A neurokinin 1 receptor antagonist decreases adhesion reformation after laparoscopic lysis of adhesions in a rat model of adhesion formation.

BACKGROUND: Up to 94% of patients experience fibrous adhesions after abdominal surgery, and a significant number of these patients require a second operation for open or laparoscopic lysis of adhesions (LOA). The authors have previously shown that inhibition of the binding of tachykinin ligands to the neurokinin 1 receptor (NK-1R) using the neurokinin 1 receptor antagonist (NK-1RA) CJ-12,255 decreases primary adhesion formation and upregulates the peritoneal fibrinolytic system in a rat model. Whereas most studies have focused on the prevention of primary adhesions, few have addressed adhesion reformation after LOA. This study aimed to determine the effects of NK-1RA administration on adhesion reformation and peritoneal fibrinolytic activity after laparoscopic LOA. METHODS: Adhesions were induced in 31 rats using our previously described ischemic button model. The rats underwent laparoscopy 7 days later, during which adhesions were scored and lysed followed by administration of the NK-1RA or saline. Then 7 days after LOA, 23 rats were killed and adhesions were scored. Eight rats also were killed 24 h after the LOA to obtain peritoneal tissue and fluid, which were analyzed for tissue plasminogen activator (tPA) mRNA expression and peritoneal fibrinolytic activity by reverse transcriptase-polymerase chain reaction (RT-PCR) and bioassay, respectively. RESULTS: At laparoscopy, 79% +/- 3% of the buttons formed adhesions. In the saline-administered control animals, 42% +/- 3.2% of the buttons reformed adhesions after LOA (p < 0.05), whereas in the animals that received the NK-1RA, 18.2% +/- 3.5% of the buttons reformed adhesions (p < 0.05). As compared with control animals, NK-1RA administration increased tPA mRNA levels by 38% and fibrinolytic activity sixfold (p < 0.05; 7.0 +/- 2.1 U/ml vs 1.2 +/- 0.54 U/ml). CONCLUSIONS: When administered during laparoscopic LOA, an NK-1RA significantly upregulates peritoneal fibrinolytic activity and decreases adhesion reformation.

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.

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.

Isolation and characterization of substance P-containing dense core vesicles from rabbit optic nerve and termini.

In neurons, neuropeptides and other synaptic components are transported down the axon to the synapse in vesicles using molecular motors of the kinesin family. In the synapse, these neuropeptides are found in dense core vesicles (DCVs), and, following calcium-mediated exocytosis, they interact with receptors on the target cell. We have developed a rapid, large-scale technique for purifying peptide-containing DCVs from specific nuclei in the central nervous system. By using differential velocity gradient and equilibrium gradient centrifugation, neuropeptide-containing DCVs can be separated by size and density from optic nerve (ON) and its termini, the lateral geniculate nuclei and the superior colliculi. Isolated DCVs contain neuropeptides (substance P and brain-derived neurotrophic factor), synaptic vesicle (SV) membrane proteins (SV2, synaptotagmins, synaptophysin, Rab3 and synaptobrevin), SV-associated proteins (alpha-synuclein), secretory markers for DCVs previously isolated (secretogranin II), and beta-amyloid precursor protein. By using electron microscopic techniques, DCV were also visualized and shown to be immunoreactive for neuropeptides, neurotrophins, and SV membrane proteins. Because of the interesting group of physiological and potentially pathophysiological proteins associated with these vesicles; this isolation procedure, applicable to other CNS nuclei, should represent an important research tool.

Widespread expression in adult rat forebrain of mRNA encoding high-affinity neurotensin receptor.

