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Biomedical subjects

M W Walker

Publications and source records attributed to M W Walker.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cloning and functional expression of a human Y4 subtype receptor for pancreatic polypeptide, neuropeptide Y, and peptide YY.

The pancreatic polypeptide family includes pancreatic polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY). Members of the PP family regulate numerous physiological processes, including appetite, gastrointestinal transit, anxiety, and blood pressure. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype has been cloned previously. We now report the cloning of an additional Y-type receptor, designated Y4, by homology screening of a human placental genomic library with transmembrane (TM) probes derived from the rat Y1 gene. The Y4 genomic clone encodes a predicted protein of 375 amino acids that is most homologous to Y1 receptors from human, rat, and mouse (42% overall; 55% in TM). 125I-PYY binding to transiently expressed Y4 receptors was saturable (pKd = 9.89) and displaceable by human PP family derivatives: PP (pKi = 10.25) approximately PP2-36 (pKi = 10.06) > PYY (pKi = 9.06) approximately [Leu31,Pro34]NPY (pKi = 8.95) > NPY (pKi = 8.68) > PP13-36 (pKi = 7.13) > PP31-36 (pKi = 6.46) > PP31-36 free acid (pKi < 5). Human PP decreased [cAMP] and increased intracellular [Ca2+] in Y4-transfected LMTK- cells. Y4 mRNA was detected by reverse transcriptase-polymerase chain reaction in human brain, coronary artery, and ileum, suggesting potential roles for Y4 receptors in central nervous system, cardiovascular, and gastrointestinal function.

Amino Acid Sequence↗
PubMed1995

Nitric oxide-induced cytotoxicity: involvement of cellular resistance to oxidative stress and the role of glutathione in protection.

A series of experiments were designed to examine the potential cytotoxicity of nitric oxide (NO), or reactive species derived from NO, in HA1 fibroblasts and H2O2-resistant variants of this cell line, designated OC14 cells. A 1-h exposure at 37 degrees C to a 1.7 mM bolus dose of NO, prepared in N2-gassed medium, significantly reduced clonogenic survival in the HA1 fibroblasts line to 60% of control cells treated with N2-gassed medium alone. The OC14 cells were found to be completely resistant (100% survival) to NO-mediated injury in comparable experiments. A second set of experiments was designed to determine the role of the intracellular antioxidant, glutathione, in protection against NO-mediated injury. Depletion of total glutathione resulted in a significant reduction in HA1 and OC14 clonogenic survival to 8% and 50% when compared with respective control cells. The effect of total glutathione depletion on NO-initiated toxicity in HA1 cells was dose- and cell-density dependent and was observed to occur within 5 min of exposure to NO. Further evidence of cytotoxicity was demonstrated by loss of trypan blue dye exclusion properties in glutathione-depleted HA1 cells after NO exposure. Other experiments demonstrated that nitrate and nitrite exposure produced no cytotoxicity in glutathione-depleted HA1 cells and that coincubation of NO-saturated medium with oxyhemoglobin inhibited NO-induced cytotoxicity in glutathione-depleted HA1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells.

Receptors with seven transmembrane domains (7TM) constitute a large family of structurally and functionally related proteins which respond to various types of ligands. We describe here the cloning and expression of a human 7TM receptor, denoted hFB22 (human Fetal Brain 22), which is the homologue (92% amino acid identity) of a bovine receptor (LCR1) reported by others to bind neuropeptide Y (NPY) with a pharmacological profile of the Y3 receptor subtype. However, upon expression in COS1 (confirmed by Northern analysis), COS7 or CHO-K1 cells, the hFB22 receptor did not confer specific 125I-Bolton-Hunter-NPY, 3H-propionyl-NPY or 125I-peptide YY (PYY) binding sites, in either intact cells or in membrane preparations. Similarly, cells transfected with the corresponding bovine clone (LCR1) did not show specific NPY/PYY binding exceeding that resulting from endogenous binding sites; mock-transfected COS7 cells, used frequently for heterologous expression of receptors, were found to have endogenous specific 125I-NPY binding sites (Bmax = 112 fmol/mg protein; Kd = 0.25 nM). Moreover, the hFB22 transfected cells, when compared to control transfected cells, did not display de novo NPY- or PYY-induced second messenger responses, i.e., (1) inhibition of forskolin-stimulated cAMP accumulation or (2) 45Ca2+ influx. The presence of hFB22 mRNA was detected in several human neuroblastoma cell lines, none of which was found to express Y3-like NPY binding sites. hFB22 displays 39% amino acid sequence identity (in the transmembrane regions) to the human interleukin-8 receptor, and 32-36% amino acid identity to the human receptors of angiotensin II, bradykinin, and n-formylpeptide, but only 23% amino acid identity to the previously described human NPY/PYY receptor of the Y1 receptor subtype. Our results show that hFB22 and LCR1 do not encode NPY receptors, and their true ligand(s) remains to be identified.

