Modulation of isolation-induced fighting by N- and C-terminal analogs of substance P: evidence for multiple recognition sites

Substance P (SP) significantly reduced fighting in mice made aggressive by prolonged isolation. The N-terminal heptapeptide fragment SP (1-7) also reduced fighting. The C-terminal fragment SP(4-11) was without activity, while the shorter C-terminal fragment analog less than E-SP(7-11) significantly increased isolation-induced fighting. The aggression-enhancing effect of less than E-SP(7-11) was antagonized by naloxone, which by itself had no significant effect. The aggression-reducing effect of SP(1-11) was significantly enhanced by naloxone, while the effect of SP(1-7) was unchanged. These results demonstrate that a behavioral effect of SP may be duplicated by an N-terminal fragment of the SP molecule, and that peptide fragments or analogs of the N- and C-terminal portions of the SP molecule can exert opposing effects on a specific behavior. These findings represent a structure/activity relationship that is strikingly different from any previously described for SP. The differing effects of naloxone on N- and C-terminal fragment analogs suggest that these two effects may be mediated by different mechanisms.     

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykinin Receptors of the NK1 Type (Substance P) Coupled Positively to Phospholipase C on Cortical Astrocytes from the Newborn Mouse in Primary Culture

Specific 125I-Bolton-Hunter substance P (125I-BHSP) binding sites are present on intact cortical astrocytes of the newborn mouse in primary culture. Therefore, these cells were used to ascertain the existence of functional substance P (SP) receptors coupled positively to phospholipase C. SP stimulated phosphoinositide breakdown with an EC50 value (4.5 x 10(-10) M) similar to its IC50 value (3.8 x 10(-10) M) for inhibiting 125I-BHSP binding. The maximal response to (10(-6) M SP for 60 min) obtained was approximately 500% of control values. The rank order of potency of tachykinins was SP greater than neurokinin (NK) A greater than NKB. Long SP C-terminal fragments were more potent than shorter ones in stimulating the accumulation of 3H-inositol phosphates. SP free acid and SP N-terminal fragments were without effect. [L-Pro9]SP and SP methyl ester, two selective agonists of NK1 receptors, were almost as potent as SP. An excellent correlation was found when the abilities of tachykinins and their analogs for stimulating phosphoinositide breakdown and for inhibiting 125I-BHSP binding were compared. Finally, when used at a concentration of 3 x 10(-6) M, spantide [( D-Arg1, D-Trp7,9, Leu11]SP), an SP antagonist, competitively reduced the stimulatory effect of SP on accumulation of 3H-inositol phosphates. These results demonstrate the presence of functional SP receptors (NK1) on cortical astrocytes from the newborn mouse in primary culture.     

Synapsin I is a highly surface-active molecule

Synapsin I is a neuron-specific phosphoprotein localized on the surface of small synaptic vesicles to which it binds with high affinity (Kd = 10 nM). Synapsin I exhibits a tendency to self-associate, suggesting that it might have amphiphilic properties. We have now found that synapsin I forms a stable monolayer at an air-water interface which can be compressed under a lateral force of up to 60 dynes/cm, indicating the presence of amphiphilic characteristics in its structure. This interpretation was also supported by circular dichroism spectra of synapsin I, which showed induction of secondary structure in the presence of trifluoroethanol. The various phosphorylated forms of synapsin I did not show any noticeable differences in the force-area isotherms. The monolayer properties of synapsin I fragments derived by cysteine-specific cleavage indicated the presence of amphiphilic characteristics throughout the entire sequence, although the C-terminal region showed less of such surfactant properties. Compositional studies of these fragments revealed that there is little interaction between the N-terminal and middle fragment regions, but that there may be some interaction between the C-terminal and middle fragment regions which affects the surface area occupied by these fragments. Based on this information, we propose a molecular topology for synapsin I consisting of amphiphilic regions and a hydrophilic region.     

Substance P enhances IL-2 expression in activated human T cells.

