Biomedical subjects

E Albrecht

Publications and source records attributed to E Albrecht.

At least 19 recordsLinked to original sources

PubMed2006

Growth- and breed-related changes of marbling characteristics in cattle.

The objective of this study was to investigate the growth- and breed-related changes of marbling characteristics in cattle. Four cattle breeds with different growth impetus and muscularity were reared and slaughtered under experimental conditions. German Angus, as a typical beef cattle; Galloway, as a smaller, environmentally resistant beef cattle; Holstein-Friesian, as a dairy-type cattle; and double-muscled Belgian Blue, as an extreme type for muscle growth, were used. These 4 breeds were expected to have differences in muscle development and i.m. fat deposition. Between 5 and 15 bulls of each breed were slaughtered at 2, 4, 6, 12, or 24 mo of age. Marbling characteristics were determined and classified in LM and semitendinosus muscle by computerized image analysis. Among breeds, differences appeared in the quantity, structure, and distribution of the marbling flecks in both muscles. The deposition of fat in the double-muscled Belgian Blue bulls remained substantially inferior to that of the other breeds, up to the age of 24 mo. Marbling in German Angus bulls particularly showed larger (P < 0.05) marbling fleck areas. Galloway cattle had the greatest (P < 0.05) number and the most regular (P < 0.05) distribution of the marbling flecks in young animals. Furthermore, for marbling characteristics in Holstein-Friesian animals, a great number and slightly finer structure were observed compared with the other breeds investigated. Postnatal growth-related changes of marbling in LM were characterized by as much as a 40-fold increase in the number of marbling flecks from 2 to 24 mo of age but also by up to a 4-fold enlargement in the area of the marbling flecks. The structure of marbling flecks was determined by 2 development trends. On the one hand, the marbling flecks became larger (P < 0.05), and the structure became coarser, which was reflected by an increasing (P < 0.01) proportion of long marbling flecks as well as an increasing (P < 0.01) maximum skeleton line length. On the other hand, continually new small, round marbling flecks appeared. This caused a decrease (P < 0.01) in the proportion of the 3 largest marbling fleck areas. The distribution of the marbling flecks became more regular (P < 0.05) with increasing proportion and number of marbling flecks. The results suggest that hyperplasia of adipocytes plays an important role in marbling during growth of muscle in cattle.

Adipose Tissue↗

PubMed2006

Computer image analysis of intramuscular adipocytes and marbling in the longissimus muscle of cattle.

The deposition of fat in muscle, recognized by the consumer as marbling, is an important meat quality trait. The objective of the study was to provide additional insights into the quantitative extent of marbling by means of computer image analysis. Fifty-one F(2) generation German Holstein and Charolais crossbreed cattle, 18 mo of age, were used to determine relationships among marbling traits, adipocyte size, and the amount of adipose tissue in different depots. Differences were recorded among the size of i.m. adipocytes in different groups of marbling flecks, divided according to the location in the muscle cross-section and to the size of the marbling flecks. The results showed positive correlation between i.m. adipocyte size and the weight of s.c. fat, intestinal fat, omental fat, and perirenal fat (r = 0.50, 0.61, 0.70, and 0.63, respectively, P < 0.001). The i.m. adipocyte size was correlated with i.m. fat content, number of marbling flecks, proportion of marbling fleck area, and total length of marbling flecks (r = 0.71, 0.44, 0.62, and 0.55, respectively, P < 0.01). The number of marbling flecks was also correlated with i.m. fat content, proportion of marbling fleck area, and total length of marbling flecks (r = 0.58, 0.62, and 0.91, P < 0.01, respectively). The ventral marbling flecks had a 5-fold larger fleck area, 4-fold more adipocytes, and larger adipocytes (P < 0.001). Larger marbling flecks contained larger adipocytes (P < 0.001). Moreover, compared with the small marbling flecks, there was a 48-fold larger fleck area and 26-fold more adipocytes in the large marbling flecks. The results indicate that i.m. fat deposition increases concurrently with the other fat depots but is still independent. Furthermore, the i.m. fat is preferentially deposited in the ventral area of LM. Although the i.m. adipocyte size has an important effect on the traits of marbling flecks, cell number plays a greater role in i.m. fat deposition than cell size.