Amino Acid Sequence↗
PubMed1993

The direct effect of graft compliance mismatch per se on development of host arterial intimal hyperplasia at the anastomotic interface.

To study the direct and sole effect of compliance mismatch on anastomotic intimal hyperplasia of the host arterial wall and to minimize possible confounding factors, dogs with a low thrombotic potential were selected as experimental subjects. Externally supported 6 cm x 5 mm Dacron grafts with a compliance value of approximately 1/300 of the host artery were implanted into the carotid arteries with end-to-end anastomoses on one side and end-to-side anastomoses on the other. The control graft was an autogenous carotid artery segment 4 cm in length transplanted into the femoral artery. Eight cases (24 grafts) were studied for 1 year and three (nine grafts) for 6 months. All were patent throughout the study period except for two noncompliant grafts with end-to-end anastomoses; thrombosis was the documented cause of occlusion. For the patent grafts, follow-up arteriograms showed no progressive narrowing of noncompliant anastomoses. Whether compliant or noncompliant, light microscopy studies showed slight intimal thickening within 1 to 2 mm of the anastomotic line, possibly the result of the normal healing response to stitch and surgical trauma. Quantitatively, 22 measurements representing longitudinal and circumferential thickness of the neointima were taken at each of the 40 patent noncompliant and 22 patent compliant control anastomoses. There was no statistically significant difference in anastomotic neointimal thickness in compliant and noncompliant grafts or for the different implantation periods. These data suggest that graft/host artery compliance mismatch does not cause arterial intimal hyperplasia at the anastomotic interface.

Anastomosis, Surgical↗
PubMed1993

Multiple areas of intestinal atresia associated with immunodeficiency and posttransfusion graft-versus-host disease.

We describe an infant with multiple segmental areas of atresia of the small and large bowel, with histologic features characteristic of the hereditary form of the disease. Posttransfusion graft-versus-host disease developed first, and then immunodeficiency was found. This report confirms the association between hereditary multiple intestinal atresia and immunodeficiency. We recommend irradiation of blood products in patients with multiple intestinal atresia pending evaluation of immune system status.

Erythrocyte Transfusion↗
PubMed1992

GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

ADP-Ribosylation Factors↗
PubMed1992

Comparative evaluation of the elasticity and flexibility of bioimpregnated knitted grafts.

Longitudinal elasticities of whole human blood clot, whole canine blood clot, Factor XIII cross-linked fibrin, glutaraldehyde-fixed human albumin, and formaldehyde-fixed collagen, gelatin and collagen/gelatin were determined and normalized to human whole blood clot. Matrices of a knitted Dacron graft were then impregnated with albumin, collagen, and collagen/gelatin and their longitudinal elasticities were determined and normalized to a preclotted graft. Comparisons were also made for the longitudinal elasticities of a virgin graft, a manipulated control for a preclotted graft, and a manipulated control for the matrix-impregnated grafts. Flexibilities were then calculated based on the weights and elasticities of these grafts and normalized to the flexibility of the preclotted graft. Fibrin had twice and 39 times more longitudinal elasticity than human blood clot and collagen, respectively. The preclotted graft has longitudinal elastic properties similar to a virgin graft, and is 2.8, 2, and 1.4 times more elastic than the albumin, collagen and collagen/gelatin grafts, respectively. The preclotted graft was 2.5, 2, and 1.4 times more flexible than the albumin, collagen and collagen/gelatin grafts, respectively.

Albumins↗
PubMed1990

Implant site: a determinant of completeness of arterial prosthesis healing in the dog and possibly in humans.

This paper compares the healing of supported knitted Dacron prostheses implanted in the descending thoracic aorta and in the subcutaneous carotid-femoral positions in each of 10 dogs. The descending thoracic aorta prostheses were 6 cm long and the carotid-femoral prostheses averaged 76 cm in length. The healing of four porous clinical axillofemoral grafts is compared to that of the experimental carotid-femoral grafts. There was little similarity between the healing observed in the descending thoracic aortic and subcutaneous positions; the descending thoracic aorta grafts healed rapidly and completely by eight weeks, with thrombus-free flow surfaces covered with confluent endothelial cells. However, the inner wall of the carotid-femoral grafts remained largely unhealed at one year, with endothelialization largely confined to a limited pannus ingrowth across the anastomoses. This indicates that the implant site in the dog is a primary determinant of the rapidity and completeness of healing. The clinical axillofemoral grafts explanted after 20 months to six and one-half years exhibited healing characteristics that were similar to those seen in the carotid-femoral position in the dog. These findings suggest that valid comparisons of interspecies healing require data obtained from similar implant locations. They present a challenge to the previously held concepts of major interspecies differences in speed and extent of tissue ingrowth based on data obtained from noncomparable implant sites.

