Localization of the peptide binding domain of the NK-1 tachykinin receptor using photoreactive analogues of substance P.
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Affinity Labels↗
Biomedical subjects
N D Boyd
Publications and source records attributed to N D Boyd.
Characterization of the substance P (NK-1) receptor in tunicamycin-treated transfected cells using a photoaffinity analogue of substance P.
Chinese hamster ovary cells expressing the N-glycosylated substance P (NK-1) receptor were treated with the glycosylation inhibitor tunicamycin and photolabeled with 125I-Bolton-Hunter-p-benzoyl-L-phenylalanine8-substance P. Two radioactive proteins of M(r) 80,000 and 46,000, representing the glycosylated and nonglycosylated substance P (NK-1) receptor, respectively, were observed. The IC50 for the inhibition of photolabeling of both receptor forms was 0.3 +/- 0.1 nM for substance P and 30 +/- 5 nM for neurokinin A (substance K). Thus, glycosylation of the substance P (NK-1) receptor has no detectable effect on the affinity of the substance P (NK-1) receptor for substance P or neurokinin A (substance K).
Animals↗
Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.
A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
Substance P receptor. Biochemical characterization and interactions with G proteins.
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Affinity Labels↗
Mercury from dental "silver" tooth fillings impairs sheep kidney function.
In humans Hg vapor is released from "silver" amalgam fillings that contain 50% Hg by weight. Previous studies show that when 12 such fillings are placed in sheep teeth, the kidneys will concentrate amalgam Hg at levels ranging from 5 to 10 micrograms Hg/g renal tissue 4-20 wk after placement. In the present study 12 occlusal fillings were placed in each of six adult female sheep under general anesthesia, using standard dental procedures. Glass ionomer occlusal fillings (12) were inserted in two control sheep. At several days before dental surgery, and at 30 and 60 days after placement of fillings, renal function was evaluated by plasma clearance of inulin and by plasma and urine electrolytes, urea, and proteins. An average plasma inulin clearance rate of 69.5 +/- 7.2 ml/min before amalgam placement was reduced to 32.3 +/- 8.1 ml/min by 30 days and remained low at 27.9 +/- 8.7 ml/min after 60 days. Inulin clearance did not change in controls. After amalgam placement urine concentration of albumin decreased from 93.0 +/- 20.5 to 30.1 +/- 15.3 mg/l and urine Na+ concentration increased steadily from 24.8 +/- 7.7 to 82.2 +/- 20.3 mmol/l at 60 days. Concentrations of K+, urea, gamma-glutamyl transpeptidase, alkaline phosphatase, and total protein did not change significantly from 0 to 60 days in urine. Plasma levels of Na+, K+, urea, and albumin remained unchanged from 0 to 60 days after amalgam. Renal histology remained normal in amalgam-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Guanine nucleotides decrease the affinity of substance P binding to its receptor.
The inhibitory effect of guanine nucleotides on the binding of 125I-Bolton-Hunter conjugated substance P (125I-BHSP) to rat submaxillary gland membranes has been confirmed and further explored. The evidence presented here indicates that this is due to a marked loss of binding affinity. In the presence of 5'-guanylyl imidodiphosphate (GppNHp) there is (1) a greater than or equal to 20-fold increase in the Kd as determined by Scatchard analysis and (2) the concentration of SP required to inhibit half of the 125I-BHSP binding (IC50) increased approximately 30-fold. Consistent with a marked decrease in affinity is an approximately 100-fold increase in the rate of dissociation of 125I-BHSP following addition of GppNHp. Complete restoration of high-affinity binding was achieved by removal of the guanine nucleotide.
Guanine Nucleotides↗
Isolation and identification of a polypeptide in the Hsp 70 family that binds substance P.
During the course of an attempt to purify the substance P (SP) receptor from horse salivary glands by substance P-affinity chromatography, a polypeptide of Mr = 78,000 was isolated. The first fifteen amino acid residues at the amino terminus were determined and, unexpectedly, were found to be identical with the amino terminus of a glucose-regulated protein (GRP) of the same molecular weight, a protein that has been identified as a member of the heat shock protein family. This finding raises the intriguing possibility that SP may interact in vivo with GRPs and other members of the heat shock protein family and play a role in modulating their biological activities.