Adipocytes↗

PubMed2006

Growth- and breed-related changes of muscle bundle structure in cattle.

The objective of this study was to investigate the changes in muscle fiber bundles of cattle of different breeds during growth. Different numbers of muscle fibers are surrounded by connective tissue to form bundles macroscopically visible as meat fibers or meat grain, a common meat quality trait. To determine the influence of breed and age on morphological characteristics of muscle fiber bundles, 4 cattle breeds with different growth impetus and muscularity were reared and slaughtered under experimental conditions. German Angus, a typical beef cattle; Galloway, a smaller beef type; Holstein Friesian, a dairy type; and double-muscled Belgian Blue, an extreme type for muscle growth, were used. Between 5 and 15 bulls of each breed were slaughtered at 2, 4, 6, 12, or 24 mo of age, and slices of semitendinosus muscle were removed. Muscle structure characteristics were determined by computerized image analysis. During growth, the muscle cross-sectional area enlarged (P < 0.001) about 5-fold in double-muscled Belgian Blue bulls and about 4-fold in the other breeds. This was a result of the enlargement (P < 0.001) of primary bundles and muscle fibers. The bundle size was similar (P > or = 0.15) in bulls of German Angus and Galloway in all age groups and was doubled (P < 0.001) in double-muscled Belgian Blue animals from 4 mo of age on. The Holstein Friesian bulls had the smallest (P < 0.001) muscle fiber bundles at 24 mo of age. The number of muscle fibers per bundle and the number of bundles per muscle remained nearly constant (P > 0.05) during growth. This supports the existing view that the structure of the muscle is already fixed in prenatal life. The double-muscled Belgian Blue bulls showed a more than 2.5-fold greater (P < 0.001) number of muscle fibers per primary bundle compared with the other breeds investigated. The larger muscle fiber bundles led to a smaller amount of connective tissue per muscle area in double-muscled cattle. The coarser grain of meat in double-muscled Belgian Blue bulls and in older animals was not related to greater shear force values.

Animals↗

PubMed2005

Protein tyrosine phosphatase kappa and SHP-1 are involved in the regulation of cell-cell contacts at adherens junctions in the exocrine pancreas.

BACKGROUND: We have previously shown that cell contacts between pancreatic acinar cells dissociate early in pancreatitis and that this is a prerequisite for the development of pancreatic oedema. Here we studied the underlying mechanism. METHODS: Employing experimental caerulein induced pancreatitis in vivo and isolated pancreatic acini ex vivo, in conjunction with protein chemistry, morphology, and electron microscopy, we determined whether cell contact regulation in the pancreas requires or involves: (1) changes in cadherin-catenin protein expression, (2) tyrosine phosphorylation of adhesion proteins, or (3) alterations in the actin cytoskeleton. RESULTS: During initial cell-cell contact dissociation at adherens junctions, expression of adhesion proteins remained stable. At time points of dissociated adherens junctions, the cadherin-catenin complex was found to be tyrosine phosphorylated and internalised. The receptor type protein tyrosine phosphatase (PTP)kappa was constitutively associated with the cadherin-catenin complex at intact cell contacts whereas following the dissociation of adherens junctions, the internalised components of the cadherin-catenin complex were tyrosine phosphorylated and associated with the cytosolic PTP SHP-1. In isolated acini, inhibition of endogenous protein tyrosine phosphatases alone was sufficient to induce dissociation of adherens junctions analogous to that found with supramaximal caerulein stimulation. Dissociation of actin microfilaments had no effect on adherens junction integrity. CONCLUSIONS: These data identify tyrosine phosphorylation as the key regulator for cell contacts at adherens junctions and suggest a definitive role for the protein tyrosine phosphatases PTPkappa and SHP-1 in the regulation, maintenance, and restitution of cell adhesions in a complex epithelial organ such as the pancreas.

Actins↗

PubMed2002

Intrinsic properties of muscle satellite cells are changed in response to long-term selection of mice for different growth traits.

