Identification of glycation at the N-terminus of albumin by gas chromatography-mass spectrometry.

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mast cell binding of neurotensin. II. Molecular conformation of neurotensin involved in the stereospecific binding to mast cell receptor sites.

Systematic substitution of the natural L-amino acids in neurotensin by their D isomers reveals that the COOH-terminal portion of this tridecapeptide is required for binding to mast cell receptors: D-amino acid replacements from Pro10 through Leu13 substantially decrease that binding. Either blockage of the COOH-terminal carboxyl group as with N-methylamidation, or formation of a cyclic structure by the inclusion of a disulfide bond, a Cys2,13 substitution, markedly reduces the specific binding to mast cell receptor sites. Modifications in the NH2-terminal portion of neurotensin do not affect the binding to mast cells. However, D-Arg8 and D-Arg9 substitutions increase binding by factors of 5- to 6-fold. The hydroxyl group at position 3 or 11 is not essential for binding since Phe3 or Phe11 is equivalent to Tyr3 or Tyr11. The COOH-terminal penta- and hexapeptides are able to displace approximately 70% 125I-neurotensin relative to the intact peptide. Of 18 other biologically active peptides tested, only xenopsin, a naturally occurring COOH-terminal analog of neurotensin, and bradykinin effectively compete in the binding assay to an extent of 60 and 100%, respectively. Histamine, diphenhydramine, and noradrenaline are ineffective in this regard.     

Binding and internalization of VIP in rat intestinal epithelial cells.

We have prepared villous cells from the jejunum of the rat small intestine and studied the effects of divalent cations and bacitracin on the binding and internalization of VIP. Villous epithelial cells (4 x 10(6) cells/ml) were suspended in a Hepes-NaCl buffer with 1.0% BSA, (pH 7.4) and the cells were incubated for varying periods of time with 125I-VIP at 24 degrees C. Specific binding of radiolabeled VIP was maximal within 10 min (10%) and slowly declined to 9.0 percent after 30 min. In the presence of 1.0 mg/ml bacitracin, however, maximal specific binding of VIP was only 2.7 percent (P less than or equal to 0.001). The addition of CA2+ or Mg2+ to the buffer significantly decreased binding of VIP in a concentration dependent manner. At 8.0, 4.0, 2.0 and 1.0 mM Ca2+, binding of 125I-VIP decreased by 70, 60, 40 and 25 percent, whereas in the presence of the same concentrations of Mg2+ binding was decreased to 50, 38, 25 and 10 percent (P less than or equal to 0.01). To determine if epithelial cells internalize VIP, we bound 125I-VIP to villous cells and then differentiated surface-bound and internalized radioactivity by treating with trypsin (150 micrograms/ml). Surface bound radioligand was the same at both 24 and 4 degrees C (5.3%), while internalized 125I-VIP was 4.0% at 24 degrees C compared to only 1.0% at 4 degrees C (P less than or equal to 0.001). At 24 and 4 degrees C, both Ca2+ (4.0 mM) and Mg2+ (8.0 mM) decreased surface bound radioligand by 60 percent (P less than or equal to 0.01) and lowered internalized radioactivity. These data demonstrate that (1) bacitracin decreases the binding of VIP to small intestinal epithelial cells, (2) both Ca2+ and Mg2+ affect the binding of VIP to its surface receptor and (3) VIP is internalized into epithelial cells.     

Mechanism of neurotensin depolarization of rabbit colonic smooth muscle

The aims of this study were (1) to measure the effect of neurotensin on the membrane potential of circular muscle of the distal colon of the rabbit and (2) to determine the mechanism by which neurotensin affects the membrane potential of this tissue. The membrane potential was measured with microelectrodes placed intracellularly and the double sucrose gap. Neurotensin (10(-11) M to 10(-7) M) dose-dependently decreased the membrane potential. The maximum decrease in membrane potential occurred with 10(-9) M neurotensin. The ED50 of neurotensin depolarization of the membrane potential was 0.87 +/- 0.33 X 10(-10) M. The frequency of the slow waves was unchanged after neurotensin. The voltage response to a constant current pulse decreased as the concentration of neurotensin increased. The amplitude of the voltage response after a 0.6 microA current pulse decreased by 6 +/- 0.5 mV after neurotensin (10(-7) M) compared to the Krebs control (P less than 0.05). Decreasing the [Na+]o to 0-23 mM did not affect the decrease in membrane potential after neurotensin. However, perfusion with a test solution containing no added Ca2+ or verapamil (10(-5) M) inhibited neurotensin depolarization of the tissue. Evidence was found that neurotensin depolarizes colonic circular smooth muscle, and the decrease in membrane potential is associated with an increase in conductance which is dependent on influx of Ca2+.     