Amino Acid Sequence↗
A guinea pig model of bovine pneumonic pasteurellosis.
The induction of pneumonic pasteurellosis in guinea pigs (Cavia porcellus) was examined. Specific pathogen free male guinea pigs were anesthetized and a tracheostomy performed to introduce 10(5), 10(4) or 10(3) Pasteurella haemolytica-A1 into the left principal bronchus. The surgical site was closed with tissue adhesive and staples and the animals were monitored for signs of respiratory tract infection. Within 24 hours after inoculation they became depressed, anorectic, pyretic and dyspneic. Fibrinous pleuropneumonia with prominent areas of necrosis and hemorrhage was present. Pericardial effusion was a frequent finding. There was infiltration of the pleura and alveoli with degenerate heterophils and macrophages, a hyperplastic mesothelium and fibrin exudation on the pleura and within alveoli. Hemorrhage, congestion, consolidation, edema and fibrin exudation were prominent in the hilar region of the lungs. Bacterial colonies were evident in all airways. More bacteria were recovered from infected lungs than were inoculated (p less than 0.05) indicating P. haemolytica was actively multiplying in the lungs. Hematological and clinical chemistry data were consistent with fibrinous pneumonia, however, blood cultures were positive for P. haemolytica in 61% (11/18) of animals sampled. Examination of pneumonic pasteurellosis in guinea pigs may be useful in studying pathogenetic and pathological features applicable to bovine pneumonic pasteurellosis (shipping fever pneumonia).
Animals↗
Serum immunoglobulin E levels in patients with primary biliary cirrhosis.
Serum IgE levels were measured in 22 symptomatic adults with primary biliary cirrhosis (PBC) and 19 asymptomatic female adults with positive serum mitochondrial antibody tests. Control populations included 45 adult patients with chronic liver disease consisting of autoimmune chronic active hepatitis (N = 15), alcoholic liver disease (N = 15), or miscellaneous cholestatic liver disorders (N = 15), and 87 healthy adult hospital personnel. Fourteen of 22 (64%) patients with PBC had low (less than 10 KU/L) or undetectable serum IgE levels, compared to 12 of 45 (27%) control patients with liver disease (p less than 0.005) and 39 of 87 (45%) healthy control subjects (p = 0.09). Low serum IgE levels were also found in most (13 of 19 patients, 68%) asymptomatic individuals with positive serum mitochondrial antibody tests (p less than 0.005 and p less than 0.05 versus liver disease and healthy control subjects, respectively). In vitro IgE production by peripheral blood mononuclear cells after pokeweed mitogen stimulation was determined in eight patients with PBC and in eight healthy control subjects. After pokeweed mitogen stimulation, one of three, patients with PBC compared to four of four healthy control subjects with detectable levels of serum IgE, produced sufficient amounts of IgE to be detected in culture supernatants. Neither the five patients with PBC nor the four healthy control subjects with undetectable serum IgE levels produced sufficient amounts of IgE in vitro to be detected in our assay system.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Regulation of substance P receptor affinity by guanine nucleotide-binding proteins.
The binding of substance P (SP) to receptors in peripheral tissues as well as in the CNS is subject to regulation by guanine nucleotides. In this report, we provide direct evidence that this effect is mediated by a guanine nucleotide-binding regulatory protein (G-protein) that is required for high-affinity binding of SP to its receptor. Rat submaxillary gland membranes bind a conjugate of SP and 125I-labeled Bolton-Hunter reagent (125I-BHSP) with high affinity (KD = 1.2 +/- 0.4 X 10(-9) M) and sensitivity to guanine nucleotide inhibition. Treatment of the membranes with alkaline buffer (pH 11.5) causes a loss of the high-affinity, GTP-sensitive binding of 125I-BHSP and a parallel loss of [35S]guanosine 5'-(3-O-thio)triphosphate ([35S]GTP gamma S) binding activity. Addition of purified G-proteins from bovine brain to the alkaline-treated membranes restores high-affinity 125I-BHSP binding. Reconstitution is maximal when the G-proteins are incorporated into the alkaline-treated membranes at a 30-fold stoichiometric excess of GTP gamma S binding sites over SP binding sites. Both Go (a pertussis toxin-sensitive G-protein having a 39,000-dalton alpha-subunit) and Gi (the G-protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated alpha-subunit of Go is without effect. The effects of added G-proteins are specifically reversed by guanine nucleotides over the same range of nucleotide concentrations that decreases high-affinity binding of 125I-BHSP to native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkalies↗
Dual-precipitation method evaluated for determination of high-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations.