Satellite cell cultures were derived from mice selected long-term over 70 generations for body weight (DU-6, growth), carcass protein amount (DU-6P, protein) and an index combining body weight and endurance treadmill performance (DU-6+LB, growth + fitness) at 42 days of age and from an unselected control line (DU-Ks). They were grown under identical environmental conditions to examine intrinsic cellular differences in proliferation, protein metabolism and responsiveness to growth factors. Growth kinetics (DNA and protein amounts) were determined over a 12-day period. During exponential growth, all growth-selected cultures grew faster than the control culture: (DU-6+LB=DU-6P)>DU-6>DU-Ks. The differences in DNA and protein levels were maintained until day 8. DU-Ks cultures reached similar levels as the growth (DU-6) and protein (DU-6P) cultures in terms of DNA at day 12 of cultivation. Thus, the cultures from the growth and protein lines, but not from the growth + fitness line, exhibited larger protein:DNA ratios (cell size) than the control cultures. Cell cultures from the selected lines were more responsive to serum and epidermal growth factor in terms of [(3)H] thymidine incorporation into DNA, whereas no stimulation by insulin or insulin-like growth factor-I was detectable in cultures from selected lines or controls. During differentiation, protein metabolism in cultures from selected lines was characterised by higher rates of protein synthesis (PS) and degradation (PD), as measured by [(3)H] phenylalanine incorporation or release, respectively, than in control cells. The ratios of the relative differences from the control in PS and PD were only >1.0 in the growth and protein lines. In conclusion, long-term selection for growth therefore modifies the intrinsic capability of satellite cells for proliferation and protein metabolism, with changes being dependent on the selection trait.

Animals↗

PubMed2001

Effect of hyperthermia on premature intracellular trypsinogen activation in the exocrine pancreas.

Hyperthermia, raising the body temperature from normal to above 40 degrees C, has been shown to prevent pancreatitis in an experimental animal model of the disease, but the underlying cellular mechanisms of this protection remain unknown. We induced controlled hyperthermia in either laboratory rats and isolated pancreatic acini or, alternatively, raised the temperature of pancreatic homogenates in vitro from 37 to 41 degrees C. In vitro controlled hyperthermia of up to 41 degrees C increased the autoactivation-induced and enterokinase-induced trypsinogen activation as well as free trypsin activity. Conversely, in whole animal studies and in living acinar cells hyperthermia reduced or abolished premature intracellular trypsinogen activation in a time- and temperature-dependent manner and this protective effect was independent of either de novo protein synthesis, interference with acinar cell signal transduction, or confirmational changes in pancreatic trypsinogen. We conclude that hyperthermia, in a manner that is independent of the synthesis of pancreatic chaperone or heat shock proteins, can directly abolish the earliest initiating event involved in the onset of pancreatitis, namely the premature and intracellular activation of digestive zymogens.

Animals↗

PubMed2001

Effects of lectins on CCK-8-stimulated enzyme secretion and differentiation of the rat pancreatic cell line AR42J.

INTRODUCTION: The peptide hormone cholecystokinin (CCK) plays an important role in the gastrointestinal tract. The rat pancreatic CCK receptor is a highly glycosylated membrane receptor that is able to bind to plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). AIMS AND METHODOLOGY: We used both lectins to block this receptor for studying the pathophysiologic relevance of its oligosaccharide side chains. In the present study we investigated the influence of WGA and UEA-I on CCK-8-induced alpha-amylase secretion of the rat pancreatic tumor cell line AR42J, which expresses both CCK-A and CCK-B receptors. RESULTS: Under the influence of WGA (25 microg/mL), the alpha-amylase release was reduced by 25% after 30 minutes compared with the hormone-stimulated controls. UEA-I (25 microg/mL) caused a reduction of 20%. The simultaneous application of the lectins with CCK antagonists L 364,718 or L 365,260 led to a reduction of secretion, but the assignment to CCK-A or CCK-B receptors was not possible. CONCLUSION: In long-term studies, both lectins revealed no toxic or apoptosis-inducing effects. On the contrary, WGA showed an inhibitory effect on cell proliferation and led to improved differentiation of cells.

Animals↗

PubMed2000

The role of intracellular calcium signaling in premature protease activation and the onset of pancreatitis.

