In vivo and in vitro effects of angiotensin II on the release of beta-endorphin-like immunoreactivity

The effect of angiotensin II (A II) on the release of beta-endorphin-like immunoreactivity (beta-END-LI) in rats was studied in vivo and in vitro. Intravenous injection of 1 microgram/100 g body weight of A II resulted in significant increase in the plasma beta-END-LI level after 10 and 20 min. Intraventricular injection of 1 ng/100 g body weight of A II also resulted in significant increase in the plasma beta-END-LI level after 10 min. A II at concentrations of 10(-12) M-10(-10) caused dose-dependent stimulation of beta-END-LI release from dispersed cells of rat anterior pituitary. On gel chromatography, the beta-END-LI released by incubation of the cells with 10(-10) M A II separated into two components which eluted in the same positions as human beta-lipotropin and human beta-endorphin, respectively. The ratio of beta-LPH to beta-END in these fractions was 5:1 on a molar basis. A II did not stimulate beta-END-LI release in Ca++-free-medium. [Sar1, Ala8]-A II at concentrations of 10(-9) M - 10(-7) M did not stimulate beta-END-LI release from the cells. Addition of [Sar1, Ala8]-A II to the incubation medium inhibited A II-induced beta-END-LI release from the cells. These results indicate that A II acts, at least in part, directly on anterior pituitary cells to stimulate beta-END-LI release and that calcium ion is involved in the mechanism of this effect.     

Absence of effect of estrogen on dopamine, norepinephrine and beta-endorphin-like immunoreactivity in human plasma

The plasma concentrations of immunoreactive norepinephrine (NE), dopamine (DA), beta-endorphin (beta-E), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined by RIA and HPLC every 6 h until 72 h after iv administration of conjugated estrogens during the midfollicular phase. The LH level showed a biphasic pattern after the injection of conjugated estrogens, i.e. significant suppression (-50%) for 6-42 h after the injection, followed by a rebound increase with a peak (+85%) at 72 h. The plasma levels of immunoreactive beta-E, NE and DA did not change significantly for 72 h after the injection.     

Adrenergic and dopaminergic regulation of circulating beta-endorphin-like immunoreactivity in hypertension.

The central alpha-2-adrenergic receptor agonist, clonidine (300 micrograms daily) significantly increased the plasma beta-endorphin-like immunoreactivity (beta ELI) in 12 patients with mild to moderate essential hypertension in a randomized, crossover study. A significant linear correlation between the increase in plasma beta ELI and the decrease in blood pressure (both systolic and diastolic) was found after clonidine administration. The role of the reduced central sympathetic tone, induced by alpha-2-adrenoceptor stimulation, in the elevation of circulating beta ELI can be suggested. The plasma beta ELI increased also significantly after the dopaminergic D-2 receptor agonist, bromocryptine treatment, (5 mg, daily) in 13 patients with borderline and mild essential hypertension in a randomized, crossover study. A significant drop in circulating noradrenaline and in arterial blood pressure and a significant linear correlation between the changes of plasma noradrenaline level and blood pressure was found after bromocryptine administration. There was no correlation between the rise in plasma beta ELI and the decrease in blood pressure after bromocryptine. The importance of the central sympathetic activity and not only a direct pituitary dopaminergic agonist effect on the beta-endorphin secretion can be stressed in the effect of bromocryptine on the immunoreactive beta-endorphin level.     

Two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes

While attempting to delineate the reason for the reported extreme variability of beta-endorphin-like immunoreactivity (beta-ir) in human plasma (eg., nondetectable to 1 ng/ml) by standard radioimmunoassay, we noted that a substantial portion of circulating beta-ir was associated with erythrocytes. That erythrocyte associated beta-ir is authentic beta-endorphin (beta-EP) was confirmed by high performance liquid chromatography (HPLC). Analysis of blood samples from rabbits, rats and mice revealed the presence of beta-ir in erythrocytes from these species as well. These results suggest that there are two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes.     

Somatostatin release from perifused rat and human gastric mucosa

In the present study the release of somatostatin-like immunoreactivity (SLI) was evaluated in vitro from isolated rat antral and fundic mucosa and from biopsy specimens of human antral mucosa. Perifusion of antral mucosa with Earle's balanced salt solution showed a pH-dependent release of SLI. SLI release did not change in response to a reduction from pH 7 during the baseline period to pH 3, whereas a significant increase occurred when the pH was changed to 2.5 or 2, respectively. Fundic SLI release remained at baseline levels during the decrease of the pH value of the buffer solutions. Atropine at doses of 10(-6) to 10(-4) M did not alter acid-induced SLI release from the isolated antral mucosa, suggesting different mechanisms in vitro compared to the acid-induced SLI release in vivo. SLI release from human mucosa was 450 +/- 217 pg/min X mg wet weight in response to perifusion with the buffer pH 2 in 7 control subjects. No significant difference was observed in patients with duodenal ulcer or acute gastritis, whereas gastric ulcer patients had significantly lower values (66 +/- 44) compared to controls and duodenal ulcer patients. These data do not support the hypothesis that impaired somatostatin production and release might be a pathogenetic factor for gastric acid hypersecretion and development of duodenal ulcer.     