Jurkat and HUT 78 T cell lines, as well as peripheral blood human T cells activated with PHA plus PMA were used to investigate the capacity of substance P (SP) neuropeptide to regulate IL-2 production. By using Northern blot analysis and dosage of the IL-2 release in cell supernatants, we show that SP can act as cosignal with PHA + PMA to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells. By using the N-terminal SP(1-4) or the C-terminal SP(4-11) fragments of the entire molecule, we show that the cosignal activity is carried by the C-terminal portion of SP. The SP and SP(4-11) optimal effects were observed at 10(-12) M and 10(-10) M when a broad range of concentrations from 10(-6) M to 10(-13) M was tested. The increase of IL-2 mRNA obtained with 10(-12) M of SP in the activated Jurkat cells was reduced by adding 10(-10) or 10(-9) M of the SP antagonist (D-Pro2,-D-Phe7,-D-Trp9)SP to the culture, indicating the specificity of SP action. The up-regulation observed when 10(-12) M of SP was applied together with the mitogens on Jurkat cells, persisted after a 16-h culture period, time at which the IL-2 mRNA signal is normally back to a minimum level when the mitogens are used alone. Furthermore, an induction of IL-2 mRNA accumulation, in a 2-h pulse, was obtained with 10(-12) M of SP on Jurkat cells previously activated with mitogens for 16 h.     

Limited proteolysis of synapsin I. Identification of the region of the molecule responsible for its association with microtubules

Synapsin I is a highly asymmetric neuronal structural phosphoprotein implicated in the regulation of neurotransmitter release probably by the multiple interactions it can contract with membranous and cytoskeletal elements of the neuronal cell. In order to locate the region(s) of synapsin I responsible for its association with microtubules, we have first studied synapsin I limited digestion by trypsin. The resulting polypeptides were localized in the synapsin I molecule by using three different criteria: their kinetics of appearance, their collagenase sensitivity, and the presence of the synapsin phosphorylation site 1 (cyclic AMP dependent). Synapsin I digestion kinetics are not affected by phosphorylation at this site. Analysis of the ability of various synapsin I tryptic fragments in mixture to cosediment with microtubules shows that a 44-kDa fragment corresponding to the NH2-terminal hydrophobic head of the molecule contains a binding site for polymerized tubulin. This fragment competes with native synapsin I for binding on microtubules. None of the polypeptides belonging to the tail region of synapsin I (COOH-terminal half of the molecule) were found to cosediment with microtubules.     

Characterization of synapsin I fragments produced by cysteine-specific cleavage: a study of their interactions with F-actin

Synapsin I is a neuron-specific phosphoprotein that is concentrated in the presynaptic nerve terminal in association with the cytoplasmic surface of synaptic vesicles. It has been demonstrated to bundle F-actin in a phosphorylation-dependent manner in vitro, a property consistent with its proposed role in linking synaptic vesicles to the cytoskeleton and its involvement in the regulation of neurotransmitter release. Synapsin I is composed of two distinct domains, a COOH terminal, collagenase-sensitive, hydrophilic, and strongly basic tail region, and an NH2 terminal, collagenase-resistant head region relatively rich in hydrophobic amino acids. To elucidate the structural basis for the interactions between synapsin I and F-actin and how it relates to other characteristics of synapsin I, we have performed a structure-function analysis of fragments of synapsin I produced by cysteine-specific cleavage with 2-nitro-5-thiocyanobenzoic acid. The fragments were identified and aligned with the parent molecule using the deduced primary structure of synapsin I and the known phosphorylation sites as markers. We have purified these fragments and examined their interactions with F-actin. Two distinct fragments, a 29-kD NH2-terminal fragment and a 15-kD middle fragment, were shown to contain F-actin binding sites. A 51/54-kD middle/tail fragment retained the F-actin binding and bundling activity of synapsin I, but the isolated tail fragment did not retain either activity. In contrast to phosphorylation of sites two and three in intact synapsin I, which abolishes F-actin bundling activity, phosphorylation of these sites in the middle/tail fragment failed to abolish this activity. In conclusion, three domains of synapsin I appear to be involved in F-actin binding and bundling.     

Effects of substance P and its fragments on microhemodynamics in animal experiments]

Action of the synthetic substance P (SP1-11) and its modified fragment (MSP1-4) on microhemodynamics was investigated in experiments. Significant hypotensive effect and reduction of blood flow velocity in rat mesenteric microvessels were produced by both substances, no changes of microvessels diameter were observed. Effective concentrations of the SP1-11 and MSP1-4 were revealed, being 10(-6) M and 10(-4) M, respectively. Organ specificity of substance P vascular effects is suggested.     