The exocrine pancreas synthesizes and secretes large amounts of digestive proteases as inactive precursor zymogens. Under physiological conditions a variety of cellular defense mechanisms protect the pancreatic acinar cell against a premature and intracellular activation of these zymogens. When these defenses fail, pancreatic autodigestion is initiated and acute pancreatitis can develop. A number of experimental observations suggest that extra- as well as intracellular calcium concentrations play an important part in the initiation of pancreatic protease activation, but the intracellular signaling events that regulate this process are unknown. Using a model system in which we used pancreatic acini (freshly prepared functional units of living acinar cells), we were able to simulate the conditions found during experimental pancreatitis in rodents. By means of a cell permeant fluorescent trypsin substrate we could demonstrate in these acini that premature protease activation is initiated at the apical acinar cell pole and occurs only in the presence of secretagogue concentrations that exceed those required for a maximum secretory response. By combining this technique with fluorescence ratio imaging for the Ca(2+)-sensitive dye fura-2, we could further show that this protease activation is highly dependent on the spatial as well as the temporal distribution of the corresponding Ca(2+) release from stores within the same subcellular compartment and that it is not propagated to neighboring acinar cells.

Animals↗

PubMed2000

The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties.

The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)) has recently been reported to act as partial agonist of the nociceptin/orphanin FQ (noc/OFQ) receptor expressed in CHO cells. In addition, this peptide acts as a competitive antagonist of noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes as well as of the noc/OFQ-evoked chronotropic effect in rat cardiomyocytes. In contrast to this antagonism, in the present study, Ac-RYYRIK-NH(2) was found to behave as an agonist at noc/OFQ receptors, affecting spontaneous locomotor activity. When administered intracerebroventricularly (i.c.v.), noc/OFQ and Ac-RYYRIK-NH(2) inhibited spontaneous locomotor activity in mice with ID(50) of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH(2) could not be clearly explained by differential metabolism, because in vivo microdialysis in rat striatum and in vitro metabolic inactivation by rat and mouse brain membranes revealed extensive inactivation of both peptides. Similar to Ac-RYYRIK-NH(2), [Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-NH(2) ([F/G]NC(1-13)NH(2)) inhibited the noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes (Schild constant 3.83 nM) and mouse brain sections, although several reports have shown that this peptide exhibits agonist activity of noc/OFQ in the CNS. Changes in the optimum conditions of the in vitro assay for GTP binding increased low partial agonism of Ac-RYYRIK-NH(2) in GTP binding response. To explain the discrepancy between the in vitro antagonism of G protein coupling of the noc/OFQ receptor and in vivo agonism of Ac-RYYRIK-NH(2) and of [F/G]NC(1-13)NH(2), it is suggested that low partial agonism of receptor/G protein coupling in native systems may be sufficient to evoke full biologic responses. The extent of partial agonism for GTP binding and of coupling reserve may vary in different systems, thus explaining why [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.

Animals↗

PubMed2000

Growth- and breed-related changes of muscle fiber characteristics in cattle.

The objective of this study was to investigate the growth- and breed-related changes of muscle fiber characteristics in cattle and their importance to meat quality. Four cattle breeds with different growth impetus and muscularity were reared and slaughtered under experimental conditions. German Angus as a beef type, Galloway as a hardy type, Holstein Friesian as a dairy type, and double-muscled Belgian Blue as an extreme type for muscle growth were used. Between 5 and 17 bulls of each breed were slaughtered at 0, 2, 4, 6, 12, 18, and 24 mo of age. Muscle fiber traits were determined and classified by computerized image analysis, and several measures of meat quality were also determined, including shear force value, meat color, and i.m. fat content. The postnatal growth of semitendinosus muscle in cattle was characterized by a nearly 10-fold increase of muscle fiber area from birth to 24 mo of age. In the first few months after birth, a transformation of type IIA fibers into IIB fibers was found, whereas type I fibers were nearly unaffected by age. The apparent total muscle fiber number of semitendinosus muscle did not increase during postnatal life. These results confirm that the fiber number is determined in embryonic development. Throughout the study, the double-muscled Belgian Blue (BBDM) bulls had almost twice the fiber number of the other breeds, emphasizing a more extensive hyperplasia of muscle fibers during embryonic development in BBDM compared with the other three breeds. The apparent number of type I fibers was, however, not affected by breed, which suggests that the additional fibers found in BBDM postnatally were type IIB and IIA fibers. We did not find significant differences in muscle fiber total number, muscle fiber type frequencies, or meat quality characteristics among breeds, with the exception of BBDM. Having pooled the four breeds, paler meat was related to a higher frequency of type IIB fibers, a lower area of type IIA and type I fiber, and a higher total muscle fiber number. These findings based on data of double muscling give us some hints for biological causes for the variation of meat quality. Further investigation, in particular within each breed, is necessary to identify the superior fiber traits for bovine meat production.