Effects of temperature and pH on hemoglobin release from hydrostatic pressure-treated erythrocytes

The release of hemoglobin from human erythrocytes hemolyzed beforehand by hydrostatic pressure, osmotic pressure, and freeze-thaw methods was examined as a function of temperature (0-45 degrees C) and pH (5.5-8.8) at atmospheric pressure. Only in the case of high pressure (2,000 bar) did the release of hemoglobin increase significantly with decreasing temperature and pH. Maleimide spin label studies showed that the temperature and pH dependences of hemoglobin release were qualitatively explicable in terms of those of the conformational changes of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins showed the diminution of band intensities corresponding to spectrin, ankyrin, and actin in the erythrocytes hemolyzed by high pressure. Cross-linking of cytoskeletal proteins by diamide stabilized the membrane structure against high pressure and suppressed hemoglobin release. These results indicate that the disruption of cytoskeletal apparatus by high pressure makes the membrane more leaky.     

Adrenocorticotropin- and beta-endorphin-like substances in brains of the freshwater snake Ptyas mucosa

Snake (Ptyas mucosa) brains (400 g) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate (5.6 g) that formed upon addition of five volumes of acetone to the extract, designated acid-acetone powder, was subjected to gel filtration on Sephadex G-25. A large unretarded peak (SB-1) with molecular weight greater than 5000 and a small retarded peak (SB-2) with molecular weight smaller than 5000 were obtained. They were then separately subjected to ion-exchange chromatography on CM-cellulose. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal cells to produce corticosterone. Opiate activity was detected in the fractions by their ability to inhibit the binding of (D-Ala2,D-Leu5)-[tyrosyl-3,5-3H]enkephalin to rat brain membranes and their cross-reactivity in a beta-endorphin radioimmunoassay. Adrenocorticotropic and opiate activities were found to be concentrated in fractions strongly adsorbed on CM-cellulose, which were eluted by combined pH and ammonium acetate concentration gradients. There appeared to be a separation between adrenocorticotropic and opiate activities, suggesting that they were due to separate molecular entities.     

The effect of cyclic nucleotides on secretin secretion in canine duodenal mucosa in vitro

We examined the effects of cyclic nucleotides and calcium on secretin release from canine duodenal mucosal explants incubated in organ culture media. Time course studies revealed that at pH 7.4, 5 and 10 mM dibutyryl cyclic adenosine monophosphate (DBcAMP) increased secretin release progressively, reaching a peak at 2 hours. Two mM of DBcAMP at pH 7.4 did not increase secretin release but at pH 4.5, all 3 doses potentiated secretin release. DBcAMP-stimulated secretin release was not dependent on the influx of extracellular calcium. Graded doses of 3-isobutyl-1-methylxanthine (IBMX) did not stimulate secretin secretion but 1 mM IBMX with 2 mM DBcAMP increased secretin secretion significantly. Dibutyryl cyclic guanosine monophosphate, cholera toxin and 5'-guanylyl-imidodiphosphate (GPP(NH)p) did not stimulate basal secretion release. The release of secretin from our explants incubated at pH 7.4 was not due to specific leakage because all of our viability studies revealed that our explants were functionally intact at the end of 2 hours. Our observations suggest that cyclic nucleotides may participate in the intracellular regulation of secretin secretion.     

Differences in the release of cholesterol from taurocholate versus taurochenodeoxycholate micellar solutions

The maximal micellar solubility, distribution and apparent monomer activity of cholesterol in taurine-conjugated cholate and chenodeoxycholate micellar solutions were studied to clarify the different modulating effect of these bile salt species on cholesterol uptake in an intestinal lumen. The maximal micellar solubility was significantly greater in taurochenodeoxycholate. The intermicellar cholesterol monomer concentration was not significantly different between the two kinds of micellar solution. However, the apparent cholesterol monomer activity determined using an artificial organic phase (polyethylene disc) was significantly higher in taurocholate than that in taurochenodeoxycholate. A linear relationship between the intermicellar cholesterol concentration and the apparent cholesterol monomer activity was found, with the slope depending upon the bile salt species. It is concluded that the difference in partitioning of cholesterol from taurocholate and taurochenodeoxycholate micelles into a fixed organic phase may contribute in part to the different regulating effects of these bile salts on the uptake of cholesterol in the intraluminal phase.     