The peptides neurotensin (NT) and neuromedin N exert effects on neurons by means of a high-affinity NT receptor (NTRH) belonging to the superfamily of G-protein-coupled receptors. In the present study, we used in situ hybridization histochemistry with sensitive riboprobe methodology to investigate the distribution of NTRH mRNA in the forebrain of adult rats. Labeled cells were abundant in the hypothalamus, epithalamus, ventral thalamus, septum, amygdala, and pallidum, including many regions where NTRH mRNA had not been detected previously. In the hypothalamus, novel sites of NTRH mRNA expression included the arcuate, periventricular, paraventricular, supraoptic, medial preoptic, anterior, ventromedial, and posterior nuclei, as well as the lateral hypothalamic area. In the thalamus, novel sites of expression included the anterodorsal nucleus, lateral habenula, and zona incerta, where labeling was much more extensive than previously reported. Novel telencephalic sites of expression included most bed nuclei of the stria terminalis, most divisions of the amygdala, the main olfactory bulb, the endopiriform nucleus, the claustrum, many parts of retrohippocampal allocortex, and limited parts of most isocortical areas. Novel sites of expression were also observed in the midbrain and pons. Taking into account expected differences in the subcellular locations of receptor mRNA and protein, the regional distribution of NTRH mRNA agrees well with that of NTRH determined previously. Our results identify many novel sites of NTRH mRNA expression in adult brain and provide a basis for investigating involvement of NT and related peptides in regulating the activity of these diverse cells, whose phenotypes remain largely undetermined.

Substance P receptor expression in intestinal epithelium in clostridium difficile toxin A enteritis in rats.

We previously reported that the inflammatory effects of Clostridium difficile toxin A on rat intestine can be significantly inhibited with a specific neurokinin-1 receptor (NK-1R) antagonist. In this study we investigated the localization and expression of NK-1R mRNA and protein in rat intestine by in situ hybridization, Northern blot analysis, and immunohistochemistry, respectively, after exposure to toxin A. Northern blot analysis showed increased mucosal levels of NK-1R mRNA starting 30 min after toxin A administration. In situ hybridization showed that toxin A increased NK-1R mRNA expression in intestinal epithelial cells after 30, 120, and 180 min. In rats pretreated with the NK-1R antagonist CP-96345 the increase in NK-1R mRNA levels after exposure to toxin A was inhibited, indicating that NK-1R upregulation is substance P (SP) dependent. One hour after exposure to toxin A many of the intestinal epithelial cells showed staining for NK-1R compared with controls. Specific 125I-SP binding to purified epithelial cell membranes obtained from ileum exposed to toxin A for 15 min was increased twofold over control and persisted for 4 h. This report provides evidence that NK-1R expression is increased in the intestinal epithelium shortly after exposure to toxin A and may be important in toxin A-induced inflammation.

Neuroimmune mechanisms of intestinal responses to stress. Role of corticotropin-releasing factor and neurotensin.

Previous studies showed that exposure of experimental animals to immobilization stress increases colonic motility and that these effects are mediated by release of corticotropin-releasing factor (CRF), Studies from our laboratory showed that 30-min immobilization stress of rats caused several not previously described colonic responses to stress, including increased colonic mucin and prostaglandin E2 (PGE2) secretion, increased colonic mucosal levels of cyclooxygenase-2 (COX-2) mRNA, and degranulation of colonic mast cells. These stress-associated colonic changes were reproduced by intravenous or intracerebral injection of CRF in conscious, nonstressed rats. Furthermore, pretreatment of rats with the CRF antagonist alpha-helical CRF9-41, hexamethonium, or the mast cell stabilizer lodoxamide inhibited our observed colon responses to immobilization stress. Our results indicate that CRF released during immobilization stress increases colonic transit via a neuronal pathway and stimulates colonic mucin release via activation of neurons and colonic mast cells. These results provide support for an important role for CRF in stress-mediated colonic responses and a link between the nervous and the immune systems.

Two distinct populations of synaptic-like vesicles from rat brain.