We evaluated a dual-precipitation method for determining cholesterol in high-density lipoprotein (HDL) and its subfractions HDL2 and HDL3. After total HDL was isolated by precipitation of very-low-density (VLDL) and low-density (LDL) lipoproteins with polyethylene glycol (Mr 8000), HDL2 was isolated from total HDL by precipitation with dextran sulfate (Mr 15,000), leaving HDL3 in the supernate. Concentration of total HDL cholesterol after precipitation of VLDL and LDL with PEG showed significant proportional and constant biases of -3.8% and 0.04 mmol/L, respectively, when compared with a phosphotungstic acid-based comparison method, although results by the two methods were correlated highly (r = 0.99, P less than 0.001). HDL2 and HDL3 cholesterol concentrations measured with the present technique were not different from those obtained by density-gradient ultracentrifugation or by combined precipitation-ultracentrifugation.
Centrifugation, Density Gradient↗
Two distinct kinetic phases of desensitization of acetylcholine receptors of clonal rat PC12 cells.
1. The desensitization of nicotinic acetylcholine receptors on the PC12 sympathetic cell line was investigated by using a 22Na+ influx assay to measure receptor activation. 2. The rate of desensitization was dependent on temperature and at 4 degrees C two distinct kinetic phases were readily discernible: a rapid phase that was characterized by rate constants that were dependent on the chemical nature and concentration of the agonist, and a slower phase that was characterized by rate constants that were less dependent on these. 3. For acetylcholine, carbamylcholine and l-nicotine, the equilibrium desensitization parameter, Kdes, the concentration that produces half-maximal desensitization, was determined and compared with the corresponding value for Kact, the concentration that results in a half-maximal increase in the permeability response. For each agonist, the value of Kdes was found to be lower than Kact, a result to be expected if desensitization is associated with a higher-affinity state of the receptor than that associated with ion channel activation. Thus, extensive receptor desensitization can occur even at agonist concentrations that do not produce appreciable channel activation. Both activation and desensitization functions exhibited positive cooperativity so that each function occurs over a narrow range of agonist concentrations. 4. Following removal of the agonist, recovery from desensitization was reversible and occurred by two distinct kinetic phases characterized by rate constants that were independent of the chemical nature and concentration of the agonist that produced the desensitization. The relative contribution of each kinetic phase of recovery was, however, dependent on the duration of prior exposure to agonist. Following short incubation periods with agonist, most of the receptors were in a rapidly recovering state. With increasing duration of exposure, progressively more of the receptors were converted to a desensitized state that recovered more slowly. 5. The rate constants associated with the two kinetic phases of recovery were dependent on the recovery temperature. Following the initial rapid phase of desensitization, recovery at 4 degrees C was characterized by a time constant, t1/2, of 1.9 min, a value that was about 3-fold greater than that observed at 22 degrees C. The rate of recovery of the desensitized state achieved following equilibrium exposures to agonists was considerably more temperature dependent: recovery of this desensitized state was characterized at 4 degrees C by a t1/2 of 62 min that was about 37-fold greater than that at 22 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Multiple actions of substance P that regulate the functional properties of acetylcholine receptors of clonal rat PC12 cells.