Substance P and behavior: opposite effects of N-terminal and C-terminal fragments

Most of the biological actions of substance P (SP) have been thought to be mediated by the carboxy-terminal portion of the peptide. Some of the behavioral effects produced by exogenous SP exhibit a strikingly different structure-activity relationship. The N-terminal heptapeptide fragment of SP, SP(1-7), inhibits nociceptive, aggressive and grooming behaviors and stimulates investigative motor behavior, but the C-terminal hexapeptide fragment analog pyroglutamyl-SP(7-11) exerts opposite effects. While the C-terminal fragment mimics the effects of administered intact SP on motor behaviors, the N-terminal fragment mimics the effects of intact SP on aggressive and nociceptive behaviors. The significant behavioral effects of SP(1-7) and the consistently opposite behavioral effects of N- and C-terminal fragments are important new findings.     

Identification, expression, and crystallization of the protease-resistant conserved domain of synapsin I.

A 35-37-kDa protease-resistant domain of synapsin Ia/ Ib, apparently produced by low levels of endogenous proteases in vapor diffusion droplets, slowly formed crystals diffracting X-rays to approximately 10 A resolution. The fragment mainly consisted of the highly conserved C domain common to the synapsin I/II family plus short N- and C-terminal flanking segments. Two constructs (SynA and SynB) of synthetic gene fragments coding for the C domain of synapsin with or without C-terminal flanking sequence were expressed in Escherichia coli as fusion proteins attached to the soluble protein glutathione-S-transferase. The fusion proteins were purified by affinity chromatography. Subsequent in situ cleavage with TEV protease resulted in the release of highly pure synapsin fragments, which were further purified by ion exchange chromatography. SynA and SynB formed crystals within three days, which diffracted to better than 3 A using a conventional X-ray source and to about 2 A using a synchrotron X-ray source. SynA crystals have the symmetry of the trigonal space groups P3(1)21 or P3(2)21 and the unit cell dimensions a = b = 77.4 A, c = 188.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. SynB crystals have the symmetry of the orthorhombic space group C222(1) with the unit cell dimension a = 104.6 A, b = 113.3 A, and c = 273.8 A.     

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.     

New Chimeric Analogues of Galanin: Synthesis and Effects on the Glucose-Induced Insulin Secretion

The solid-phase synthesis and in vitro assays on the glucose-induced insulin secretion from rat pancreatic islets of Langerhans with six new chimeric peptides were performed. All the peptides were built up of the N-terminal galanin (GAL) fragment or its analogues, linked to the C-terminal portion of substance P (SP) analogues or scyliorhinin I (SCY-I) analogues. Two strong antagonists of the inhibitory effect of galanin on the glucose-induced insulin release were found: [cycloleucine⁴]GAL(1-13)-SP(5-11)-amide and GAL(1-13)-[L-norleucine¹⁰]SCY-I(3-10)-amide.     

Activation of Metabotropic Glutamate Receptors Inhibits Synapsin I Phosphorylation in Visceral Sensory Neurons

Activation of glutamate metabotropic receptors (mGluRs) in nodose ganglia neurons has previously been shown to inhibit voltage-gated Ca⁺⁺ currents and synaptic vesicle exocytosis. The present study describes the effects of mGluRs on depolarization-induced phosphorylation of the synaptic-vesicle-associated protein synapsin I. Depolarization of cultured nodose ganglia neurons with 60 mm KCl resulted in an increase in synapsin I phosphorylation. Application of mGluR agonists 1-aminocyclopentane-1s-3r-dicarboxylic acid (t-ACPD) and L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) either in combination or independently inhibited the depolarization induced phosphorylation of synapsin I. Application of the mGluR antagonist (RS)-α-Methyl-4-carboxyphenylglycine (MCPG) blocked t-ACPD-induced inhibition of synapsin phosphorylation but not the effects of L-AP4. In addition, application of either t-ACPD or L-AP4 in the absence of KCl induced depolarization had no effect on resting synapsin I phosphorylation. RT-PCR analysis of mGluR subtypes in these nodose ganglia neurons revealed that these cells only express group III mGluR subtypes 7 and 8. These results suggest that activation of mGluRs modulates depolarization-induced synapsin I phosphorylation via activation of mGluR7 and/or mGluR8 and that this process may be involved in mGluR inhibition of synaptic vesicle exocytosis in visceral sensory neurons of the nodose ganglia. Received 28 June 2000/Revised: 11 September 2000     