In nonneuronal cells, several plasma membrane proteins such as exofacial enzymes, receptors, and ion channels recycle between their intracellular compartment(s) and the cell surface via an endosomal pathway. In neurons, however, this pathway has not been extensively characterized. In particular, it remains unclear whether or not it is related to the recycling of small synaptic vesicles, the major pathway of membrane traffic in nerve terminals. To approach this problem, we purified and studied a vesicular fraction from rat brain synaptosomes. Two distinct populations of vesicles with different buoyant densities and sedimentation coefficients were detected in this fraction by sucrose gradient centrifugation and Western blot analysis of the individual proteins. Both populations contain proteins that are markers of synaptic vesicles, namely, SV2, synaptotagmin, synaptophysin, secretory carrier membrane proteins (SCAMPs), synaptobrevin, and rab3a. A striking difference between the two populations is the presence of arginine aminopeptidase activity (a previously suggested marker for the regulated endosomal recycling pathway) exclusively in the lighter less-dense vesicles. The same two vesicular populations were also detected in the preparation of clathrin-coated vesicles isolated from whole rat brain or purified synaptosomes after removal of their clathrin coats by incubation at pH 8.5. We conclude, therefore, that both types of vesicles recycle in synaptosomes via a clathrin-mediated pathway. These data present experimental evidence for biochemical heterogeneity of synaptic-like vesicles in rat brain.

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.

A substance P antagonist, RP-67,580, ameliorates a mouse meningoencephalitic response to Trypanosoma brucei brucei.

Mice infected with the protozoan parasite Trypanosoma brucei brucei and treated subcuratively with the trypanocidal drug diminazene aceturate develop an acute inflammatory meningoencephalitis with associated astrocytic proliferation. This reaction is very similar to that seen in the fatal posttreatment reactive encephalopathies that can occur in human African trypanosomiasis. The 11-amino acid neuropeptide substance P (SP) has recently been identified as a mediator in many inflammatory responses, and the development of potent, highly specific, nonpeptide SP antagonists has provided a new opportunity to investigate the possible involvement of SP in a variety of pathological conditions. We therefore postulated that SP may play a role in the development of the posttreatment inflammatory encephalopathy found in this experimental mouse model of African trypanosomiasis. In the present study RP-67,580, a SP antagonist that binds specifically to NK-1 receptors, was given intraperitoneally at a dose of 2 mg/kg twice daily to mice in which a severe meningoencephalitis had been produced. A significant reduction in both the severity of the inflammatory response (P = 0.0001) as well as the degree of astrocyte activation (P < 0.001) was found in the brains of these animals as compared with control mice that had not received RP-67,580. An inactive enantiomer of this SP antagonist, RP-68,651, had no effect on the central nervous system inflammatory reaction. We conclude from these findings that the neuropeptide SP plays a key role in the development of the severe central nervous system inflammatory response associated with African trypanosomiasis.

Increased substance P responses in dorsal root ganglia and intestinal macrophages during Clostridium difficile toxin A enteritis in rats.

Previously we reported that pretreatment of rats with the substance P (SP) antagonist CP-96,345 inhibits the enterotoxic responses following administration of toxin A from Clostridium difficile into ileal loops, indicating that SP participates in the intestinal responses to this toxin. We now report that injection of toxin A into rat ileum causes a rapid increase in SP content in lumbar dorsal root ganglia (DRG) and mucosal scrapings 30-60 min after toxin A administration. Toxin A-mediated fluid secretion, mannitol permeability, and ileal histologic damage is significantly increased only after 2 hr. Toxin A also causes an increase in the abundance of SP mRNA in lumbar DRG and ileal mucosa as measured by reverse transcription-PCR. Lamina propria macrophages (LPMs) obtained from toxin A-injected loops release greater amounts of tumor necrosis factor alpha (TNFalpha) and SP as compared with LPMs isolated from buffer-injected loops (P < 0.01). Pretreatment of rats with the SP antagonist CP-96,345 inhibits toxin A-mediated TNFalpha release from isolated LPMs, whereas an inactive enantiomer (CP-96,344) of the SP antagonist has no effect. LPMs obtained from toxin A-injected ileal loops incubated in vitro with SP (10(-8) to 10(-9) M) show enhanced TNFalpha secretion, whereas LPMs isolated from buffer-injected loops do not respond to SP. In addition, LPMs obtained from toxin A-injected ileal loops incubated in vitro with CP-96,345 showed a diminished TNFalpha release. Our results indicate that activated LPMs secrete SP during toxin A enteritis that can lead to secretion of cytokines, suggesting an autocrine/paracrine regulation of cytokine secretion by SP from LPMs during intestinal inflammation.