Animal Husbandry↗

PubMed2000

[Nphe(1)]NC(1-13)NH(2) selectively antagonizes nociceptin/orphanin FQ-stimulated G-protein activation in rat brain.

[Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-amide ([F/G]NC(1-13)NH(2)) and acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)), two peptidic ligands of the nociceptin/orphanin FQ (noc/OFQ) receptor, have been shown to exert both agonist and antagonist activity in different in vitro and in vivo systems. This is despite the observation that both peptides competitively antagonized the coupling of the activated receptor to G-proteins in brain preparations, measured in GTPgamma(35)S binding assays. In this study, [Nphe(1)]NC(1-13)-amide ([Nphe(1)]NC(1-13)NH(2)), a new noc/OFQ analog recently characterized as a pure and selective noc/OFQ receptor antagonist in several in vitro and in vivo assay systems, was shown to competitively inhibit the noc/OFQ-stimulated GTPgamma(35)S binding to rat cerebral cortex membranes with pA(2) of 7.76 (Schild analysis). This antagonism of noc/OFQ receptor G-protein coupling was selective because the peptide inhibited the noc/OFQ-evoked GTPgamma(35)S binding to rat brain membranes but not that evoked by selective agonists of the mu-, delta-, and kappa-opioid receptors. In rat cortical membranes, the effects of [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) on the binding of GTPgamma(35)S were clearly differentiated from the effect of [Nphe(1)]NC(1-13)NH(2) when the concentration of GDP, competing with GTPgammaS for binding, was lowered from 100 microM (assay optimum) to 5 microM. At 5 microM GDP, the former peptides showed clear partial agonist activity, whereas [Nphe(1)]NC(1-13)NH(2) did not. These data indicate that only [Nphe(1)]NC(1-13)NH(2) was a pure antagonist of noc/OFQ receptor G-protein coupling. Furthermore, it is suggested that the variable behavior of [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) (agonist, partial agonist, and antagonist) in different in vitro and in vivo systems may be explained by different partial GTP binding agonism and the existence of a GTP binding stimulus/response reserve (coupling reserve).

Amino Acid Sequence↗

PubMed1999

[Violence our medical trauma in specialized multidisciplinary consultation].

In this article a particular patient/physician relationship is described and analyzed: The described interaction between patient and physician during a consultative investigation by several specialists differs markedly from the common trustful relation between a patient and his family doctor. In this context the term and phenomenon pain is discussed and the necessity for an understandable, patient-oriented presentation of diagnosis and hypotheses considering the patient's individual bio-psycho-social dimension is stressed. Consequences for student education are mentioned.

Adaptation, Psychological↗

PubMed1999

Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ.

For the further elucidation of the central functions of nociceptin/orphanin FQ (noc/OFQ), the endogenous ligand of the G protein-coupled opioid receptor-like receptor ORL1, centrally acting specific antagonists will be most helpful. In this study it was found that the hexapeptide acetyl-RYYRIK-NH2 (Ac-RYYRIK-NH2), described in literature as partial agonist on ORL1 transfected in CHO cells, antagonizes the stimulation of [35S]-GTPgammaS binding to G proteins by noc/OFQ in membranes and sections of rat brain. The antagonism of the peptide was competitive, of high affinity (Schild constant 6.58 nM), and specific for noc/OFQ in that the stimulation of GTP binding by agonists for the mu-, delta-, and kappa-opioid receptor was not inhibited. The hexapeptide also fully inhibited the chronotropic effect of noc/OFQ on neonatal rat cardiomyocytes. It is suggested that Ac-RYYRIK-NH2 may provide a promising starting point for in vivo tests for antagonism of the action of noc/OFQ and for the further development of highly active and specific antagonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1999

The effect of estrogen on aromatase and vascular endothelial growth factor messenger ribonucleic acid in the normal nonhuman primate mammary gland.