Modulation by neurotensin and neuromedin N of adherence and chemotaxis capacity of murine lymphocytes.

The action of neurotensin and related peptides has not been yet studied on lymphocytes, although there are studies indicating the stimulative action of neurotensin, a peptide first isolated from bovine hypothalamus, on different functions of phagocytic immune cells. The present study demonstrates that neurotensin and a related peptide, neuromedin N, increased significantly the adherence and chemotaxis capacity of murine peritoneal lymphocytes, when they were incubated in the presence of neuropeptide concentrations between 10(-9) M and 10(-12) M. With respect to their adherence capacity, neuromedin N showed a slightly higher stimulation than neurotensin at a shorter time. However, both neuropeptides stimulated the chemotaxis capacity in a similar percentage. The study of the action mechanisms of these neuropeptides showed that intracellular cAMP levels were not modified by neurotensin or neuromedin N, but using an extracellular calcium chelator, EGTA (1 mM), and a blocker of calcium channels in endoplasmic reticulum, ryanodine (0.5 mM), we observed that neurotensin and neuromedin N could produce their effects through an augmentation of the intracellular Ca2+ concentration. As adherence and chemotaxis are initial processes of immune response, the results show that both neuropeptides may be physiological modulators of the lymphocyte function.     

Contulakin-G, an O-glycosylated invertebrate neurotensin.

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10 contulakin-G was approximately an order of magnitude lower than that of neurotensin1-13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.     

Na+ and K+ binding by glycerinated muscle fibers at equal concentrations of the chlorides of these cations in the medium

The Na+ and K+ equilibrium distribution between the medium and glycerinated muscle fibres of the frog has been investigated under equal concentrations of NaCl and KCl in solutions. Concentrations of NaCl and KCl varied from 0.5-1.5 mkM till 50 mM. Ion strength (0.11) was constant owing to the imidazol--HCl buffer. The binding of Na+ and K+ by model fibres occurred in accordance with the Langmur equation. Two kinds of cation-binding sites were found. The one with a low limiting ion sorption (A infinity approximately 1.3 mmol/kg dry weight of fibres) and high affinities (-delta F0 approximately 4.3 kcal/mol) was saturated at 0.5 mM concentrations (Na+ = K+) in the medium, and the other--with A infinity exceeding the previous one by an order and low -delta F0 (2.5 kcal/mol) was discovered at Na+, K+-1-10 mM. At ion concentrations equal to 0.5-1 mM the Langmur-binding is disturbed. At Na+-K+ less than or equal to 1 mM Na+ bound:K+ bound approximately to 1:1. At higher concentrations of cations Na+ bound:K+ bound approximately equal to 3:2. It is concluded that at least part of the sites in model fibres is capable of interacting only with Na+, but not with K+. It is supposed that at equal concentrations of Na+ and K+ in the medium the cations are bound by Na+, K+-ATPase of glycerinated muscle fibres.     