Animals↗
PubMed1990

The evolution of a clinically relevant experimental model for the realistic evaluation of long-length (80-cm) arterial prostheses.

A new carotid-femoral experimental model has been developed which has significant clinical relevance to human limb salvage bypass grafting in the areas of graft length, hemodynamics and tissue environment. More significantly, the graft healing pattern observed with this model is similar to what is seen in humans. Therefore, this experimental model provides a challenging and critical site for dynamic healing studies and comparisons of different types of vascular prostheses.

Animals↗
PubMed1989

Characterization of amphiphilic secondary structures in neuropeptide Y through the design, synthesis, and study of model peptides.

Neuropeptide Y (NPY) has the potential to form two amphiphilic secondary structures: a polyproline II-like helix in residues 1-8, and an alpha-helix in residues 13-32. NPY dimerizes in aqueous solution and forms stable monolayers at the air-water interface, suggesting that these amphiphilic conformations are stabilized at interfaces. Furthermore, the negative molar ellipticity of monomeric NPY at 222 nm (-8500 degree cm2/dmol), suggests that hydrophobic interactions with the NH2-terminal amphiphilic structure may stabilize the alpha-helix in residues 13-32 before it binds to cell surfaces, even at physiological concentrations. In order to investigate the role of these amphiphilic structures, five NPY models with multiple substitutions in positions 13-32 have been synthesized and studied. Our data demonstrate that the surfactant properties of NPY result from its potential to form amphiphilic secondary and tertiary structures and not from specific amino acid sequences in this region. However, specific residues on the hydrophilic face of the amphiphilic alpha-helix that have been substituted in the models appear to be required to reproduce the full potency of NPY in our pharmacological assays. A possible role for the amphiphilic structures in NPY in presenting such specific determinants to cell surface receptors in the correct conformation is suggested.

Amino Acid Sequence↗
PubMed1989

Intermittent naloxone attenuates the development of physical dependence on methadone in rhesus monkeys.

Rhesus monkeys that received 15 daily injections of methadone (2 mg/kg i.m.) exhibited a characteristic opiate withdrawal syndrome after injection of naloxone (0.5 mg/kg i.m.) on the 16th day. In comparison, injection of naloxone (0.5 mg/kg i.m.) once every 2 days during a similar 15 day methadone treatment period in these same monkeys significantly attenuated the severity of the opiate withdrawal syndrome exhibited after naloxone injection on the 16th day. Each naloxone administration during the 15 day methadone treatment period elicited an opiate withdrawal syndrome that did not significantly differ on each of the 7 days it was given and was less severe than the syndrome precipitated by naloxone following 15 days of methadone without intermittent naloxone. The lack of increments in the withdrawal response to the seven naloxone injections during the 15 days of methadone treatment and the attenuation of the withdrawal response to naloxone on day 16 after intermittent naloxone administration during the 15 day methadone treatment period support the hypothesis that naloxone modifies opiate receptor mechanisms so that they revert to an agonist-naive state following antagonist exposure. These findings suggest that various agonist and antagonist opiate drug combinations or mixed agonist-antagonist drug could be clinically useful in the management of situations where physical dependence on opiates is a problem.

Animals↗
PubMed1989

Central actions of adenosine on pituitary secretion of prolactin, luteinizing hormone and thyrotropin.

Pituitary hormones were measured in plasma in unanesthetized male and female rats prepared with venous and ventricular cannulae following ventricular infusions of adenosine and two adenosine receptor agonists. Adenosine (10, 100, and 200 nmol) injected intraventricularly caused dose-related rises in plasma prolactin (PRL) in males; in females, only the 100- and 200-nmol doses increased PRL. Similar doses of adenosine had little effect on plasma levels of luteinizing hormone (LH) and thyrotropin (TSH) in the same animals. The adenosine receptor agonists L-N6-phenylisopropyladenosine (L-PIA) and 5'-N-ethyl-carboxamideadenosine (NECA) potently stimulated prolactin secretion at doses of 10 and 50 nmol when administered into the lateral ventricle, and at a dose of 2.5 nmol when administered into the third ventricle. The secretion of PRL was antagonized by the adenosine receptor antagonist theophylline (25 nmol), when theophylline was coadministered with NECA and L-PIA. TSH levels were reduced slightly but significantly following the 10- and 50-nmol infusions of L-PIA into the lateral ventricle. The less potent D-isomer of PIA (D-PIA) did not significantly stimulate PRL release. Coupled with studies indicating the presence of adenosine in the basal hypothalamus, our observations indicate a potential neuroendocrine role for this purine in prolactin secretion.

