaxing effect of SP and BK. [Leu8]-des-Arg9-BK (1.0 x 10(-6) M), indomethacin (2.8 x 10(-5) M), and lioresal (4.7 x 10(-5) M) are also inactive. When the dog common carotid artery is desensitized with high concentrations of SP, BK, eledoisin, and physalaemin a cross-desensitization is observed only between SP and physalaemin. These results support the conclusion that SP and kinins act on different receptors. The order of potency of kinins is the following: BK = [Tyr(Me)8]-BK greater than des-Arg9-BK, suggesting that the receptor for kinins is of the B2 type. The order of potency of peptides related to SP is SP greater than C-terminal 4-11 greater than C-terminal hexapeptide 6-11, similar to that observed in other vascular preparations. The results summarized in this paper indicate that the dog common carotid artery is a preparation sensitive to SP and BK and useful for studying the relaxant effect of these two peptides on vascular smooth muscles.     

Characterization of a metal-chelating substance in coffee

A metal-chelating substance in brewed coffee was separated and characterized by its chemical structure. This substance was a brown polymer. The contents of sugars, amino acids and phenolics in the substance were evaluated. This polymer contained small amounts of sugars and amino acids in its partial structure. After being decomposed by alkaline fusion, the decomposition products were identified by HPLC and GC-MS. Several phenolics were detected in the decomposed products. To characterize this substance, various types of model compounds were prepared by roasting chlorogenic acid, sucrose, and (or) protein with cellulose powder. Among these model compounds, the polymer-forming ability was highest in the model prepared from all four of materials, but the metal-chelating ability was the highest in the model prepared from chlorogenic acid and cellulose. These results suggest that this metal-chelating substance was a melanoidin-like polymer formed by the decomposition and polymerization of sugars, amino acids and phenolics.     

Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1

Removal of glutamate from the synaptic cleft by (Na(+) + K(+))-coupled transporters prevents neurotoxicity due to elevated concentrations of the transmitter. These transporters exhibit an unusual topology, including two reentrant loops. Reentrant loop II plays a pivotal role in coupling ion and glutamate fluxes. Here we used cysteine-scanning mutagenesis of the GLT-1 transporter to test the idea that this loop undergoes conformational changes following sodium and substrate binding. 15 of 22 consecutive single cysteine mutants in the stretch between Gly-422 and Ser-443 exhibited 30-100% of the transport activity of the cysteine-less transporter when expressed in HeLa cells. The transport activity of 11 of the 15 active mutants including five consecutive residues in the ascending limb was inhibited by small hydrophilic methanethiosulfonate reagents. The sensitivity of seven cysteine mutants, including A438C and S440C, to the reagents was significantly reduced by sodium ions, but the opposite was true for A439C. The non-transportable analogue dihydrokainate protected at almost all positions throughout the loop, and at two of the positions, the analogue protected even in the absence of sodium. Our results indicate that reentrant loop II forms part of an aqueous pore, the access of which is blocked by the glutamate analogue dihydrokainate, and that sodium influences the conformation of this pore-loop.     

A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained approximately 32% of wild type transport activity and approximately 56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na(+) and at 4 degrees C, their ability to protect Cys-357 required Na(+) and was prevented at 4 degrees C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K(+) ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity.     

Modeling a conformationally sensitive region of the membrane sector of the fungal plasma membrane proton pump

A molecular model for transmembrane segments 1 and 2 from the fungal proton pumping ATPase has been developed, and this structure is predicted to form a helical hairpin loop structure in the membrane. This region was selected because it is highly conformationally active and is believed to be an important site of action for clinically important therapeutics in related animal cell enzymes. The hairpin loop is predicted to form an asymmetric tightly packed structure that is stabilized by an N-cap between D140 and V142, by hydrogen bonding between residues in the turn region and the helices, and by - interactions between closely apposed aromatic residues. A short four-residue S-shaped turn is stabilized by hydrogen bonding but is predicted to be conformationally heterogeneous. The principal effect of mutations within the hairpin head region is to destabilize the local close packing of side groups which disrupts the pattern of hydrogen bonding in and around the turn region. Depending on the mutation, this causes either a localized or a more global distortion of the primary structure in the hairpin region. These altered structures may explain the effects of mutations in transmembrane segments 1 and 2 on ATP hydrolysis, sensitivity to vanadate, and electrogenic proton transport. The conformational sensitivity of the hairpin structure around the S-turn may also account for the effects of SCH28080 and possibly ouabain in blocking ATPase function in related animal cell enzymes. Finally, the model of transmembrane segments 1 and 2 serves as a template to position transmembrane segments 3 and 8. This model provides a new view of the H⁺-ATPase that promotes novel structure/function experimentation and could serve as the basis for a more detailed model of the membrane sector of this enzyme.     

Sequence analysis of cloned cDNA for rat substance P precursor: existence of a third substance P precursor

The sequence of the mRNA for the rat substance P precursor (preprotachykinin A) has been elucidated by molecular cloning and sequence analysis. The deduced amino acid sequence of rat preprotachykinin A indicates that it contains both substance P and substance K but differs in the sequence organization from either bovine alpha- or beta-preprotachykinin A reported previously. The existence of the bovine mRNA for the third preprotachykinin A has thus been examined and evidenced by the isolation of the corresponding cDNA clone. This mRNA, named gamma-preprotachykinin A mRNA, deletes the sequence precisely corresponding to the exon 4 sequence of the preprotachykinin A gene. Thus, alternative RNA splicing in the expression of the single preprotachykinin A gene results in the generation of three different forms of the preprotachykinin A mRNAs.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Interaction of substance P antagonists with substance P receptors on dispersed pancreatic acini

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.     

Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat

Immunohistochemical and radioimmunoassay studies revealed that both CGRP- and SP-like immunoreactivity in the caudal spinal trigeminal nucleus and tract, the substantia gelatinosa and the dorsal cervical spinal cord as well as in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglion is markedly depleted by capsaicin which is known to cause degeneration of a certain number of primary sensory neurons. Higher brain areas and the ventral spinal cord were not affected by capsaicin treatment. Furthermore CGRP and substance P-like immunoreactivity were shown to be colocalized in the above areas and to coexist in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglia. It is suggested that CGRP, like substance P, may have a neuromodulatory role on nociception and peripheral cardiovascular reflexes.