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Biomedical subjects

R A Murphy

Publications and source records attributed to R A Murphy.

At least 73 records · Page 4Linked to original sources
PubMed1992

Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i and uncoupling stress from myosin phosphorylation.

Elevations in guanosine 3',5'-cyclic monophosphate concentration ([cGMP]) are proposed to induce arterial smooth muscle relaxation by either 1) decreasing myoplasmic [Ca2+] ([Ca2+]i), 2) decreasing the [Ca2+]i sensitivity of phosphorylation, or 3) uncoupling force from myosin phosphorylation. We evaluated the importance of each of these mechanisms by measuring changes in [cGMP], aequorin- and fura-2-estimated [Ca2+]i, myosin light chain phosphorylation, and stress in histamine-stimulated swine carotid arteries. In tissues submaximally stimulated with 3 microM histamine, nitroprusside (NP) induced a proportional decrease in myoplasmic [Ca2+] and myosin phosphorylation, suggesting that the relaxation was at least partially induced by decreases in [Ca2+]i without a change in the [Ca2+]i sensitivity of phosphorylation. In tissues maximally stimulated with 10 microM histamine, NP and nitroglycerin produced significant relaxations that were not associated with significant sustained reductions in [Ca2+]i or myosin phosphorylation. With both submaximal and maximal histamine stimulation, nitrovasodilators produced more substantial relaxation than that expected from the nitrovasodilator-induced reduction in myosin phosphorylation. These results suggest that nitrovasodilators relax histamine-stimulated swine arterial smooth muscle by at least two mechanisms: 1) reducing [Ca2+]i, an effect observed in submaximally stimulated tissues, and 2) uncoupling of stress from myosin phosphorylation.

Animals↗
PubMed1992

Evaluation of the cariogenic potential of the intense natural sweeteners stevioside and rebaudioside A.

Stevioside and rebaudioside A, two intense natural sweeteners, that are constituents of the South American plant Stevia rebaudiana, were tested for cariogenicity in albino Sprague-Dawley rats. Sixty rat pups colonized with Streptococcus sobrinus were divided into four groups and fed stevioside, rebaudioside A or sucrose added to basal diet 2000 as follows: group 1, 30% sucrose; group 2, 0.5% stevioside; group 3, 0.5% rebaudioside A, and group 4, no addition. All four groups were sacrificed after 5 weeks. S. sobrinus counts were made and caries was evaluated according to Keyes' technique. There were no differences in food and water intake and weight gains between the four groups. There were significant differences in sulcal caries scores (p < 0.02) and S. sobrinus counts (p < 0.05) between group 1 and the other three groups. There were no significant differences between the stevioside, rebaudioside A and no-addition groups. It was concluded that neither stevioside nor rebaudioside A is cariogenic under the conditions of this study.

Animals↗
PubMed1992

Spatiotemporal increases in epidermal growth factor receptors following peripheral nerve injury.

Non-neuronal cells of peripheral nerve respond to axonal injury with a series of cellular changes that facilitate neuronal regeneration. To characterize the potential role of the epidermal growth factor (EGF) family of proteins in this response, we monitored the expression of EGF receptor mRNA and protein in the injured rat sciatic nerve. EGF receptor mRNA is synthesized in both primary cultured fibroblasts and Schwann cells, and Schwann cells express EGF receptor-like immunoreactivity. In situ hybridization and immunocytochemistry revealed that EGF receptor mRNA and protein are expressed in Schwann cells and fibroblasts of the sciatic nerve in vivo, and that receptor levels increase following nerve injury. Thirty-six hours postlesion, EGF receptors were expressed in gradients along the nerve both proximal and distal to the lesion, with the highest levels localized adjacent to the transection site. By 72 hr, receptor levels were maintained in a gradient in the proximal segment, but were uniformly increased throughout the portions of the distal segment that were analyzed. These changes were similar to those observed for low-affinity NGF receptor mRNA and protein, with transection causing increased expression in both Schwann cells and fibroblasts. Northern blots confirmed that primary cultured fibroblasts express low-affinity NGF receptor mRNA. To determine whether spatiotemporal gradients were a general characteristic of the nerve injury response, we monitored expression of the mRNA encoding the major myelin protein P0. Levels of P0 mRNA decreased initially in cells immediately adjacent to the transection site and, by 72 hr, were uniformly decreased throughout the distal segment. These data suggest that members of the EGF family of proteins may play a role in the peripheral nerve response to injury, and demonstrate a generalized gradient of cellular responses that commence at the transection site and progress distally in the nerve in the absence of intact axons.

Animals↗
PubMed1991

Generation of the truncated form of the nerve growth factor receptor by rat Schwann cells. Evidence for post-translational processing.