Partial IGF affinity of circulating N- and C-terminal fragments of human insulin-like growth factor binding protein-4 (IGFBP-4) and the disulfide bonding pattern of the C-terminal IGFBP-4 domain

Within the IGF axis, the insulin-like growth factor-binding proteins (IGFBPs) are known to play a pivotal role in cell proliferation and differentiation. Defined proteolysis of the IGFBPs is proposed to be an essential mechanism for regulating IGF bioavailability. The generated IGFBP fragments in part exhibit different IGF-dependent and -independent biological activities. Characterizing naturally occurring forms of IGFBPs in human plasma, we identified both a N- and a C-terminal fragment of IGFBP-4 by means of immunoreactivity screening. As a source for peptide isolation, we used large amounts of human hemofiltrate obtained from patients with chronic renal failure. Purification of the IGFBP-4 peptides from hemofiltrate was performed by consecutive cation-exchange and reverse-phase chromatographic steps. Mass spectrometric and sequence analysis revealed an M(r) of 13 233 for the purified N-terminal fragment spanning residues Asp(1)-Phe(122) of IGFBP-4 and an M(r) of 11 344 for the C-terminal fragment extending from Lys(136) to Glu(237). Proteolytic digestion and subsequent biochemical analysis showed that the six cysteines of the C-terminal IGFBP-4 fragment are linked between residues 153-183, 194-205, and 207-228 (disulfide bonding pattern, 1-2, 3-4, and 5-6). Plasmon resonance spectroscopy, ligand blot analysis, and saturation and displacement studies demonstrated a very low affinity of the C-terminal IGFBP-4 fragment for the IGFs (IGF-II, K(d) = 690 nM; IGF-I, K(d) > 60 nM), whereas the N-terminal fragment retained significant IGF binding properties (IGF-II, K(d) = 17 nM; IGF-I, K(d) = 5 nM). This study provides the first molecular characterization of circulating human IGFBP-4 fragments formed in vivo exhibiting an at least 5-fold decrease in the affinity of the N-terminal IGFBP-4 fragment for the IGFs and a very low IGF binding capacity of the C-terminal fragment.     

Structure-activity relationships of monomeric and dimeric synthetic ACTH fragments in perifused frog adrenal slices

The effect of synthetic monomeric and dimeric ACTH fragments on spontaneous and ACTH(1-39)-evoked steroidogenesis in frog interrenal tissue was studied in vitro. Infusion of ACTH fragment 11-24 (10(-6) M) or its dimeric conjugates, attached either by their N-terminal, Glu(11-24)2, or their C-terminal amino acid, (11-24)2Lys, had no effect on the spontaneous release of corticosteroids. The monomer ACTH(11-24) and the dimer Glu(11-24)2 were also totally devoid of effect on the steroidogenic response to ACTH(1-39) (10(-9)M). In contrast, the (11-24)2Lys conjugate (10(-6)M) significantly decreased ACTH-induced stimulation of corticosterone and aldosterone (-63 and -62%, respectively). The dimeric conjugate of the fragment ACTH(7-24), linked through the C-terminal ends, (7-24)2Lys (10(-6)M), was also completely devoid of effect on basal steroidogenesis but caused a marked decrease of ACTH-evoked corticosterone and aldosterone release (-72 and -80%, respectively). Conversely, infusion of the dimer (1-24)2Lys gave rise to a dose-related stimulation of corticosterone and aldosterone release. The time-course of the steroidogenic response to the dimer was similar to that of ACTH(1-24). The 1-24 conjugate was 70 times less potent than the monomers ACTH(1-24) and ACTH(1-39). These results suggest that amphibian adrenocortical cells contain only one class of ACTH receptor which recognizes the 11-24 domain of ACTH with an affinity which depends on the presence of a strong potentiator segment, located at the N-terminus end of ACTH(1-39). Since the ACTH-dimers are thought to induce cross-linking of the receptors, our results suggest that aggregation of ACTH receptors causes a down-regulation of the receptors.     

