Biomedical subjects

J E Maggio

Publications and source records attributed to J E Maggio.

At least 55 records · Page 3Linked to original sources

PubMed1994

Agonist-induced photoincorporation of a p-benzoylphenylalanine derivative of substance P into membrane-spanning region 2 of the Torpedo nicotinic acetylcholine receptor delta subunit.

The neuropeptide substance P acts, at micromolar concentrations, as a noncompetitive antagonist of nicotinic acetylcholine receptors (AChRs) of both neuronal and muscle subtypes. The mechanism of this inhibition has been shown to be most consistent with stabilization of a nonconducting desensitized state of the AChR, via binding to a site distinct from both the agonist site and the high affinity noncompetitive antagonist site. We have used a radioiodinated photoreactive analogue of substance P, containing the amino acid p-benzoyl-L-phenylalanine in place of the Phe8 residue of substance P, to identify the sites of interaction of substance P within the Torpedo california AChR. AChR-rich membrane suspensions were photolabeled in the absence or presence of the agonist carbamylcholine and/or nonradioactive substance P, and incorporation into AChR subunits was assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of agonist 125I incorporation was detected in each subunit and was insensitive to substance P, whereas in the presence of carbamylcholine there was a 2-fold increase in photoincorporation into the AChR delta subunit that was inhibited by the addition of an excess of substance P. The sites of specific photoincorporation in the delta subunit were initially mapped by use of Staphylococcus aureus V8 protease to a 14-kDa fragment extending from delta Ile-192 to Glu-280. Further fragmentation of this 14-kDa fragment with trypsin and S. aureus V8 protease established that the sites of specific incorporation were restricted to the region delta Ser-253 to Glu-280, which contains the membrane-spanning region 2 that is known to form the lining of the ion channel. These results establish that in the presence of agonist at least a part of the undecapeptide substance P binds within the ion channel in the desensitized state of the AChR, and it is likely that the binding of substance P to this site is responsible for the action of substance P as a noncompetitive AChR antagonist.

Affinity Labels↗

PubMed1993

Neuropeptide Y/peptide YY receptor binding sites in the heart: localization and pharmacological characterization.

[125I]Peptide YY was used to localize and characterize peptide YY and neuropeptide Y receptor binding sites in the heart. In the rat and rabbit heart, nearly every artery and arteriole that could be histologically identified also expressed saturable binding sites for [125I]peptide YY. In the arteries, these [125I]peptide YY binding sites were primarily associated with the smooth muscle layer. Pharmacological experiments demonstrated that peptide YY and neuropeptide Y were equipotent in competing for [125I]peptide YY binding in the heart. In another competition series, [Leu31,Pro34]-neuropeptide Y (a Y1 receptor-specific agonist when used with [125I]peptide YY) was significantly more potent than neuropeptide Y (a Y2 receptor-specific agonist when used with [125I]peptide YY) in competing for [125I]peptide YY binding from coronary arteries, suggesting that the receptor binding sites on cardiac arteries and arterioles are of the Y1 subtype. These results demonstrate that smooth muscle cells of the atrial and ventricular arteries and arterioles in rat and rabbit heart express Y1 receptors and suggest a possible direct effect of neuropeptide Y on coronary blood vessels to induce vasoconstriction.

Animals↗

PubMed1993

Aluminum, iron, and zinc ions promote aggregation of physiological concentrations of beta-amyloid peptide.

A major pathological feature of Alzheimer's disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human beta-amyloid peptide beta A4, a 40-mer whose neurotoxicity is related to its aggregation. Certain metals have been proposed as risk factors for AD, but the mechanism by which the metals may exert their effects is unclear. Radioiodinated human beta A4 has been used to assess the effects of various metals on the aggregation of the peptide in dilute solution (10(-10) M). In physiological buffers, 10(-3) M calcium, cobalt, copper, manganese, magnesium, sodium, or potassium had no effect on the rate of beta A4 aggregation. In sharp contrast, aluminum, iron, and zinc under the same conditions strongly promoted aggregation (rate enhancement of 100-1,000-fold). The aggregation of beta A4 induced by aluminum and iron is distinguishable from that induced by zinc in terms of rate, extent, pH and temperature dependence. These results suggest that high concentrations of certain metals may play a role in the pathogenesis of AD by promoting aggregation of beta A4.

Aluminum↗

PubMed1993

Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion.