Regulation of mast cell histamine release by neurotensin

Neurotensin (NT), a neuropeptide found both centrally and peripherally, stimulated release of histamine from rat peritoneal mast cells in a dose-dependent manner. Release was evident by 10 nM and reached a plateau of 15-20% total cellular histamine by 10(-7)-10(-6) M NT. Optimal conditions for stimulation occurred at pH 6.5-7.5, 37 degrees C and at calcium concentrations of less than 1 mM. Release was complete within 2 minutes of peptide addition. Studies of histamine release by NT analogues indicted that the C-terminus is the biologically active portion of the molecule in this system, as is true of all other systems responsive to NT (1). D-Trp11-NT, which acts as a NT antagonist in several peripheral NT-sensitive tissues (2,3), also inhibited NT action on mast cells. Manipulations involving Ca2+ availability suggest that the mechanism of NT stimulation may involve use of intracellular Ca2+ to a greater extent than extracellular Ca2+. Lowering the extracellular Ca2+ concentration or blocking influx of extracellular Ca2+ with lanthanum (La3+), had little effect on NT-induced release, whereas Ca2+ depletion by treatment with ethylenediaminetetracetic acid (EDTA) or blockade of intracellular Ca2+ mobilization by N,N-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), inhibited the response to NT. Increasing cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), by treatment with 8-bromo-cAMP or stimulation with prostaglandin E2 (PGE2) in the presence of isobutylmethylxanthine (IBMX), served to reduce histamine release by NT, indicating that cAMP may play a role in NT stimulation.     

The effect of caffeine, adenosine, and buffer ionic composition on the induction of cell-surface cAMP binding during starvation of Dictyostelium discoideum

We have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell-surface cAMP receptor in suspension-starved Dictyostelium discoideum cells by measuring ammonium sulfate-stabilized binding of [3-H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+ phosphate buffer, pH 6.5, plus 1 mM CaCl2 and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/10(7) cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine-treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30-40 mM was found to significantly inhibit the induction of cAMP receptors.     

Preparation of neurotensin selectively iodinated on the tyrosine 3 residue. Biological activity and binding properties on mammalian neurotensin receptors

[Monoiodo- Tyr3 ]neurotensin, a neurotensin analogue that contains a single iodine atom on the side chain of Tyr 3 was prepared and purified. This analogue can be labeled at any specific radioactivity between 0 and 2000 Ci/mmol; its binding and biological properties on rat and guinea pig neurotensin receptors are identical to those of the parent peptide. These properties make [monoiodo- Tyr3 ]neurotensin the best suitable radioligand for detection and characterization of neurotensin receptors in various tissues and species.     

Regulation of neurotensin secretion in a mammalian C cell line: effect of dexamethasone

We established in culture two colony clones of rMTC 44-2 cells, rMTC 44-2B and 44-2C which secrete substantially greater quantities of neurotensin (NT) than the parent cell line. We describe here the effects of the synthetic glucocorticoid, dexamethasone, on NT and cAMP release. Medium and intracellular levels of NT and cAMP were measured by specific RIAs. Long-term release experiments were performed in Dulbecco's Modified Eagle's Medium supplemented with 15% horse serum (DMEM). Short-term release experiments were performed in Krebs-Ringer-bicarbonate-glucose buffer (KRBG) supplemented with 1.0 mm Ca²⁺. Dexamethasone stimulated NT release and increased intracellular NT levels. The ED₅₀ values for stimulation of NT release following 24 or 48 h incubation of cells in DMEM with dexamethasone were 5 · 10^?9 and 7 · 10^?9 M, respectively. Dexamethasone markedly enhanced intracellular levels of NT in rMTC 44-2 cells while it decreased cell growth. Cells pretreated with dexamethasone for 48 h released greater amounts of NT in response to Ca²⁺ (1.0 mM) with or without K⁺ (50 mM) or NE (10^?6 M) following a 10 min incubation with these substances in KRBG. This experimental paradigm was also used to measure the efflux of cAMP following a brief (10 min) exposure of cells to NE. We conclude that the rMTC 44-2B and 44-2C cells are useful tools for studying the effects of dexamethasone on the regulation of cell growth, as well as the secretion of NT and cAMP.     

Inhibitory and excitatory mechanisms of neurotensin action in canine intestinal circular muscle in vitro.