1. The effects of substance P (SP) on each of the kinetic components of reversible desensitization (measured at 4 degrees C) and also on irreversible deactivation (measured at 22 degrees C) of the nicotinic acetylcholine receptor on PC12 cells were examined by 22Na+ influx measurements of the functional state of the receptor. 2. In the absence of agonists, SP converts the acetylcholine receptors in a time- and concentration-dependent manner, to a state that is not responsive to agonist. Upon removal of the peptide, this effect was reversible and the kinetics of the recovery of the permeability response were analysed to provide further characterization of the non-responsive state. Following exposure of cells to SP (10 microM) for 3 or more min, recovery was by a first-order process (time constant, t1/2 = 2.1 min), the same value, within experimental error, as that observed for recovery measured after the initial rapid phase of agonist-mediated desensitization. 3. In the presence of agonist, SP caused a strong enhancement of both the rate and extent of agonist-mediated desensitization. This effect was observed even at concentrations of peptide which produced only a small extent of desensitization when incubated alone. For 500 microM-carbamylcholine, the equilibrium level of desensitization (approximately 85% loss of the permeability response) was achieved at 4 degrees C in about 20 min by a biphasic process, while in the presence of 1.0 microM-SP, complete (100%) desensitization occurred by a single rapid exponential phase characterized by a t1/2 of 20 s. 4. The concentration of carbamylcholine required to produce half-maximal desensitization at equilibrium, Kdes, was 94 microM and was reduced by 6-fold in the presence of 0.3 microM-SP. 5. A mechanistic model is presented in which the receptor is viewed as existing in a dynamic conformational equilibrium between an activatable state Rc and the initial desensitized state Rd. It is proposed that SP binds preferentially to the Rd state and thus can allosterically (1) stabilize the receptor in the absence of agonist in that state, and (2) enhance, in an even lower concentration range, both the rate and extent of agonist-mediated stabilization of the receptor in the Rd state. 6. The second, slower component of agonist-mediated desensitization is, in contrast, inhibited by SP. This desensitization step appears to involve a covalent modification of the initial desensitized state (Rd) and is dependent on Ca2+. SP may exert this inhibitory effect by limiting the access of Ca2+ to an intracellular site of action.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Effect of cationic proteins on the glomerular deposition of anionic proteins and immune complexes.
When 3 mg of cationized human IgG (cIgG, pI greater than 9) was injected intravenously into mice and followed 15 min later by 3 mg anionized bovine serum albumin (aBSA, pI = 4), deposits were detected along the capillary loops and in the mesangium. Additional infusion of immunoaffinity-purified rabbit anti-BSA antibodies 1 h later led to their deposition at the same location. Intravenous injection of an equivalent amount of preformed BSA-anti-BSA or aBSA-anti-BSA immune complexes into mice with glomerular planted cIgG gave rise to few deposits. Similarly, the perfusion of preformed aBSA-anti-BSA complexes in isolated rat kidneys with planted cIgG led to granular deposition of both, but the intensity of staining was less than when sequential perfusions were used. Cationic macromolecules bound to the glomerulus may lead to the deposition of anionic macromolecules and anionic immune complexes.
Animals↗
Location of a polypeptide sequence within the alpha-subunit of the acetylcholine receptor containing the cholinergic binding site.
Proteolytic fragments of the alpha-subunit of the acetylcholine receptor of Torpedo electric organ were generated by digestion with Staphylococcus aureus V8 protease, and their ability to bind alpha-bungarotoxin was assessed following resolution on polyacrylamide gels and transfer to nitrocellulose. The position of the smallest fragment (Mr = 17,000) with toxin-binding activity was located within the primary sequence of the alpha-subunit by isolation and chemical characterization. The amino acid sequence at its amino terminus is Val-Asn-Gln-Ile-Val-Glu, which is identical to a unique sequence on the alpha-subunit beginning at Val 46. Based on considerations of the apparent molecular weight of this polypeptide fragment, the enzyme specificity of V8 protease, analysis of the partial amino acid composition, and the position of various identifying landmarks of the primary sequence of the alpha-subunit, the carboxy terminus is restricted to an acidic amino acid residue between Asp 152 and Asp 180. The apparent affinities of the 17-kDa polypeptide for alpha-bungarotoxin (IC50 = 100 nM) and d-tubocurarine (IC50 = 0.4 mM) were not significantly different from the values obtained for the undigested alpha-subunit, suggesting that the 17-kDa polypeptide contains all of the essential elements present on the alpha-subunit that are necessary for the binding of both of these cholinergic ligands. A second toxin-binding fragment (Mr = 19,000) was also identified. Although the amino terminus of this polypeptide fragment has not been determined directly, its position relative to Asn 141, the site of N-glycosylation, was established, and it appears that its amino terminus cannot be located any closer to the amino terminus of the alpha-subunit than Asp 152. If this tentative assignment is correct, then the region of the alpha-subunit involved in binding cholinergic ligands is more closely defined as occurring within the segment of the primary sequence of the alpha-subunit that the 17- and 19-kDa polypeptide fragments have in common, i.e., an overlapping sequence maximally bounded by Asp 152 and Asp 180.
