Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes.

Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects received 5,000 IU rHuEpo every second day for 14 days, and subsequently a single dose of 5,000 IU weekly for 12 weeks. Muscle biopsies were obtained before and after 14 weeks of rHuEpo treatment. The treatment increased hematocrit (from 44.7 to 48.8%), maximal oxygen uptake by 8.1%, and submaximal performance by approximately 54%. Membrane transport systems and carbonic anhydrases involved in pH regulation remained unchanged. Of the Na(+), K(+)-pump isoforms only the density of the alpha2 subunit was decreased (by 22%) after treatment. The marker enzymes cytochrom c and hexokinase remained unchanged with the treatment. In conclusion, changes in muscle membrane transport proteins and selected muscle enzymes do not contribute to the Epo-induced improvement in performance.

Prolonged administration of recombinant human erythropoietin increases submaximal performance more than maximal aerobic capacity.

The effects of recombinant human erythropoietin (rHuEpo) treatment on aerobic power (VO2max) are well documented, but little is known about the effects of rHuEpo on submaximal exercise performance. The present study investigated the effect on performance (ergometer cycling, 20-30 min at 80% of maximal attainable workload), and for this purpose eight subjects received either 5,000 IU rHuEpo or placebo every second day for 14 days, and subsequently a single dose of 5,000 IU/placebo weekly/10 weeks. Exercise performance was evaluated before treatment and after 4 and 11 weeks of treatment. With rHuEpo treatment VO2max increased (P<0.05) by 12.6 and 11.6% in week 4 and 11, respectively, and time-to-exhaustion (80% VO2max) was increased by 54.0 and 54.3% (P<0.05) after 4 and 11 weeks of treatment, respectively. However, when normalizing the workload to the same relative intensity (only done at time point week 11), TTE was decreased by 26.8% as compared to pre rHuEpo administration. In conclusion, in healthy non-athlete subjects rHuEpo administration prolongs submaximal exercise performance by about 54% independently of the approximately 12% increase in VO2max.

K+ as a vasodilator in resting human muscle: implications for exercise hyperaemia.

AIM: Potassium (K(+)) released from contracting skeletal muscle is considered a vasodilatory agent. This concept is mainly based on experiments infusing non-physiological doses of K(+). The aim of the present study was to investigate the role of K(+) in blood flow regulation. METHODS: We measured leg blood flow (LBF) and arterio-venous (A-V) O(2) difference in 13 subjects while infusing K(+) into the femoral artery at a rate of 0.2, 0.4, 0.6 and 0.8 mmol min(-1). RESULTS: The lowest dose increased the calculated femoral artery plasma K(+) concentration by approx.1 mmol L(-1). Graded K(+) infusions increased LBF from 0.39 +/- 0.06 to 0.56 +/- 0.13, 0.58 +/- 0.17, 0.61 +/- 0.11 and 0.71 +/- 0.17 L min(-1), respectively, whereas the leg A-V O(2) difference decreased from 74 +/- 9 to 60 +/- 12, 52 +/- 11, 53 +/- 9 and 45 +/- 7 mL L(-1), respectively (P < 0.05). Mean arterial pressure was unchanged, indicating that the increase in LBF was associated with vasodilatation. The effect of K(+) was totally inhibited by infusion (27 micromol min(-1)) of Ba(2+), an inhibitor of Kir2.1 channels. Simultaneous infusion of ATP and K(+) evoked an increase in LBF equalled to the sum of their effects. CONCLUSIONS: Physiological infusions of K(+) induce significant increases in resting LBF, which are completely blunted by inhibition of the Kir2.1 channels. The present findings in resting skeletal muscle suggest that K(+) released from contracting muscle might be involved in exercise hyperaemia. However, the magnitude of increase in LBF observed with K(+) infusion suggests that K(+) only accounts for a limited fraction of the hyperaemic response to exercise.