We have previously demonstrated that the venom of the scorpion Leiurus quinquestriatus blocks small-conductance Cl- channels, derived from epithelial cells, when applied to the cytoplasmic surface. We have now purified to near homogeneity, and characterized, the component responsible for this blocking activity. It is a small basic peptide of 4,070 Da. The primary amino acid structure shows considerable homology to a class of previously described putative short insectotoxins. A brief characterization of the kinetics of Cl- channel block as well as a demonstration of toxicity to arthropods is also presented.

Amino Acid Sequence↗

PubMed1992

Reversible in vitro growth of Alzheimer disease beta-amyloid plaques by deposition of labeled amyloid peptide.

The salient pathological feature of Alzheimer disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of victims. The plaques are predominantly composed of human beta-amyloid peptide (beta A4), a 40-mer whose neurotoxicity is related to its aggregation. Radioiodinated human beta A4 is rapidly deposited in vitro from a dilute (less than 10 pM) solution onto neuritic and diffuse plaques and cerebrovascular amyloid in AD brain tissue, whereas no deposition is detectable in tissue without performed plaques. This growth of plaques by deposition of radiolabeled beta A4 to plaques is reversible, with a dissociation half-time of approximately 1 h. The fraction of grey matter occupied by plaques that bind radiolabeled beta A4 in vitro is dramatically larger in AD cortex (23 +/- 11%) than in age-matched normal controls (less than 2%). In contrast to the human peptide, rat/mouse beta A4 (differing at three positions from human beta A4) does not affect the deposition of radiolabeled human beta A4. beta A4 has no detectable interaction with tachykinin receptors in rat or human brain. The use of radioiodinated beta A4 provides an in vitro system for the quantitative evaluation of agents or conditions that may inhibit or enhance the growth or dissolution of AD plaques. This reagent also provides an extremely sensitive method for visualizing various types of amyloid deposits and a means for characterizing and locating sites of amyloid peptide binding to cells and tissues.

Aged↗

PubMed1992

Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia.

Sympathetic ganglia are innervated by neuropeptide-containing fibers originating from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and in some cases, myenteric neurons. In the present report receptor autoradiography was used to determine whether sympathetic ganglia express receptor binding sites for several of these neuropeptides including bombesin, calcitonin gene-related peptide-alpha, cholecystokinin, galanin, neurokinin A, somatostatin, substance P, and vasoactive intestinal polypeptide. The sympathetic ganglia examined included the rat and rabbit superior cervical ganglia and the rabbit superior mesenteric ganglion. High levels of receptor binding sites for cholecystokinin, galanin, somatostatin, substance P, and vasoactive intestinal polypeptide were observed in all sympathetic ganglia examined, although only discrete neuronal populations within each ganglion appeared to express receptor binding sites for any particular neuropeptide. These data suggest that discrete populations of postganglionic sympathetic neurons may be regulated by neuropeptides released from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and myenteric neurons.

Animals↗

PubMed1991

Alterations in receptors for sensory neuropeptides in human inflammatory bowel disease.

Glutamate and several neuropeptides are synthesized and released by subpopulations of primary afferent neurons. These sensory neurons play a role in regulating the inflammatory and immune responses in peripheral tissues. We have explored what changes occur in the location and concentration of receptor binding sites for sensory neurotransmitters in two human inflammatory diseases, ulcerative colitis and Crohn's disease, using quantitative receptor autoradiography. The sensory neurotransmitter receptors included bombesin, calcitonin gene-related peptide-alpha, cholecystokinin, galanin, glutamate, somatostatin, neurokinin A (substance K), substance P, and vasoactive intestinal polypeptide. Of the nine receptor binding sites examined only binding sites for substance P and vasoactive intestinal peptide were significantly altered in the inflamed tissue. These data suggest that substance P is involved in regulating the inflammatory and immune responses in human inflammatory diseases and indicate a specificity of efferent action for each sensory neurotransmitter in peripheral tissues.

Adolescent↗

PubMed1990

Pharmacological analysis of [3H]-senktide binding to NK3 tachykinin receptors in guinea-pig ileum longitudinal muscle-myenteric plexus and cerebral cortex membranes.