Role of substance P in blood pressure regulation in salt-dependent experimental hypertension.

The participation of substance P in the pathogenesis of five models of experimental hypertension, ie, DOCA-salt, subtotal nephrectomy, one-kidney-one clip renovascular, two-kidney-one clip renovascular, and spontaneous hypertension, was evaluated via an acute infusion of a newly synthesized potent, specific nonpeptide antagonist of substance P at the NK-1 receptor, the agent CP 96,345. In conscious unrestrained rats, CP 96,345 induced significant and sustained increases in mean arterial pressure of DOCA-salt, subtotal nephrectomy, and one-kidney-one clip renovascular hypertensive rats but only small and nonsignificant changes in blood pressure of two-kidney-one clip renovascular and spontaneously hypertensive rats. CP 96,345 had no effect on the blood pressure of sham-treated controls and Wistar-Kyoto rats. This NK-1 receptor antagonist did not significantly affect the heart rate of any experimental model studied. The data suggest that endogenous substance P may act as a partial counterregulatory mechanism against vasoconstriction in models of salt-dependent hypertension.

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.

A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats.

We previously reported that short-term immobilization stress of rats causes increased colonic mucin release, goblet cell depletion, prostaglandin E2 secretion, and colonic mast cell activation, as well as increased colonic motility. The purpose of this study was to investigate whether neurotensin (NT), a peptide expressed in both brain and digestive tract, participates in these responses. Rats were pretreated with SR 48692 (1 mg/kg, i.p.), an NT antagonist, 15 min before immobilization (30 min). The administration of the antagonist significantly inhibited stress-mediated secretion of colonic mucin, prostaglandin E2, and a product of rat mast cells, rat mast cell protease II (P < 0.05), but did not alter the increase in fecal pellet output caused by immobilization stress. Immobilization stress also resulted in a quantifiable decrease in the abundance of NT receptor mRNA in rat colon compared with that in colonic tissues from nonimmobilized rats as measured by densitometric analysis of in situ hybridization studies (P < 0.03). We conclude that the peptide NT is involved in colonic goblet cell release and mucosal mast cell activation after immobilization stress.

Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.

Streptozotocin-induced diabetes produces a decrease in pituitary substance P content and preprotachykinin mRNA.

Complications arising from diabetes mellitus include hormonal dysfunctions such as impairment in the regulation of gonadatroph and corticotroph secretion. Preprotachykinin (PPT) mRNA encoding the peptide substance P (SP), has been localized in the anterior pituitary. The goal of this study was to determine if streptozotocin (STZ)-induced diabetes affects the SP content or PPT mRNA level in the pituitary of male rats. We injected STZ (55 mg/kg) to 6-week-old rats which developed hyperglycemia (blood glucose > 400 mg/dl) by 6 weeks post-injection. SP-like immunoreactivity in the pituitary dropped 54%. In situ hybridization was performed using a PPT-specific oligonucleotide with signal intensity differences semi-quantified using an image analysis system. Normal pituitary had a regional distribution of PPT mRNA, with no detectable signal in the posterior or intermediate lobes, while the anterior lobe displayed a distinctive pattern of labeled cells arranged in clusters. In diabetic rats there was a 23% decrease in the PPT-mRNA hybridization signal compared to controls (P < 0.05). The changes observed in PPT gene expression and SP content may be additional factors participating in the hormonal complications seen in diabetes mellitus.