These studies were initiated to determine whether the soluble, truncated form of the nerve growth factor (NGF) receptor arises from post-translational processing of the intact, membrane-bound receptor or from an alternatively spliced mRNA. Pulse-chase analysis of cultured primary rat Schwann cells coupled with immunoprecipitations using antibodies to the intracellular and extracellular domains of the receptor were used to monitor receptor production. Three forms of the NGF receptor (80, 83, and 85 kDa) displaying a precursor product relationship were detected over the 2-h chase period; only the 85-kDa species was detected on the cell surface. Truncated receptors (50 and 52 kDa) were detected in conditioned media 5 h after cell labeling but were never observed intracellularly. Polymerase chain reaction and RNase protection analyses of NGF receptor mRNA targeted toward the coding region for the transmembrane domain detected no splice variants that could generate truncated receptor, and media conditioned by fibroblasts transfected with rat receptor cDNA, in which splicing cannot occur, nonetheless contained the truncated receptor protein. Taken together, these results suggest that the truncated NGF receptor does not arise as a distinct translation product but rather from a post-translational modification of the intact, surface-bound form of the protein.

Amino Acid Sequence↗
PubMed1991

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle.

Smooth muscle cells express isoforms of actin and myosin heavy chains (MHC). In early postnatal animals the nonmuscle (NM) actin and MHC isoforms in vascular (aorta) smooth muscle were present in relatively high percentages. More than 30% of the MHC and 40% of the actin isoforms were NM. The relative percentage of the NM isoforms decreased significantly as the animals reached maturity, with NM MHC less than 10% and NM actin less than 30% of the totals. Concurrent with this decrease in NM isoforms was an increase in the smooth muscle (SM) isoforms. The relative changes and time frame in which these changes occurred were very similar for the actin and MHC isoforms. In arterial tissue there were species differences for changes with development in the two SM MHC isoforms (SM1 and SM2). The ratio of SM1:SM2 in young rat aorta was approximately 0.5, while this same ratio was approximately 3 in young swine carotid. Both adult rats and swine had a SM1:SM2 MHC ratio of approximately 1.2. Rat bladder smooth muscle showed no significant change in NM vs SM ratio between young and old rats, while the SM1:SM2 ratio decreased from 2.7 to 1.7 between these age groups. The shifts in alpha and beta actin were similar to those in the vascular tissue, but of much smaller magnitude.

Actins↗
PubMed1991

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF.

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.

Animals↗
PubMed1991

Cyclic AMP relaxes swine arterial smooth muscle predominantly by decreasing cell Ca2+ concentration.

1. Our objective was to evaluate the mechanism of cyclic AMP-dependent arterial smooth muscle relaxation. Cyclic AMP-dependent relaxation has been proposed to result from either (a) a decrease in intracellular [Ca2+] or (b) a decrease in [Ca2+] sensitivity of myosin light chain kinase by protein kinase A-dependent phosphorylation of myosin kinase. 2. We evaluated these proposed mechanisms by examining forskolin-induced changes in aequorin-estimated myoplasmic [Ca2+], [cyclic AMP], myosin phosphorylation and stress generation in agonist-stimulated or KCl-depolarized swine common carotid media tissues. 3. Forskolin, an activator of adenylyl cyclase, increased [cyclic AMP] and reduced [Ca2+], myosin phosphorylation and stress in tissues pre-contracted with phenylephrine or histamine. This relaxation was not associated with an alteration of the [Ca2+] sensitivity of phosphorylation, nor the dependence of stress on phosphorylation. 4. Forskolin pre-treatment attenuated, but did not abolish, agonist-induced increases in [Ca2+] and stress. 5. These results suggest that cyclic AMP-induced relaxation of the agonist-stimulated swine carotid media is primarily caused by cyclic AMP-mediated decreases in myoplasmic [Ca2+].

Animals↗
PubMed1991

Localization of isoactins in isolated smooth muscle thin filaments by double gold immunolabeling.

Smooth muscle cells contain both smooth muscle-specific and cytoplasmic actin isoforms. We tested the hypothesis that these two classes of isoactins are segregated into separate "contractile" vs. "cytoskeletal" populations of thin filaments. Isolated thin filaments from adult swine stomach were double-labeled with isoactin-specific peptide antibodies with the use of two different size protein A-gold markers to localize the isoactins on individual filaments. The large and small gold beads bound to individual filaments were counted on electron micrographs, and the results were analyzed statistically. Both classes of actin isoforms were present in every filament, and the relative proportions of smooth muscle vs. cytoplasmic actin isoforms in each filament showed no significant deviation from a single random population. Furthermore, the distribution of the isoactins along each filament was also random; no significant clustering of isoforms was observed. Controls containing added skeletal muscle F-actin showed that the techniques used could have detected nonrandom isoform distributions if they had been present.

Actin Cytoskeleton↗
PubMed1990

Synthesis and characterization of iodobenzamide analogues: potential D-2 dopamine receptor imaging agents.