In the present study, the baboon was used as a model to investigate the effects of steroid hormones on vascular endothelial growth factor (VEG/PF) and aromatase expression and on proliferation of the normal mammary gland. Immunocytochemistry revealed that both aromatase and VEG/PF were expressed in the epithelial cells of the terminal ductal lobular units. Mammary tissue biopsies were obtained from female baboons during the follicular and luteal phases of the menstrual cycle, 4 weeks after ovariectomy (OVX), and after 2 weeks of treatment with estradiol benzoate (E2B; 500 microg/day, i.m.). Although there was little apparent difference in aromatase messenger ribonucleic acid (mRNA) in tissue from follicular and luteal phases or after ovariectomy, aromatase mRNA was decreased in tissue from ovariectomized (OVX) animals treated for 2 weeks with E2B. Furthermore, aromatase activity in tissue from these animals was markedly reduced compared to activity in tissue from the OVX animals before treatment (P < 0.001). In one animal in which mammary aromatase activity was measured sequentially during the follicular and luteal phases, aromatase activity was increased significantly after OVX and was reduced to the level in the intact animal by subsequent treatment with E2B. This effect on both aromatase activity and mRNA occurred rapidly 2 and 4 h after injection with E2B. In contrast to its effect on aromatase, E2B treatment of OVX animals stimulated VEG/PF mRNA 2 and 4 h after injection. In histoculture of mammary biopsies from these animals in the follicular and luteal phases of the menstrual cycle or after OVX, [3H]thymidine incorporation was increased significantly by incubation with testosterone (T) as well as estrogen (P < 0.01). The effect of T was blocked by aromatase inhibitor, 4-hydroxyandrostenedione, suggesting that the tissue is responsive to E produced by aromatization of T in the tissue. When mammary tissue from OVX animals was cultured with T, there was a significantly greater increase in [3H]thymidine incorporation than in histocultures of tissue from intact animals (P < 0.01). However, in histocultures of tissue from the OVX animals treated with E2B (500 microg) for 2 weeks, [3H]thymidine incorporation was similar to the level in tissue of intact animals incubated with T. No significant changes occurred in [3H]thymidine incorporation with the nonaromatizable androgen dihydrotestosterone or progesterone alone. These findings suggest that estrogens produced locally by aromatization of T have a functional role in mammary tissue. Aromatase expressed in the mammary gland could be important in maintaining local estrogen concentrations, particularly after menopause. Estrogen appears to regulate transcription of both aromatase and VEG/PF in the mammary gland, suggesting a regulatory loop by which local estrogens could stimulate VEG/PF production. Thus, paracrine/autocrine mechanisms that can enhance the proliferation of malignant cells and their metastatic spread already exist before transformation.

Animals↗

PubMed1998

Cellular memory: neutrophil orientation reverses during temporally decreasing chemoattractant concentrations.

Cell directional orientation or shape polarization is the first cellular step in neutrophil locomotion. To better understand how chemoattractants interact with cells, we studied neutrophil polarization (or shape changes) during exposure to a temporally decreasing chemoattractant signal of N-formyl-methionyl-leucyl-phenylalanine (FMLP) in the absence of a spatial concentration gradient. To accomplish this objective, we used a manifold of differing FMLP concentrations attached to a stopped-flow microscope chamber. Spatial gradients of a fluorescent chemotactic peptide could not be detected in the chamber by using microfluorometry. When FMLP was injected at continually increasing concentrations at 10-s intervals, the shape and relative direction of the neutrophil persisted. However, when temporally decreasing FMLP concentrations were injected, approximately 80% of the cells changed their direction with 44% of the total cells swinging about to 180 degrees +/- 15 degrees. Most of these directional changes involved dissolution of both the lamellipodium and uropod and reformation of these structures 180 degrees from their original positions. This research suggests that neutrophils reverse their morphological polarity when exposed to temporally decreasing ligand concentrations by "remembering" their ligand exposure history and relative direction.