The effect of neurotensin on canine ileal circular muscle devoid of myenteric plexus was investigated using single and double sucrose gap techniques. Similar results were obtained with microelectrode techniques. Neurotensin caused a temperature-sensitive and dose-dependent biphasic response, an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase, usually consisting of spike discharge and tonic and phasic contractions, for which depolarization was not required. Neither response was affected by tetrodotoxin, phentolamine, propranolol, or atropine. The hyperpolarization was associated with decreased membrane resistance, blocked by 10(-7) M apamin, and converted to tonic depolarization by apamin (10(-6) M). Tachyphylaxis to neurotensin occurred when the stimulation interval was less than 20 min. After Ca2+ depletion, depolarization was observed instead of the hyperpolarization; this depolarization was not affected by nitrendipine and was gradually abolished with repetitive stimulation at 20-min intervals. When Ca2+ was present, nifedipine did not alter the hyperpolarizing phase of the response but inhibited spiking and blocked all contractions. The excitatory phase of the response was enhanced by Bay K-8644. Neuromedin N elicited a response identical with that of neurotensin. The responses of the two peptides were completely cross tachyphylactic. Inhibitory junction potentials were not affected by neurotensin tachyphylaxis. It is concluded that neurotensin and neuromedin N activate apamin-sensitive, calcium-dependent potassium channels in circular muscle, causing membrane hyperpolarization and inhibition of muscle contraction. Release of intracellular calcium is involved in the activation of these potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

Inhibition of germinant binding by bacterial spores in acidic environments.

Commitment to germinate occurred in both Clostridium botulinum and Bacillus cereus spores during 0.5 min of exposure to 100 mM L-alanine or L-cysteine, measured by the inability of germination inhibitors (D form of amino acid) to inhibit germination. Spore germination at pH 4.5 was inhibited because the germinant did not bind to the trigger sites. C. botulinum spores exposed to 100 mM L-alanine or L-cysteine at pH 4.5 remained sensitive to D-amino acid inhibition at pH 7, indicating that no germinants had bound to the trigger site at pH 4.5. Inhibition of germinant binding at pH 4.5 was reversible but lagged in commitment to germinate upon transfer to pH 7. Spores sequentially exposed to pH 4.5 buffer and pH 7 buffer with the germinant also demonstrated a lag in commitment to germinate. The pH at which binding was inhibited was not significantly affected by composition of the buffer or by reduced germinant concentrations (10 mM). Nonspecific uptake of L-[3H]alanine by C. botulinum spores was not inhibited at pH 4.5. Inhibition of germinant binding in acidic environments appeared to be due to protonation of a functional group in or near the trigger site. This may represent a general mechanism for inhibition of spore germination in acidic environments.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic and nucleotide requirements for microtubule polymerization in vitro.

The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, withmaximal assembly occurring at physiological concentrations of 150 mM. Maximal extent of assembly occurred at pH 6.8 and optimal rate at pH 6.6. Inhibition of polymerization was half-maximal at added calcium concentrations of 1.0 mM and magnesium concentrations of 10.0 mM. EGTA (ethylene glycol bis(beta-aminoethyl ether)tetraacetic acid), which chelates Ca-2+, had no effect on polymerization over a concentration range of 0.01-10.0 mM. In contrast, EDTA (ethylenediaminetetraacetic acid), which chelates both Mg-2+ and Ca-2+, inhibited assemble half-maximally at 0.25 mM and totally at 2.0 mM. As determined from experiments using Mg-2+-EDTA buffers, magnesium was required for polymerization. Magnesium promoted the maximal extent of assembly at substoichiometric levels relative to tubulin, but was maximal for both rate and extent at stoichiometric concentrations. Elemental analyses indicated that approximately 1 mol of magnesium was tightly bound/mol of tubulin dimer. Viscosity development was dependent upon hydrolyzable nucleoside triphosphate, and stoichiometric levels of GTP were sufficient for maximal polymerization. The effect of magnesium in increasing the rate of GTP-dependent polymerization suggests that a Mg-2+-GTP complex is the substrate required for a step in assembly.     