(S)-N-[(1-Ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-3-iodo-6- methoxybenzamide ([123I]IBZM) is a central nervous system (CNS) D-2 dopamine receptor imaging agent. In order to investigate the versatility of this parent structure in specific dopamine receptor localization and the potential for developing new dopamine receptor imaging agents, a series of new iodinated benzamides with fused ring systems, naphthalene (INAP) and benzofuran (IBF), was synthesized and radiolabeled, and the in vivo and in vitro biological properties were characterized. The best analogue of IBZM is IBF (21). The specific binding of [125I]IBF (21) with rat striatal tissue preparation was found to be saturable and displayed a Kd of 0.106 +/- 0.015 nM. Competition data of various receptor ligands for [125I]IBF (21) binding show the following rank order of potency: spiperone greater than IBF (21) greater than IBZM greater than (+)-butaclamol greater than (+/-)-ADTN,6,7 greater than ketanserin greater than SCH-23390 much greater than propranolol. The in vivo biodistribution results confirm that [125I]IBF (21) concentrated in the striatal area after iv injection into rats. The study demonstrates that [123I]IBF (21) is a potential agent for imaging CNS D-2 dopamine receptors.

Animals↗
PubMed1990

Crossbridge phosphorylation and regulation of vascular smooth muscle contraction.

A model is proposed to explain crossbridge regulation in smooth muscle and the latch state (high force with very low crossbridge cycling rates). The model predicts that low levels of [Ca2+] and myosin phosphorylation can induce high values of force. In this analysis we show that reductions in myosin light chain phosphatase activity make stress more proportional to phosphorylation, with abolition of the latch state. This may explain some of the differences in the phosphorylation dependence of force between intact and skinned smooth muscle.

Animals↗
PubMed1990

Latch-bridge model in smooth muscle: [Ca2+]i can quantitatively predict stress.

Ca2+ concentration ([Ca2+])-dependent cross-bridge phosphorylation by myosin light chain kinase is postulated to be the primary regulator of stress development in smooth muscle. A four-state model of cross-bridge function, regulated only by [Ca2+]-dependent changes in myosin kinase activity, has been proposed to explain contraction and the latch state of smooth muscle (high force with reduced cross-bridge cycling and ATP consumption). A key test of this model is to determine whether changes in myoplasmic [Ca2+], per se, can quantitatively predict changes in myosin kinase activity, cross-bridge phosphorylation, and therefore force production. We find that changes in aequorin-estimated myoplasmic [Ca2+] can quantitatively predict the time course of phosphorylation and isometric stress production in response to stimulation with histamine and angiotensin II and during adenosine 3',5'-cyclic monophosphate-mediated relaxation when [Ca2+] is not changing rapidly. These results suggest that changes in myoplasmic [Ca2+] and activation of myosin light chain kinase may be sufficient to explain both contraction and relaxation of agonist stimulated swine carotid arterial smooth muscle.

Animals↗
PubMed1990

Muscle length, shortening, myoplasmic [Ca2+], and activation of arterial smooth muscle.

The effect of muscle length on smooth muscle contraction was evaluated by measuring myoplasmic [Ca2+] (with aequorin), myosin light chain phosphorylation, length, and isometric stress in histamine-stimulated swine carotid media preparations. Tissues were equilibrated at the optimal length for stress development (Lo). Isometric contractions at short tissue lengths (0.7 Lo) were associated with a decrease in maximal stress development. Isometric contraction at 0.7 Lo also reduced the sensitivity to histamine as measured by steady-state increases in [Ca2+], phosphorylation, or stress. This suggests that decreased agonist sensitivity at shorter lengths is caused by reduced Ca2+ mobilization. Isotonic shortening also led to decreases in histamine sensitivity. Isometric contractions at 1.2 Lo were not associated with significant changes in histamine-induced increases in [Ca2+]. The [Ca2+] dependence of phosphorylation was not altered at 0.7 or 1.2 Lo. Sinusoidal length changes from 0.95 to 1.05 Lo at 1 Hz were not associated with significant changes in the resting or histamine-stimulated [Ca2+]. These results suggest that Ca2+ mobilization and the resulting contraction is relatively independent of length changes near Lo. Inactivation occurs at lengths substantially below Lo where Ca2+ mobilization by agonists is impaired.

Aequorin↗
PubMed1990

The characterization of IBF as a new selective dopamine D-2 receptor imaging agent.

The in vivo and in vitro studies of a new iodinated benzamide analog, [125I]IBF,5-iodo-7-N-[(1-ethyl-2-pyrrolidinyl)methyl]carboxamido-2,3- dihydrobenzofuran as a potential central nervous system (CNS) D-2 dopamine receptor imaging agent were investigated. In vivo biodistribution of IBF in rat indicated that this agent concentrated in the striatum region and displayed a remarkably high target-to-nontarget ratio (striatum/cerebellum = 48 at 120 min post-injection). The in vitro binding studies suggested that IBF binds selectively to D-2 dopamine receptors with high affinity and low nonspecific binding (Kd = 0.106 +/- 0.015 nM, Bmax = 448 +/- 18.2 fmole/mg protein). Ex vivo autoradiography results in rats further confirmed the high uptake and retention of this agent in the basal ganglia region. The planar images of monkey brains (lateral view of the head) after i.v. injection of [123I]IBF clearly demonstrated that D-2 dopamine receptors can be visualized. With the excellent in vivo stability to deiodination and high target-to-nontarget ratio, [123I]IBF may be useful as a CNS D-2 dopamine receptor imaging agent for single photon emission computed tomography (SPECT) in humans.

Animals↗
PubMed1989

Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands.

In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.

Amino Acid Sequence↗