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

[Optimizing the distribution and test locations in statistical perimetry. Preliminary results]. / Optimierung der Verteilung und der Anzahl Testorte in der statischen Perimetrie. Vorläufige Resultate.

BACKGROUND: Are short programs useful to glaucoma-screening? We investigated the concept of Program G1x with Octopus 1-2-3, which permits examination of various numbers of test locations i.e., 16, 32, 45, or all 59 test locations. PATIENTS AND METHODS: Using 99 visual fields of glaucomatous or glaucoma-suspect right eyes, we compared mean defect (MD) and loss variance (LV) of the global visual field with MD and LV of the stages of G1x-program (standard strategy). RESULTS: The results showed that stage 1 of Program G1x underestimated the glaucomatous visual field damage present in the entire field. CONCLUSIONS: Thus, the use of only the first stage (16 test locations) of G1x is not advisable. At least 32 test locations, i.e., stages 1 and 2 of program G1x, are recommended for clinical perimetry.

Corticosteroids reduce regenerative repair of epithelium in experimental gastric ulcers.

The association between corticosteroid treatment and gastric ulcer healing is controversial. The effects of corticosteroids on experimental ulcer healing in the rat were studied and the effect of coadministration of a prostaglandin E1 analogue was determined. Forty male adult rats were divided into five groups and pretreated for 10 days as follows: (a) control, (b) prednisolone (10 mg/kg), (c) prednisolone and misoprostol (300 micrograms/kg), (d) misoprostol, and (e) indomethacin (2 mg/kg) Gastric ulcer was induced by applying a cryoprobe to the serosal surface of the stomach. Healing was assessed by determining the ulcer size at three and six days. Mucosal regenerative activity at the ulcer edge was assessed by quantitating DNA synthesis in cells by immunohistochemical techniques using monoclonal antibodies to detect expression of proliferating cell nuclear antigen (PCNA) and incorporated 5-bromo-5-iododeoxyuridine (BrdU). Compared with control rats, the rate of healing and the mucosal regenerative activity were significantly reduced in rats treated with prednisolone and indomethacin (p < 0.05). Coadministration of misoprostol and corticosteroids reversed the delay in healing and the reduction in mucosal regeneration induced by corticosteroids alone. With misoprostol alone, the ulcer size and the number of epithelial cells that actively synthesised DNA did not differ from control animals. A comparison between the two immunohistochemical markers of cell proliferation showed a highly significant correlation between the two techniques (r = 0.64, p < 0.005), indicating that the simpler PCNA technique should prove valuable in assessing regeneration in experimental ulcer disease.

Agonist-stimulated phosphorylation of the carboxyl-terminal tail of the secretin receptor.

The secretin receptor belongs to a recently recognized family of G protein-coupled receptors that lack the sequence motifs typical of the beta-adrenergic receptor family. Because our understanding of the regulatory mechanisms for these receptors is largely based on the latter group, we have begun to explore these mechanisms in the secretin receptor. In the present study, we focused on receptor phosphorylation, a key mechanism of receptor desensitization. Secretin receptor phosphorylation was demonstrated in intact transiently transfected COS cells and a stable receptor-bearing Chinese hamster ovary cell line in response to stimulation with native agonist. Secretin phosphoreceptor migrated on a sodium dodecyl sulfate-polyacrylamide gel at M(r) 57,000-62,000 in its native state and at M(r) 42,000 after deglycosylation, similar to the receptor that had been affinity-labeled with 125I-[Tyr10,p-NO2-Phe22]-secretin-27. Phosphorylation occurred rapidly in a secretagogue concentration-dependent manner, with 0.1 microM secretin eliciting a 7.2-fold increase in phosphorylation after 2 min. One-dimensional phosphopeptide mapping after cyanogen bromide cleavage revealed a single band of M(r) 9400, corresponding in size to the carboxyl-terminal tail domain. This identification was confirmed with a truncation mutant in which potential sites of phosphorylation in the tail were eliminated and no agonist-stimulated phosphorylation was observed. Phosphoamino acid analysis of the secretin phosphoreceptor demonstrated predominance of phosphothreonine over phosphoserine (3.2:1), with no phosphotyrosine observed. Three distinct carboxyl-terminal truncation mutants were constructed to each eliminate a subset of potential phosphorylation sites, and differential levels of phosphorylation were observed. Appropriate biosynthetic processing, expression on the cell surface, and signaling for each of these constructs were ensured by demonstration of ligand binding and cAMP responsiveness. Thus, receptors in the recently described secretin receptor family are phosphorylated in response to agonist stimulation in a manner analogous to the beta-adrenergic receptor, likely representing an important molecular mechanism for receptor desensitization.

Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell.

Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell could be particularly effective to protect the cell from processes which might initiate pancreatitis, while providing for the rapid resensitization of this receptor to ensure appropriate pancreatic secretion to aid in nutrient assimilation for the organism.

Dual pathways of internalization of the cholecystokinin receptor.

Receptor molecules play a major role in the desensitization of agonist-stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agonist-stimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403-2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and peak of receptor phosphorylation after agonist occupation correlates best with a plasmalemmal localization, while stimulated receptor phosphatase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjacent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.

Clinical implications of liver biopsy findings in collagen-vascular disorders.

We surveyed over a 5-year period liver abnormalities in all patients with collagen-vascular disorders in whom liver histology was obtained, including 46 patients with systemic lupus erythematosus, rheumatoid arthritis, primary Sjogren's syndrome, periarteritis nodosa, mixed cryoglobulinemia, Wegener's granulomatosis, systemic sclerosis, and other conditions. Histological appearances diagnostic of the primary condition were only found in three patients, each of whom had periarteritis nodosa. Significant chronic liver disease was found in 11 patients (24%), in five of whom a strong clinical suspicion of severe chronic liver disease already existed. Clinically inapparent but potentially significant chronic liver disease was found predominantly in patients with mixed cryoglobulinemia or sicca syndrome. Seventeen percent of all biopsies suggested drug induced hepatitis. In patients with collagen-vascular diseases and abnormal liver function tests, histological examination of the liver is most frequently of value in indicating drug-induced liver damage. Significant chronic liver disease is common but usually clinically apparent. In patients with periarteritis nodosa and mixed cryoglobulinemia, liver biopsy may be of value diagnostically, revealing serious liver disease with prognostic and therapeutic implications.

[Problems of instituting insulin therapy in advanced age]. / Probleme bei der Realisierung einer Insulintherapie im höheren Lebensalter.

The population of diabetics of a district was analysed concerning the age structure, the proportion of the insulin-dependent patients as well as the insulin-dependent diabetics older than 60 years were analysed with regard to the degree of the realization of insulinisation. 13.8% of all diabetics with an age older than 60 years are insulin-dependent. In 71.5% of them the insulinisation is realized. Only in 43% of all insulin-dependent older diabetics an autoinjection of insulin is possible. The results are discussed and propositions for changing the situation are submitted.

[Significance and scope of interdisciplinary cooperation in the specialty of internal medicine (inpatients)]. / Bedeutung und Umfang der interdisziplinären Zusammenarbeit im Fachgebiet Innere Medizin (stationär).

In a department of internal medicine of a district hospital all consultations given by representatives of non-internal medical specialties and by specialized or highly specialized representatives of the subject of internal medicine in a period of five months were registered. In 29% of all patients attended in the period of observation consultation achievements were demanded. 65% of these achievements could be realized by physicians working in hospitals or outpatient departments of the district. 23% of the consultations were carried out by specialists from institutions conducted by county authorities. 11% of the consultations were carried out by highly specialized physicians of university clinics and academy institutes. The investigation confirms the high significance of the inter-disciplinary cooperation for the subject of internal medicine. cooperation for the subject of internal