1. The binding properties and pharmacological specificity of the selective NK3 tachykinin receptor agonist [3H))-senktide [( 3H]-succinyl[Asp6,MePhe8] substance P (6-11] have been examined in homogenates of guinea-pig ileum longitudinal muscle-myenteric plexus (LM/MP) and cerebral cortex. 2. Scatchard analysis of saturation binding studies in guinea-pig ileum LM/MP and cerebral cortex membranes indicated that [3H]-senktide bound to a single site with apparent high affinity, KD = 2.21 +/- 0.65 nM; Bmax = 13.49 +/- 0.04 fmol mg-1 protein in ileum and KD = 8.52 +/- 0.45 nM; Bmax = 76.3 +/- 1.6 fmol mg-1 protein in cortex (values are means +/- ranges; n = 2). 3. The pharmacological profile for tachykinins and analogues in displacing [3H]-senktide from ileum membranes was: [MePhe7] neurokinin B greater than neurokinin B (NKB) congruent to senktide greater than eledoisin greater than substance P (SP) greater than neurokinin A(NKA) greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than [Nle10]NKA(4-10) = [Glp6,L-Pro9]-SP(6-11) greater than substance P methyl ester, consistent with [3H]-senktide binding to an NK3 subtype of tachykinin receptor. A similar rank order of affinity was obtained for these peptides in displacing [3H]-senktide from cortex membranes. 4. Several tachykinin receptor agonists were tested for their ability to displace [3H]-senktide from ileal and cortical NK3 binding sites and were found to be either weak displacers (pIC50 less than 5.00) or inactive. 5. The binding of [3H]-senktide to cortex membranes was inhibited by GTP (p1C,0 = 6.49)and GTP-gamma- S (p1C,0 = 6.67) with ATP being at least three orders of magnitude less potent (pIC50 = 3.55). 6. These results indicate that both central and peripheral NK3 receptors share a similar pharmacological specificity and that they may be labelled selectively with the NK3 receptor agonist [3H]-senktide.

Animals↗

PubMed1989

Substance P receptor binding sites are expressed by glia in vivo after neuronal injury.

In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, we examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.

Animals↗

PubMed1989

Substance P receptors on canine chief cells: localization, characterization, and function.

Saturable binding sites for 125I-Bolton-Hunter substance P were observed in frozen sections of the oxyntic mucosa of the canine stomach using quantitative autoradiography. The cell type possessing substance P binding sites in this region was identified as the chief cell in 2 ways. First, the saturable binding of radioiodinated substance P correlated with chief cell content (and not with parietal cell content, for example) in dispersed oxyntic mucosal cells fractionated by centrifugal elutriation. Second, saturable binding of radioiodinated substance P was localized to dispersed chief cells by autoradiography using emulsion-coated preparations of isolated cells affixed to glass slides. Parietal and mucous cells did not bind substance P. In studies of enriched chief cell preparations, the binding of radiolabeled substance P was found to be time- and cell number-dependent, specific, saturable, reversible, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent Kd of 105 pM and a Bmax of 3000 receptors per cell. In competitive displacement studies, the order of potency of analogs for inhibition of the saturable binding of radiolabeled substance P to chief cells was substance P = physalaemin greater than substance K greater than neuromedin K; thus, the chief cell has a substance P-preferring tachykinin binding site. Bombesin, cholecystokinin, and somatostatin had no effect on substance P binding. Substance P stimulated pepsinogen secretion from isolated canine oxyntic glands in dose-dependent fashion with a half-maximal response occurring at a substance P dose of about 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Autoradiographic localization and characterization of tachykinin receptor binding sites in the rat brain and peripheral tissues.

Quantitative receptor autoradiography using several radiolabeled tachykinins was used to localize and characterize tachykinin peptide receptor binding sites in rat CNS and peripheral tissues. Autoradiographic localization and displacement experiments using several radiolabeled tachykinins indicate that in the rat there are at least 3 distinct tachykinin receptor binding sites. One of these is present in both the CNS and peripheral tissues, one is present only in the CNS, and one is present only in peripheral tissues. The first tachykinin receptor binding site, which is detectable in both the CNS and peripheral tissues, appears to prefer substance P (SP) as an endogenous ligand. Areas expressing high concentrations of this binding site include the medial septum, superior colliculus, inferior olive, inner plexiform layer of the retina, external muscle of the bladder, and the muscularis externa of the esophagus. The second type of tachykinin receptor binding site, which is detectable only in the CNS appears to prefer either neuromedin K (NK) and/or substance K (SK) as the endogenous ligand. This receptor binding site is labeled by Bolton-Hunter conjugates of NK, SK, eledoisin, or kassinin and is found in high concentrations in laminae 4 and 5 of the cerebral cortex, the ventral tegmental area, laminae 1 and 2 of the spinal cord, and the inner plexiform layer of the retina. The third type of tachykinin receptor binding site is detectable only in peripheral tissues and appears to prefer SK as the endogenous ligand. This receptor binding site is labeled by SK, eledoisin, or kassinin radioligands and tissues that express high concentrations include the muscularis mucosae of the esophagus, the circular muscle of the colon, and the external muscle of the bladder. These data suggest that SP receptors are expressed in the brain and peripheral tissues, NK receptors are expressed in the CNS, and SK receptors are expressed in peripheral tissue. These data fit well with radioimmunoassay data that suggest that, whereas in the CNS SP, SK and NK are present in high concentrations, in peripheral tissues only SP and SK are present in detectable concentrations. The present classification of tachykinin receptors places a lower limit on the number of mammalian tachykinin receptor types and provides a functional/morphological framework for exploring the diverse actions of tachykinin peptides in both the CNS and peripheral tissues.