Cell Adhesion↗

PubMed1998

Nociceptin (orphanin FQ): high-affinity and high-capacity binding site coupled to low-potency stimulation of guanylyl-5'-O-(gamma-thio)-triphosphate binding in rat brain membranes.

G protein activation by the agonist-occupied nociceptin- (orphanin FQ-) receptor in rat cerebral cortex was studied by characterizing the nociceptin-stimulated binding of the radiolabeled guanylyl triphosphate (GTP) analog 35S-guanylyl-5'-O-(gamma-thio)-triphosphate (GTPgammaS). Using 3H-Tyr14- and 125I-Tyr14-nociceptin in saturation and displacement receptor binding studies, a single high-affinity (Kd 21.6-116.7 pM) and high-capacity binding site for nociceptin (orphanin FQ) in membranes and sections of rat cerebral cortex was identified. Stable GTP analogs and NaCl lowered the affinity only moderately by 2- to 3-fold, but under these conditions nociceptin stimulated the binding of 35S-GTPgammaS to G proteins in the membranes with a potency about 100-fold lower (EC50 9.11 nM). It was estimated that this stimulation was due to a 29-fold increase in the affinity from Kd 45. 8 to 1.57 nM of only about 6.5% of the basal binding sites for GTPgammaS, and that at least 10 G protein binding sites could be stimulated by one receptor site. The link of this nociceptin-stimulated binding of GTP to the nociceptin receptor was further evidenced by the specificity of stimulation, as seen with nociceptin, nociceptin(1-13), D-Ala7-nociceptin and nociceptin(1-9), which paralleled that of their receptor affinities. Furthermore, the distribution in rat brain regions of the binding of 35S-GTPgammaS stimulated by nociceptin differed from that stimulated by the mu opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol)]-enkephalin. Especially, no stimulation by nociceptin was observed in caudate putamen, where also the absence of ORL1 receptors had been reported. The putative coupling of the high-affinity nociceptin receptor to the low-potency stimulation of GTPgammaS binding in rat cerebral cortex might be explained by the switch of a low part of occupied nociceptin binding sites to a very low-affinity state being stabilized at high peptide concentrations and catalytically stimulating the GTP binding.

Analgesics, Opioid↗

PubMed1997

Influence of continuous levels of fentanyl in rats on the mu-opioid receptor in the central nervous system.

The highly potent and efficacious mu-opioid agonist fentanyl was SC infused into rats with submaximal analgesic doses (0-1.14 mumol/kg/day) continuously for 8 days, checked by the constant daily urinary recovery of intact drug (0.43 +/- 0.031% of the daily dose). Tail-flick latencies measured at 24 (day 1) and 48 h (day 2) after starting the infusion were increased in a dose-dependent fashion compared with those before the infusion (day 0). However, at day 8, the latencies were increased only weakly, not significantly, revealing tolerance to the antinociceptive activity of fentanyl. Fentanyl at all doses showed no significant effect on the capacity (Bmax) and affinity (Kd) of the mu-opioid receptor binding of DAMGO to whole brain (Bmax 126.2 +/- 3.00 fmol/mg protein, Kd 1.00 +/- 0.04 nM) and spinal cord (Bmax 48.24 +/- 2.71 fmol/mg protein, Kd 1.93 +/- 0.13 nM) membranes gained from the rats after killing them at day 8. Gpp(NH)p increased the Kd for brain and spinal cord sites by 3.09 and 2.65, respectively, independent of the fentanyl dose. The infusion with fentanyl did not after the basal and forskolin-stimulated adenylate cyclase activity in the whole brain membranes, nor did it change the inhibition of the forskolin-stimulated activity by DAMGO. It is concluded that, in rats, constant long-term body levels of highly potent mu-agonists result in a tolerant state that, however, does not produce overall changes in the parameters of their specific receptor sites in the CNS, i.e., receptor capacity and affinity, and in the events closely related to them, i.e., their regulation by GTP and of adenylate cyclase. This does not exclude such possible changes to be restricted to specific regions in the CNS.

Adenylyl Cyclases↗

PubMed1996

The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes.

Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.

Antibodies↗