Effects of ouabain on frog gastric mucosa in vitro

The effect of the addition of ouabain to the nutrient solution was determined on resistance, potential difference (p.d.) and H⁺ secretion rate. In NaCl media, 10^?3 M ouabain decreased significantly the p.d. from 25.6 mV to 16.1 mV in 30 min and to 11.0 mV in 60 min. NO significant changes occurred in resistance and H⁺ secretion rate. In Na₂SO₄ (Cl^?-free) media, ouabain produced a biphasic effect on p.d. The p.d. changed from ?28.0 mV (nutrient-negative) to a nadir of ?37.4 mV in 7 min and then increased to ?16.4 mV in 60 min. At the nadir there was no significant change in resistance or H⁺ secretion rate but at 60 min, unlike Cl^? media, resistance increased by 36% and H⁺ secretion rate decreased by 43%. To decide whether the ouabain-caused decrease in H⁺ rate in Na₂SO₄ media was due to an effect on the H⁺ pump or on resistance of the return pathways, the voltage was clamped at 0 and 40 mV. Clamping the voltage showed that in the case of a marked decrease in the H⁺ secretion rate, the H⁺ transport mechanism itself was inhibited (and not the parallel pathway). The decrease in p.d. due to ouabain in Cl^? and SO₄^2? media indicates that the (Na⁺ + K⁺)-ATPase mechanism may be electrogenic.     

Effects of remifentanyl and fentanyl on LPS-induced cytokine release in human whole blood in vitro

Aim The present study sought insight into the effects of remifentanyl and fentanyl on LPS-induced release of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-10 in human whole blood. Methods Whole blood was incubated in the presence and absence of remifentanyl and fentanyl. Effects of remifentanyl and fentanyl on spontaneous and endotoxin (lipopolysaccharide; 100 ng ml⁻¹)-stimulated cytokine release were studied in whole blood from volunteers (n = 10) cultured for 6 h. Results IL-6, TNF-α and IL-10 concentrations in groups added with LPS were significantly higher than those in control group (P < 0.01). IL-6, TNF-α and IL-10 concentrations in activation groups treated with remifentanyl or fentanyl were significantly lower than those in LPS treated group (P < 0.05). There were no significant differences on IL-6,TNF-α and IL-10 concentrations in drug-alone groups compared with control group (P > 0.05). Conclusion Remifentanyl or fentanyl alone has no effects on IL-6, TNF-α and IL-10 production, but could attenuate LPS-induced IL-6,TNF-α and IL-10 production in human whole blood. Remifentanyl and fentanyl could inhibit the expressions of IL-6, TNF-α and IL-10 induced by LPS.     

Prostaglandin release from the human umbilical artery in vitro

Release of prostaglandins from human umbilical artery preparations into the surrounding bathing fluid was studied by radioimmunoassay using PGF_2α antibodies. A significant release of prostaglandins was found under conditions where a spontaneous tone of the artery could be maintained. Indometacin reduced the prostaglandin release and the spontaneous tone of the artery. Intramural synthesis of prostaglandins in the human umbilical arteries is postulated.     

The effect of pH on the kinetics of iron release from human transferrin

The rate of iron release from the N-terminal and C-terminal monoferrictransferrins (Fe_N-transferrin and Fe_C-transferrin, respectively) has been studied at 37°C over the pH range 3.5–10.6 using EDTA as the accepting chelate. Fe_N-transferrin is the more facile except above pH 8.2. Plots of log₁₀k_obs against pH showed a deviation for both monoferrictransferrins between pH 5.6 and 6.0 and studies above and below this transition point indicated that iron release occures by different mechanisms. At low pH (< 5.6) the rate of release from Fe_N-transferrin is independent of the presence of EDTA or NaClO₄, whereas Fe_c-transferrin shows a small but significant increase with increasing EDTA concentration. Rapid protonation of both monoferrictransferrins is followed by relatively slow release of Fe³⁺ which is subsequently chelated by EDTA. The slower release from Fe_c-transferrin is probably due to its greater binding strength for iron and the greater conformational stability of the C-terminal domain. Above pH 6.0 iron release from both monoferrictransferrins increases as the concentration of EDTA is increased. Direct attacks of EDTA probably occurs giving Fe-transferrin (HCO₃). EDTA as a transition state or intermediate. The factors which may lead to the observed pH dependence of the rate include (i) protonation of groups directly bound to the iron, (ii) conformers which differ in degree of protonation and (iii) the degree of protonation of the attacking chelating agent. It is suggested that an increase in conformational fluctuations as the pH is lowered may play a very important role. Studies with differrictransferrin at pH 4.53 and 7.40 showed that when iron is released to EDTA the rate is independent of the occupancy of the other site; that is, the two sites are exhibiting non-co-operativity.     

Keratinocyte differentiation of human buccal mucosa in vitro

The process of differentiation in keratinocytes of human buccal mucosa is accompanied by a number of specific membrane and cytoplasmic changes. Using simple tissue culture techniques, it has been possible to establish an in vitro model for keratinocyte differentiation which closely resembles the in vivo situation. A multi-layered structure is formed with maturation towards the surface. The superficial cells are characterized by a thickening of the plasma membrane, an increased concentration of tonofilaments, loss of intercellular adhesions and the presence of membrane-coated granules. It is concluded that this represents a suitable in vitro model for the biological and pathological study of both normal and abnormal oral epithelium.