Specificity of antisera obtained to substance P and its C-terminal hexapeptide

Synthetic fragments and analogs were used to characterize specificity of antisera to SP and SP6-11. [Tyr8] SP and [Lys6] SP6-11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP7-11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP6-11 by His or Gly showed that all but Glu6 take part in the structure of the antigenic determinant.     

Substance P augmentation of CSF-1-stimulated in vitro myelopoiesis. A two-signal progenitor restricted, tuftsin-like effect

The inflammatory neuropeptide substance P acted as a costimulant for macrophage CSF-1-induced clonal proliferation of murine marrow-derived two signal-dependent mononuclear phagocyte progenitors. Substance P had no effect on clonal proliferation by progenitors responding solely to CSF-1. Substance P fragment 2-11 had no costimulatory activity; however, SP fragment 1-4 retained the full activity of the parent undecapeptide. Fragment 1-4 (ARG-PRO-LYS-PRO), a peptide containing a PRO residue between two positive charges, is a tuftsin-like (THR-LYS-PRO-ARG) tetrapeptide, and tuftsin exerted an identical costimulatory effect. Substance P, SP:1-4, and tuftsin were optimally effective as costimulants at 10(-7) to 10(-6) M. (ALA1)-tuftsin, an inhibitory analog of tuftsin, was a potent negative regulator of two signal-dependent colony formation. (ALA1)-tuftsin at concentrations less than or equal to 10(-9) M exerted dose-dependent inhibition of the positive effects of optimal concentrations of all of the co-stimulants tested, including bacterial LPS. The inhibitory tetrapeptide was equivalent in activity to ferritin, an established inhibitor of two signal-dependent colony formation. The results indicated that SP may influence myelopoiesis in addition to its other inflammatory and immunopotentiating properties. In addition, a potentially valuable modulator of SP and LPS responses in this system, (ALA1)-tuftsin, was identified.     

Comparative study of substance P and its fragments: analgesic properties, effect on behavior and monoaminergic processes

The effect of substance P (SP) and of its fragments 5-11, 8-11, 9-11, 10-11 administered into the brain ventricles in doses of 5, 25 and 50 nM on the behavior and content of biogenic monoamines of the rat brain was studied. The analgetic properties of the substances under consideration and those of fragment SP 10-11 in doses of 5, 25, 50 and 100 nM were also subjected to examination. It was found that SP and fragment 5-11 stimulate and enhance the locomotor activity in rats, while fragments 8-11 and 9-11 provoke hypoactivity. The substances under study increase the serotonin and dopamine turnover, whereas SP and fragment 8-11 lower the serotonin content as well. After administration of SP and fragment 5-11 analgesia was seen to transform to hyperalgesia depending on the dose. Fragments 8-11 and 9-11 produce analgetic effect. It is suggested that both SP fragments and the whole SP molecule can influence the neurochemical process that regulate behavior and pain perception.     

The development and application of a novel N-terminal directed substance P antiserum

Most of the substance P (SP) antisera currently used for radioimmunoassay cross-react to a greater or lesser extent with C-terminal fragments of SP. With the availability of SP free acid it has been possible to produce an antiserum specific for the N-terminal part of the molecule. The amino groups on Arg¹ and Lys³ were protected by trifluoracetylation and the peptide was then conjugated through its C-terminal carboxyl group to bovine serum albumin by 1-ethyl-3-(3-dimethyl-amino propyl)-carbodiimide (EDC). After conjugation, the amino protective groups were removed under alkaline conditions and the resulting SP-albumin conjugate was used for immunization of rabbits. The SP antiserum thus produced was found to possess only 0.1% and 0.01% cross-reactivity with SP(2–11) and SP(3–11) respectively, and none at all in the presence of 1000 fold molar excess of other smaller C-terminal fragments of SP. There was no significant difference in the brain content of SP like immunoreactivity (SPLI) measured with either the N- or C-terminal directed antiserum. Using this novel antiserum together with a C-terminal directed antiserum, it was shown that deamidation is unlikely to be a rate limiting step in SP inactivation by rat brain slices.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.