Animals↗

PubMed1988

Receptor binding sites for substance P and substance K in the canine gastrointestinal tract and their possible role in inflammatory bowel disease.

The mammalian tachykinins, substance P, substance K (neurokinin A) and neuromedin K (neurokinin B), are putative peptide neurotransmitters in both the brain and peripheral tissues. We used quantitative receptor autoradiography to localize and quantify the distribution of binding sites for radiolabeled substance P, substance K and neuromedin K in the canine gastrointestinal tract. Substance P binding sites were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, neurons in the myenteric plexus, mucosal epithelial cells, exocrine cells and lymph nodules. Substance K binding sites were distributed in a pattern distinct from substance P binding sites and were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, and neurons of the myenteric plexus. Neuromedin K binding sites were not observed in any area of the canine gastrointestinal tract although they were localized with high specific/non-specific binding ratios in the canine spinal cord. These results indicate that there are at least two distinct types of tachykinin receptor binding sites in the canine gastrointestinal tract, one of which probably recognizes substance P and the other substance K as endogenous ligands. In correlation with previous physiological data, these substance P and substance K receptor binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including gastric motility, mucosal ion transport, hemodynamics, digestive enzyme secretion and neuronal excitability. In addition these results demonstrate that receptor binding sites for substance P and substance K are expressed by cells involved in mediating inflammatory and immune responses. These data, together with our studies on surgical specimens from patients with inflammatory bowel disease, suggest that in a pathophysiological state tachykinins and their receptors may play a role in inflammatory bowel disease and should permit a rational approach to designing neuropeptide antagonists which may prove effective in treating inflammatory diseases.

Animals↗

PubMed1988

Receptor binding sites for substance P, but not substance K or neuromedin K, are expressed in high concentrations by arterioles, venules, and lymph nodules in surgical specimens obtained from patients with ulcerative colitis and Crohn disease.

Several lines of evidence indicate that tachykinin neuropeptides [substance P (SP), substance K (SK), and neuromedin K (NK)] play a role in regulating the inflammatory and immune responses. To test this hypothesis in a human inflammatory disease, quantitative receptor autoradiography was used to examine possible abnormalities in tachykinin binding sites in surgical specimens from patients with inflammatory bowel disease. Surgical specimens of colon were obtained from patients with ulcerative colitis (n = 4) and Crohn disease (n = 4). Normal tissue was obtained from uninvolved areas of extensive resections for carcinoma (n = 6). In all cases, specimens were obtained less than 5 min after removal to minimize influences associated with degradation artifacts and were processed for quantitative receptor autoradiography by using 125I-labeled Bolton-Hunter conjugates of NK, SK, and SP. In the normal colon a low concentration of SP receptor binding sites is expressed by submucosal arterioles and venules and a moderate concentration is expressed by the external circular muscle, whereas SK receptor binding sites are expressed in low concentrations by the external circular and longitudinal muscle. In contrast, specific NK binding sites were not observed in any area of the human colon. In colon tissue obtained from ulcerative colitis and Crohn disease patients, however, very high concentrations of SP receptor binding sites are expressed by arterioles and venules located in the submucosa, muscularis mucosa, external circular muscle, external longitudinal muscle, and serosa. In addition, very high concentrations of SP receptor binding sites are expressed within the germinal center of lymph nodules, whereas the concentrations of SP and SK binding sites expressed by the external muscle layers are not altered significantly. These results demonstrate that receptor binding sites for SP, but not SK or NK, are ectopically expressed in high concentrations (1000-2000 times normal) by cells involved in mediating inflammatory and immune responses. These data suggest that SP may be involved in the pathophysiology of inflammatory bowel disease and might provide some insight into the interaction between the nervous system and the regulation of inflammation and the immune response in human inflammatory disease.