Peripheral inactivation of neurotensin. Isolation and characterization of a metallopeptidase from rat ileum

A peptidase that inactivated neurotensin by cleaving the peptide at the Pro10-Tyr11 bond, generating the biologically inactive fragments neurotensin(1-10) and neurotensin(11-13) was purified from whole rat ileum homogenate. The purified enzyme behaved as a 70-75-kDa monomer as determined by SDS-PAGE analysis in reducing or non-reducing conditions and gel permeation on Ultrogel AcA34. The peptidase was insensitive to thiol-blocking agents and acidic and serine protease inhibitors but could be strongly inhibited by 1,10-phenanthroline, EDTA, dithiothreitol and heavy metal ions such as zinc, copper and cobalt. Zinc was the only divalent cation able potently to reactivate the apoenzyme. This enzyme could be distinguished from endopeptidases EC 3.4.24.15 and EC 3.4.24.11, angiotensin-converting enzyme, proline endopeptidase, aminopeptidase and pyroglutamyl-peptide hydrolase since it was not affected by micromolar concentrations of their specific inhibitors. The peptidase displayed a high affinity for neurotensin (1.6 microM). Studies concerning the specificity of the enzyme towards the sequence of neurotensin established the following. (a) Neurotensin(9-13) was the shortest partial sequence that fully inhibited tritiated neurotensin degradation; shortening the C-terminal part of the neurotensin molecule led to inactive fragments. (b) Amidation of the C-terminal end of the peptide did not prevent the recognition by the peptidase. (c) There existed a strong stereospecificity of the peptidase for the residues in positions 8, 9 and 11 of the neurotensin molecule. (d) Pro-Xaa dipeptides (where Xaa represented aromatic or hydrophobic residues) were the most potent inhibitors of tritiated neurotensin degradation while all the Xaa-Pro dipeptides tested were totally ineffective. (e) The neurotensin-related peptides: neuromedin N, xenopsin and [Lys8-Asn9]neurotensin(8-13), as well as angiotensins I and II and dynorphins(1-8) and (1-13) were as potent as neurotensin in inhibiting [3H]neurotensin hydrolysis.     

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used.

The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 microM) potentiated the response to carbachol at 6 mM K+ when Ca2(+)-free, but not when 2.52 mM Ca2+ assay buffer was used. In Ca2(+)-free buffer, verapamil (10 microM) inhibited the response to carbachol at both 6 and 18 mM K+ but higher concentrations (30-300 microM) were needed when 2.52 mM Ca2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 microM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K+ at 1.3 mM Ca2+, but was without effect on the basal rate at other K+ and Ca2+ concentrations. In the presence of NMDA (100 microM) or quisqualate (100 microM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K+ and Ca2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.     

Terbium binding to rat brain acetylcholinesterase. A fluorescence probe of anionic sites

Studies are in progress to characterize the nature of ligand interactions at peripheral anionic sites on mammalian brain AChE, including the beta-anionic or "accelerator" anionic sites where enzyme activity is increased upon Ca2+ binding. Terbium was studied as a fluorescence probe of Ca2+ binding sites in partially purified AChE from whole rat brain. Scatchard analysis of Tb3+ binding in low ionic strength (2 mM) Pipes buffer revealed at least two populations of sites: high affinity sites with Kd(app) approximately 7.6 microM and low-affinity sites with a Kd(app) approximately 49.6 microM. Low-affinity binding was selectively inhibited by 50 mM NaCl; high-affinity binding was completely inhibited by 2 mM CaCl2; and all the bound Tb3+ could be displaced by 1 mM EDTA. The heterogeneity of Tb3+ binding sites is consistent with the multiple, concentration-dependent effects of Tb3+ on enzyme activity.     

Characterization and visualization of neurotensin binding to receptor sites in human brain

The binding of monoiodo [125I-Tyr3]-neurotensin to human brain was characterized and visualized using radioreceptorassay and autoradiographic techniques. Specific binding to homogenates of human substantia nigra at 25 degrees C was maximal at 20 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two populations of binding sites with Kd values of 0.26 nM and 4.3 nM. Corresponding binding capacities were 26 and 89 fmol/mg of protein. Neurotensin analogs inhibited the binding of iodinated neurotensin with relative potencies that demonstrated the crucial role of the C-terminal hexapeptide portion of neurotensin for binding to its receptors. Autoradiography of human substantia nigra sections incubated with iodinated neurotensin revealed high levels of specific binding in the nucleus paranigralis and substantia nigra, pars compacta, and low levels in the substantia nigra, pars reticulata.