Adenosine↗
PubMed1989

Neuropeptide Y receptor binding sites in rat brain: differential autoradiographic localizations with 125I-peptide YY and 125I-neuropeptide Y imply receptor heterogeneity.

Neuropeptide Y (NPY) receptor binding sites have been localized in the rat brain by in vitro autoradiography using picomolar concentrations of both 125I-NPY and 125I-peptide YY (PYY) and new evidence provided for differentially localized receptor subtypes. Equilibrium binding studies using membranes indicate that rat brain contains a small population of high-affinity binding sites and a large population of moderate-affinity binding sites. 125I-PYY (10 pM) is selective for high-affinity binding sites (KD = 23 pM), whereas 10 pM 125I-NPY labels both high- and moderate-affinity sites (KD = 54 pM and 920 pM). The peptide specificity and affinity of these ligands in autoradiographic experiments match those seen in homogenates. Binding sites for 125I-PYY are most concentrated in the lateral septum, stratum oriens, and radiatum of the hippocampus, amygdala, piriform cortex, entorhinal cortex, several thalamic nuclei, including the reuniens and lateral posterior nuclei, and substantia nigra, pars compacta, and pars lateralis. In the brain stem, 125I-PYY sites are densest in a variety of nuclei on the floor of the fourth ventricle, including the pontine central grey, the supragenual nucleus, and the area postrema. 125I-NPY binding sites are found in similar areas, but relative levels of NPY binding and PYY binding differ regionally, suggesting differences in sites labeled by the two ligands. These receptor localizations resemble the distribution of endogenous NPY in some areas, but others, such as the hypothalamus, contain NPY immunoreactivity but few binding sites.

Animals↗
PubMed1988

125I-neuropeptide Y and 125I-peptide YY bind to multiple receptor sites in rat brain.

We describe the preparation of monoiodinated neuropeptide Y (Tyr1-125I-NPY) and monoiodinated peptide YY (Tyr36-125I-PYY). Using these ligands, we detected high, moderate, and low affinity receptor populations in rat brain. Only high and moderate affinity binding sites were suggested by saturation binding studies. Tyr1-125I-NPY bound to 8 +/- 3% of the sites with a Kd of 54 pM (Bmax = 19.4 fmol/mg of protein) and to 92 +/- 3% of the sites with a Kd of 0.92 nM (Bmax = 220.0 fmol/mg of protein). Tyr36-125I-PYY bound to 14 +/- 3% of the sites with a Kd of 23.5 pM (Bmax = 36.4 fmol/mg of protein) and to 86 +/- 3% of the sites with a Kd of 1.9 nM (Bmax = 220.1 fmol/mg of protein). The fragments NPY 13-36 and PYY 13-36 were able to compete with 10 pM Tyr1-125I-NPY for essentially all the binding sites. The fragments were 1 to 2 orders of magnitude less potent than the native peptides. Approximately 50% of the moderate affinity sites, but not the high affinity sites, were reversibly "lost" in the presence of 5'-guanylyl imidophosphate [Gpp(NH)p], a nonhydrolyzable analog of GTP. Kinetic studies revealed that Tyr1-125I-NPY dissociation could be best described by three dissociation rates. The proportions of slow and intermediate dissociation matched the proportions of moderate and high affinity binding sites, respectively, as suggested by equilibrium studies. There also existed a phase of fast dissociation. When Gpp(NH)p was added during dissociation, the proportion of slow dissociation decreased to the same extent that the fast dissociation was increased. However, the proportion of intermediate dissociation did not change. We propose that rat brain contains a minor population of high affinity NPY binding sites with an intermediate dissociation rate and no sensitivity to Gpp(NH)p. There is also a major population of moderate affinity binding sites with a slow dissociation rate. A component of these sites can convert to a low affinity state with a fast dissociation rate. Gpp(NH)p enhances conversion by stabilizing the low affinity state, thereby producing a "loss" of moderate affinity binding.