Autoradiography↗

PubMed1988

Tachykinins.

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Animals↗

PubMed1988

A study of tachykinin-immunoreactive neurons in monkey cerebral cortex.

Immunocytochemical methods were used to localize tachykinin-like immunoreactivity within neurons of the monkey cerebral cortex. Three primary antibodies were used: polyclonal antisera raised against fragments of substance P and substance K that excluded the carboxyl termini of these peptides, and a monoclonal antibody that recognized the carboxyl terminus of the tachykinin family. Each antibody stained 2 populations of cortical nonpyramidal neurons: (1) A small number of large, intensely stained cells that give rise to long, coarsely beaded processes; (2) a relatively large number of small, lightly stained cells that are embedded in dense plexuses of stained punctate profiles. The large, dark cells are present in a superficial band that includes layers II and III, and in a deep band that includes layer VI and the subjacent white matter. The smaller, pale cells are present in the middle layers of cortex (layers IV and/or V). Colocalization studies indicate that virtually all the small tachykinin-immunoreactive neurons also display GABA immunoreactivity. The larger cells are not GABA-positive, but display both somatostatin-like and neuropeptide Y-like immunoreactivity. The immunocytochemically stained beaded processes and punctate profiles from plexuses that vary in density and laminar distribution among different areas of monkey cortex. The coarsely beaded processes form a basic quadrilaminar pattern, with relatively dense plexuses in layers I and VI and in 2 middle layers, usually III and V. However, this pattern varies considerably from area to area. Electron microscopically, the large cells contain a rich collection of cytoplasmic organelles, particularly Golgi complex, while the small cells contain relatively few organelles. Both types of cells, including large neurons in the white matter, receive symmetric and asymmetric synaptic contacts on their somata and proximal dendrites. The numbers of these axosomatic contacts are low. Virtually all synaptic contacts formed by immunoreactive terminals possess symmetric membrane thickenings. In 2 areas examined in detail (areas 2 and 4), pyramidal cell somata and dendrites are the major targets of the immunoreactive synaptic terminals.

Animals↗

PubMed1986

Mild footshock stress dissociates substance P from substance K and dynorphin from Met- and Leu-enkephalin.

Mild footshock stress selectively activates ventral tegmental area dopamine neurons innervating the prefrontal cortex. The same stressor rapidly dissociates ventral tegmental substance P from its preprotachykinin-derived co-transmitter substance K, and preproenkephalin B-derived dynorphin B from preproenkephalin A-derived Met-enkephalin-Arg-Gly-Leu and Leu-enkephalin. Mild footshock stress may provide a paradigm for studying both peptidergic modulation of brain dopaminergic neurons and the dynamic regulation of tachykinin and opioid peptide transcription, processing and utilization.

Animals↗

PubMed1986

Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis.

Skin secretions from the South African frog Xenopus laevis have been chromatographed by high performance liquid chromatography (HPLC), fractionated, and analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The HPLC chromatograms showed the secretion to be a complex mixture with over 30 components at similar levels to the four peptides previously isolated from X. laevis skin, i.e. xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa. FAB-MS analysis of the HPLC fractions gave numerous protonated molecular ions ranging from m/z 491 to 2662. Preliminary assignments of these components were made by comparing these experimental molecular weights to those predicted for regions within the xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa precursors. These results suggested that many of these skin secretions were peptides originating from additional processing of the xenopsin, caerulein, and PGLa precursors, primarily involving cleavage at single arginine residues, and a novel cleavage at the NH2-terminal side of single lysines. These assignments were subsequently confirmed by Edman degradation, FAB-MS peptide sequencing, and amino acid analysis. All of these peptides contain one or more lysines and would be expected to have amphiphilic structures. As yet, nothing is known about their activity, although they resemble in composition the mast cell degranulating peptides melittin and the bombolitins. These precursor fragments were also found to have limited sequence homology to bombinin, a hemolytic amphibian peptide isolated from the European Bombina toad.

Amino Acid Sequence↗

PubMed1986

The localization and characterization of substance P and substance K in striatonigral neurons.

Specific substance P and substance K radioimmunoassays coupled to high-performance liquid chromatography were used to characterize striatal and nigral tachykinin immunoreactivity. Using these assays, authentic substance P and substance K accounted for nearly all substance P and substance K immunoreactivity, respectively. A series of coronal knife cuts of the striatum caused parallel depletions in nigral substance P and substance K, consistent with the possible colocalization of these tachykinins in striatonigral neurons.

Animals↗