In vivo somatosensory stimuli evoked the release of substance P from primary afferent neurons that terminate in the spinal cord and stimulated endocytosis of substance P receptors in rat spinal cord neurons. The distal dendrites that showed substance P receptor internalization underwent morphological reorganization, changing from a tubular structure to one characterized by swollen varicosities connected by thin segments. This internalization and dendritic structural reorganization provided a specific image of neurons activated by substance P. Thus receptor internalization can drive reversible structural changes in central nervous system neurons in vivo. Both of these processes may be involved in neuronal plasticity.

Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60%o of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR-i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo.GTP-binding regulatory protein-coupled (G protein-coupled) receptors are a large family of receptors that is widely distributed in the mammalian central nervous system (CNS). This family includes the adrenergic, dopaminergic, and all known peptidergic receptors. Although these receptors exhibit substantial diversity in the ligands with which they interact and the types of neurons and glia that express them, receptors in this family share several common structural and functional features (1, 2). All members of this family contain seven transmembrane domains and activate second-messenger systems via G proteins. Functionally, the majority of receptors in this family undergo desensitization after receptor signaling. Studies on cultured cells transfected with cDNA encoding G protein-coupled receptors indicate that after binding, several types of G protein-coupled receptors undergo phosphorylation, endosomal internalization, dissociation from the ligand in the endosome, dephosphorylation, and finally receptor recycling to the plasma membrane (3-7). Whether G proteincoupled receptors undergo a similar sequence of events in vivo is not known.Substance P (SP) belongs to the tachykinin neuropeptide family which also includes neurokinin A and neurokinin B, and all tachykinins are characterized by the C-terminal sequence -Phe-Xaa-Gly-Leu-Met-NH2 (8). In the CNS, SP is widely distributed and is present in h...

The salient pathological feature of Azhelmer (1-4).Radloiodination. Peptides containing tyrosine were radiolabeled by oxidative radioiodination using Na125I and chloramine T and separated from free iodide by reverse-phase adsorption. Peptides not containing tyrosine were first acylated with the N-hydroxysuccinimide ester of 4-hydroxyphenylpropionic acid and then oxidatively radioiodinated as above. Labeled peptides containing methionine were then reduced from sulfoxide to native form with 2-mercaptoethAbbreviations: AD, Alzheimer disease; ,BA4, ,B-amyloid peptide; RP-HPLC, reversed-phase HPLC; 125I-PA4-(1-40), 1251-labeled ,BA4-(1-40). tTo whom reprint requests should be addressed. 5462The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Antibodies to neuropeptide receptors can be used to localize and characterize the receptors in tissues and cell lines. Two strategies were used to study the rat substance P receptor (SPR, NK-1) by immunological methods. First, a polyclonal antiserum was raised by immunizing rabbits with a peptide corresponding to the 15 amino acid residues (KTMTESSSFYSNMLA, SPR393-407) at the intracellular C-terminus of the rat SPR coupled to bovine thyroglobulin. An antiserum was obtained with a titer for half-maximal binding of 125I-SPR393-407 of 1:70,000. Nonradioactive SPR393-407 inhibited 50% of binding at a concentration of 10 pM. Binding of 125I-SPR393-407 to the antiserum was also displaced in a parallel manner by membrane proteins from tissues expressing high levels of the SPR (brain and submaxillary gland). Second, a chimeric SPR construct of a hydrophilic Flag peptide (DYKDDDDK) genetically engineered in sequence with the extracellular N-terminus of rat SPR was generated by polymerase chain reaction. The Flag-SPR chimera was expressed in rat kidney epithelial cells (KNRK) and judged to be fully functional, assessed by binding of 125I-substance P (apparent Kd of 5.63 nM) and calcium mobilization in response to substance P (EC50 of 0.66 nM). Antibodies to SPR393-407 and the Flag peptide stained the plasma membrane of KNRK cells expressing the native SPR or the Flag-SPR chimera. Staining was abolished by preincubation with SPR393-407 or the Flag peptide. Cells transfected with vector alone were unstained. The SPR antiserum recognized a broad protein band on Western blots of membranes prepared from cells expressing SPR but not from cells transfected with vector alone. The signal was quenched by preincubation of the antiserum with SPR393-407. By immunohistochemistry, the SPR antiserum was found to bind to neurons in the dorsal horn of the rat spinal cord and to ganglion cells in the myenteric plexus of the rat ileum near substance P-immunoreactive nerve fibers. Staining was abolished by preabsorption of the antiserum with SPR393-407. These antibodies can be used to localize the SPR in tissues and cells and to examine the function of the receptor in cell lines.