Animals↗
PubMed1988

Neuropeptide Y modulates neurotransmitter release and Ca2+ currents in rat sensory neurons.

Using 125I-labeled neuropeptide Y (NPY) and peptide YY (PYY), we demonstrated the existence of specific receptors for these peptides on rat dorsal root ganglion (DRG) cells grown in primary culture. Scatchard analysis of membrane homogenates indicated that the peptides bound to 2 populations of sites, with approximate affinities of 0.08 and 6.5 nM. Only low levels of binding were detected on sympathetic neurons cultured from the same animals or on a variety of neuronal clonal cell lines. The binding of 125I-NPY and 125I-PYY to DRG cell membranes was considerably reduced by the nonhydrolyzable analog of GTP, Gpp(NH)p. The major effect of Gpp(NH)p was to reduce the number of lower-affinity NPY binding sites without altering the number of high-affinity binding sites. NPY potently inhibited Ca2+ currents recorded under voltage clamp in rat DRG cells. Both the transient and sustained portions of the Ca2+ current were inhibited. The inhibitory effects of NPY were completely blocked following treatment of the cells with pertussis toxin. Depolarization elicited a large influx of Ca2+ into DRG neurons as assessed using fura-2-based microspectrofluorimetry. This influx of Ca2+ could be partially inhibited by NPY. Furthermore, NPY effectively inhibited the depolarization-induced release of substance P from DRG cells in vitro. Thus, NPY may be an important regulator of sensory neuron function in vivo.

Animals↗
PubMed1988

The effect of down regulation of protein kinase C on the inhibitory modulation of dorsal root ganglion neuron Ca2+ currents by neuropeptide Y.

Dorsal root ganglion (DRG) neurons cultured from neonatal rats contained high concentrations of protein kinase C (PKC). Normally, the majority of the enzyme activity was found in the cytosol and considerably less was associated with the membrane fraction. Upon incubation with the phorbol ester phorbol dibutyrate (PDBu, 10(-6) M) for 20 min, PKC activity increased in the membrane-associated fraction and decreased in the cytoplasmic fraction. Longer incubations with phorbol ester also induced a decline in membrane-associated PKC activity. If incubations were continued for periods of over 10 hr, both membrane and cytosolic PKC activity declined essentially to zero. Down-regulation of PKC had no effect on the number or affinity of 125I-neuropeptide Y (NPY) binding sites on DRG cells or on the absolute magnitude of the DRG Ca2+ current. However, the ability of NPY to inhibit the DRG Ca2+ current was greatly reduced. When sustained Ca2+ currents were evoked from depolarized holding potentials (-40 mV), all concentrations of NPY (10(-10)-10(-7) M) were less effective. In contrast, higher concentrations of NPY still blocked the transient portion of the DRG Ca2+ current evoked from hyperpolarized holding potentials. These results support the suggestion that PKC is involved in the inhibitory modulation of DRG Ca2+ currents by neurotransmitters. The precise role of PKC may vary depending on the type of Ca2+ channel involved.

Animals↗
PubMed1988

Differences in reendothelialization after balloon catheter removal of endothelial cells, superficial endarterectomy, and deep endarterectomy.

The process of reendothelialization was studied in a deendothelialized 3 cm segment of the canine descending thoracic aorta from which peripheral endothelial cell ingrowth had been prevented by impervious prosthetic graft sleeves. Three preparations were studied: (1) removal of only the endothelial cells, accomplished by flow surface drying and balloon catheter denudation, (2) removal of the superficial portion of the nonvasal media, accomplished by endarterectomy to a depth of 200 to 300 micron, and (3) removal of the entire inner (nonvasal) media, accomplished by endarterectomy to a depth of 500 to 600 micron to reach the outer (vasal) media. A total of 44 specimens were studied after implantation periods of 7, 14, 28, and 56 days. In all cases endothelial cell ingrowth from the host aorta into the test specimen was prevented by the impervious graft sleeves. In the deep endarterectomy group there were scattered areas of reendothelialization at 1 week, extensive reendothelialization at 2 weeks, almost complete reendothelialization at 4 weeks, and confluence by 8 weeks. However, in the superficial endarterectomy group scanning electron microscopy showed scattered areas of factor VIII/von Willebrand factor (FVIII/vWF)-negative, endothelial-like cells at 4 weeks, whereas at 8 weeks most of the surface was covered by endothelial cells identified by FVIII/vWF. In those specimens subjected to balloon catheter removal of endothelial cells only, reendothelialization was not seen, even at 8 weeks.

Animals↗