Acetylcholine↗
Role of fibrinogen in the glomerular permeability of the perfused, isolated dog kidney.
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Animals↗
Desensitization of membrane-bound Torpedo acetylcholine receptor by amine noncompetitive antagonists and aliphatic alcohols: studies of [3H]acetylcholine binding and 22Na+ ion fluxes.
Measurements of the kinetics of binding of [3H]acetylcholine ([3H]AcCh) to membrane-bound nicotinic AcCh receptors from Torpedo electric tissue have been used to characterize the effects of a series of amine and alcohol noncompetitive antagonists on receptor conformational equilibria. The receptor exists in multiple, interconvertible conformations distinguished by agonist binding affinity. In the absence of cholinergic ligands, certain aromatic amines including proadifen, dimethisoquin, and lidocaine, as well as propanol and butanol, produce a dose-dependent increase in the fraction of receptors (f) in a high-affinity conformation from a value of fmax approximately 0.17 in the absence of drug to fmax approximately 0.9. Not all noncompetitive antagonists produce that same value of fmax. For histrionicotoxin (HTX), fmax approximately 0.3, and the aromatic amine adiphenine did not alter f while tetracaine actually decreased f to 0.1. The high-affinity receptor conformation stabilized by noncompetitive antagonists was characterized by (1) the rate constant (krec) for receptor reisomerization upon removal of stabilizing ligand and (2) the rate constant (kdis) for dissociation of [3H]AcCh-receptor complexes. On the basis of these criteria, the high-affinity receptor conformation stabilized by amine and alcohol noncompetitive blockers is the same as that stabilized by agonist. At 4 degrees C, krec = (2.2 +/- 0.2) X 10(-3) s-1 and kdis = 4 X 10(-2) s-1. Since HTX and adiphenine produced only a small conformational perturbation, their effects on the actions of proadifen and 2-propanol were examined. HTX and adiphenine antagonized the conformational perturbation caused by proadifen, while mixtures of HTX and 2-propanol produced additive effects. Effects of noncompetitive blockers were also assayed in terms of the inhibition of agonist-induced efflux of 22Na+ from Torpedo vesicles. Exposure to proadifen in the absence of agonist produced a reversible inhibition (desensitization) of the flux response, and recovery from desensitization occurred at the same rate as the reisomerization from the high-affinity receptor state.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholine↗
Stimulation of luteinizing hormone release after stereotaxic microinjection of neurotensin into the medial preoptic area of rats.
Neurons immunoreactive to luteinizing hormone-releasing hormone (LHRH) are localized to the medial preoptico-septal region of the rat hypothalamus, an area known to be essential for the regulation of the surge of luteinizing hormone (LH) that precedes ovulation. The effect of neurotensin (NT) on circulating LH levels following its discrete microinjection into this region was evaluated. Within 30 min after the stereotaxic microinjection of 40 ng of NT in 50 nl into the medial preoptic area (MPOA) just rostral to the organum vasculosum of the lamina terminalis in ovariectomized (OVX), anesthetized rats, plasma LH levels rose from 417 +/- 36 to 923 +/- 113 ng/ml (p less than 0.001). There was also a significant increase (p less than 0.001) in plasma LH levels following the microinjection of 50 ng of norepinephrine in 50 nl in this same site. Similarly, levels of LH increased from 48 +/- 7 to 110 +/- 19 ng/ml (p less than 0.004) following the microinjection of NT into the rostral MPOA on the morning of proestrus in anesthetized rats. In contrast, saline, substance P, leu-enkephalin and LHRH microinjected in the rostral MPOA of OVX rats had no significant effect on circulating levels of LH. The microinjection of NT 0.4 mm rostral or caudal to this area was also without effect. The presence of two binding sites with KdS of 4.2 and 29 nM and site concentrations of 0.11 and 0.68 pmol/mg in tissue homogenates of the rostral MPOA were also established. These data suggest a role for NT in the central regulation of LH release.
Animals↗