Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Identification of collagen alpha1(I) trimer in embryonic chick tendons and calvaria

Collagen with a molecular composition [α₁(I)]₃ has been identified in acetic acid extracts from lathyritic chick embryo tendons and calvaria. These molecules characteristically have greater solubility than Type I collagen at neutral pH and contain increased amounts of hydroxylysine residues. It is suggested that these molecules represent a separate gene product of embryonic cells which may be important in the process of maturation and development.     

The size of collagen fibrils that stimulate platelet aggregation in human plasma.

Suspensions of collagen fibrils of different size were prepared from solutions of radioactive tropocollagen type I by either differential centrifugation or differential incubation at elevated temperature. The fractions were compared with respect to their ability to stimulate human blood platelet aggregation in plasma, their binding to human platelets, and their morphology, as seen in the electron microscope. Although small particles with a sedimentation coefficient as low as 4.5 S bound to platelets, aggregation was not observed in the presence of collagen multimers and protofibrils without visible cross-bands in stained specimens. The onset of platelet-aggregating activity before the appearance of turbidity in collagen solutions incubated at elevated temperature is due to the formation of a few banded fibrils; this early onset and the fibrils do not appear in collagen solutions that have been ulctracentrifuged before incubation.     

Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss mouse 3T3 cells

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F_2α alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF_2α stimulation. In contrast tunicamicin does not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF_2α     

Immune recognition of affinity-labelled cholecystokinin receptor

The present study was undertaken to characterize the immune recognition of pancreatic cholecystokinin receptor by an anti-cholecystokinin antibody. Cholecystokinin receptor from pancreatic plasma membranes was photoaffinity labelled using the specific, cleavable probe 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK(25-33) [CCK(25-33) is the C-terminal nonapeptide of the 33-amino-acid form of cholecystokinin]. Labelled receptor was then solubilized and subsequently prepurified on immobilized wheat-germ agglutinin. The C-terminal-directed anti-cholecystokinin serum (8E) specifically immunoprecipitated a fraction of affinity-labelled cholecystokinin receptor which was identified at Mr 85,000 - 100,000 on SDS/PAGE. The binding affinity of antiserum 8E for covalently labelled cholecystokinin receptor was lower (Kd 0.11 +/- 0.02 nM) than for cholecystokinin (Kd 3.65 +/- 0.55 pM). The compound L364-718, an A-subtype cholecystokinin-receptor antagonist did not interfere with the immune recognition of cholecystokinin. However, the recognition of affinity-labelled cholecystokinin receptor was enhanced as a result of an increasing availability of cholecystokinin molecules. Indeed, the amount of immunoprecipitated receptor was doubled in the presence of 10 microM L364-718. This study offers the possibility of using an anti-cholecystokinin antibody for cholecystokinin-receptor purification and demonstrates that prepurified affinity-labelled cholecystokinin receptor retains A-subtype specificity.     

Ischemic reperfusion injury in rabbit spinal cord: protective effect of superoxide dismutase on neurological recovery and spinal infarction

The potential role of superoxide dismutase (SOD), a specific superoxide anion radical scavenger, in treating spinal cord ischemia was investigated in rabbits subjected to aortic occlusion for 20 min. SOD treatment, targeted to the early reperfusion period, reduced both motor dysfunction and incidence of spinal infarcts at 7 days after ischemia. Present results suggest that oxygen-derived free radicals play a role in the pathogenesis of infarcts developing in the spinal cord after ischemia and reperfusion injuries.     

Induced release and metabolism of arachidonic acid from myeloid cells by purified colony-stimulating factor

The in vitro incubation of cells from turpentine-induced rat myeloid hyperplastic marrow and peritoneal monocyte/macrophage with 14C-arachidonic acid resulted in the incorporation of the radiolabel into the particulate phospholipids. Challenge of the radiolabeled cells with a highly purified type I CSF (CSF I) from human pancreatic carcinoma cells in continuous culture resulted in the hydrolysis and release of the 14C-arachidonic acid from the cellular phospholipids. The simultaneous challenge of the prelabeled cells with CSF-I and its specific antibody (anti-CSF-I antibody) inhibited the CSF-I induced hydrolysis of 14C-arachidonic acid from the cells. These results confer a specificity on the CSF-I induced release of arachidonic acid from the cellular phospholipids. Our data also demonstrated that the 14C-arachidonic acid released from the cellular phospholipids was further transformed into products of the cyclooxygenation and lipoxygenation pathways by cellular enzyme systems in both populations of cells. Interestingly, our data also indicate that the challenge of the granulocytic hyperplastic marrow cells and the monocyte/macrophage cells with purified CSF-I resulted in a higher generation of lipoxygenase products in the predominantly granulocytic cell population than in the population rich in monocyte/macrophage cells. The biological significance of this observation remains to be further explored. Thus, the CSF-I induced release of cellular arachidonic acid explains, at least in part, the presence of prostaglandins and other metabolites of arachidonic acid that are found in the media of hemopoietic cells incubated with a variety of CSF preparations.     

Epidermal growth factor initiates DNA synthesis after a time-dependent sequence of regulatory events in Swiss 3T3 cells—interactions with hormones and growth factors

Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14–15 hours. The final rate of initiation follows apparent first-order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4–6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases k, but after a delay of 4–6 hours. In contrast, both hydrocortisone and prostaglandin E₁ (PGE₁) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k. Prostaglandin F_2α (PGF_2α), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF_2α, nevertheless, act through different regulatory events.     

Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.