Microwaves and the cell membrane. III. Protein shedding is oxygen and temperature dependent: evidence for cation bridge involvement

Microwaves (2450 MHz, 60 mW/g) are shown to result in the release or shedding of at least 11 low-molecular-weight proteins (less than or equal to 31,000 Da) from rabbit erythrocytes maintained in physiological buffer. Protein release was detected by gel electrophoresis of cell-free supernatants using sensitive silver staining. This release is oxygen dependent and occurs in 30 min for exposures conducted within the special temperature region of 17-21 degrees C, which is linked to a structural or conformational transition in the cell membrane. Shedding of 26,000 and 24,000 Da proteins is unique to microwave treatment, with enhanced release of 28,000 and less than or equal to 15,000 Da species during microwave compared to sham exposures. Two-dimensional isoelectric focusing further reveals that proteins of less than or equal to 14,000 Da shed during microwave treatment exhibit a pI of 6.8-7.3 not seen in sham-treated cells. Treatment of erythrocytes with a serine-directed protease inhibitor does not prevent release of proteins. However, when erythrocytes are maintained at 17-21 degrees C by conventional heating in the absence of divalent cations, release of 28,000-31,000 and less than or equal to 14,000 Da components is detected. This indicates that cation-bridge stability may be important for release of these proteins. The above results provide evidence that microwaves alter erythrocyte protein composition at temperatures linked to a transition in the cell membrane and that destabilization of salt bridges may play a role in an interaction mechanism for protein release.     

In vivo targeting of inflamed areas by electroloaded neutrophils.

Due to their spontaneous accumulation in inflamed or infected areas, blood phagocytes are potent drug vectors with specific targeting. Drug like molecule loading was obtained by use of cell electropermeabilization in which the impermeability of their plasma membrane is transiently impaired. Electrical conditions were used which allow electroloading of a drug like molecule (propidium iodide) in 70% of leukocytes in a whole blood sample while preserving in vitro functional properties. Slow release of entrapped hydrophilic molecules was observed with a half lifetime longer than 4 hours at 4 degrees C and at 37 degrees C. With an in vivo assay, using a rat model of inflammation, we showed that, as for non-pulsed cells, pulsed neutrophils accumulate 10 times more in an inflamed area than they do in control areas. Phagocyte electropermeabilization is therefore a very efficient way of drug targeting. Accumulation of electropulsed neutrophils in an area of inflammation gives targeted release of the electroloaded drug.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Correction of defective protein trafficking of a mutant HERG potassium channel in human long QT syndrome. Pharmacological and temperature effects.

The chromosome 7-linked form of congenital long QT syndrome (LQT2) is caused by mutations in the human ether-a-go-go-related gene (HERG) that encodes the rapidly activating delayed rectifier potassium channel. One mechanism for the loss of normal channel function in LQT2 is defective protein trafficking, which results in the failure of the channel protein to reach the plasma membrane. Here we show that the N470D LQT2 mutant protein is trafficking-deficient when expressed at 37 degrees C in HEK293 cells, whereas at 27 degrees C its trafficking to the plasma membrane and channel function are markedly improved. We further show that the antiarrhythmic drug E-4031, which selectively blocks HERG channels, also corrects defective protein trafficking of the N470D mutant and can restore the generation of HERG current. Similar findings were obtained with the drugs astemizole and cisapride, as well as with high concentrations of glycerol. The effect of E-4031 on HERG protein trafficking was concentration-dependent and required low drug concentrations (saturation present at 5 microM), developed rapidly with drug exposure, and occurred post-translationally. These findings suggest that protein misfolding leading to defective trafficking of some HERG LQT mutations may be corrected by specific pharmacological strategies.     

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.     

Thyroid function in a lizard, a tortoise and a crocodile, compared with mammals

1. Thyroid activity was examined in the lizard, Trachydosaurus rugosus, the tortoise Chelodina longicollis and the crocodile, Crocodylus johnstoni, acclimated to 20-22 degrees C and 30-32 degrees C. Thyroidal uptake and release of 125I, plasma concentrations of T3 and T4 were measured as was resting oxygen consumption (at 30 degrees C) before and after both thyroidectomy and thyroxine injections. 2. All three species showed 125I uptake at both temperatures and showed no thyroidal release of 125I at 20-22 degrees C but exhibited thyroidal release of 125I (and presumably hormone secretion) at 30-32 degrees C. 3. Plasma concentrations of thyroxine ranged from 0.55 nM to 3.24 nM and triiodothyronine from 0.14 nM to 0.51 nM. 4. Neither thyroidectomy nor thyroxine injections had any effect on metabolic rate in 20-22 degrees C acclimated lizards. Thyroidectomy resulted in a significant decrease in metabolic rate in 30-32 degrees C acclimated lizards and tortoises and thyroxine injections resulted in significant increases in metabolism in 30-32 degrees C acclimated lizards, tortoises and crocodiles. 5. A comparison of thyroid parameters in reptiles and mammals concluded that although the reptilian thyroid is active at high temperatures it is still considerably less active than it is in mammals.     

Microwave-induced changes in nerve cells: effects of modulation and temperature

Helix aspersa neurons were irradiated with continuous-wave (CW) and noise-amplitude-modulated microwaves (carrier frequency 2450 MHz, 20% AM, 2 Hz-20 kHz) in a specially designed waveguide exposure system. Continuous-wave microwave irradiations were conducted at 8 degrees, 21 degrees, and 28 degrees C, while noise-modulated irradiation was performed at 21 degrees C. The results showed that exposure of snail neurons to CW microwaves for 60 min at 12.9 W/kg inhibited spontaneous activity and reduced input resistance at 8 degrees and 21 degrees C but not at 28 degrees C. The relative decrease in resistance at 21 degrees C was half that at 8 degrees C. Exposure of neurons to noise-modulated microwaves at 6.8 and 14.4 W/kg predominately caused excitatory responses characterized by augmented membrane resistance and the appearance of greater activity. The effect differed qualitatively from the inhibition observed with continuous, unmodulated microwave irradiation.     

Effects of temperature and oxygen availability on circulating catecholamines in the toad Bufo marinus.

The release of catecholamines during hypoxia has received limited attention in amphibians and the adrenergic regulation of cardio-pulmonary functions is, therefore, not well understood at the organismic level. To describe the changes in plasma catecholamine concentrations, we exposed toads (Bufo marinus) to different levels of hypoxia at two temperatures (15 and 25 degrees C). In addition, blood oxygen binding properties were determined in vitro at 15 and 25 degrees C at two different pH values. Hypoxia elicited a significant increase in plasma catecholamines (adrenaline and noradrenaline) at both temperatures, in spite of a respiratory alkalosis. At 15 degrees C, the increase was from 2.6+/-1.0 in normoxia to 4.8+/-1.4 ng ml(-1) at an inspired oxygen fraction of 0.05. At 25 degrees C, the hypoxic release of catecholamines was significantly higher (maximum levels of 44.8+/-11.6 ng ml(-1)). Plasma noradrenaline concentration was elevated at the most severe hypoxic levels, suggestive of an adrenal release. The arterial oxygen threshold for catecholamine release were approximately 1.0 mmol O(2) l(-1) blood or a PaO(2) of 30 mmHg. The P(50) values at 15 degrees C were 23.5+/-0.7 and 28.9+/-1.0 mmHg at pH 7.98+/-0.01 and 7.62+/-0.02, respectively, and increased to 36.5+/-0.6 and 43.0+/-1.1 mmHg at pH 8.04+/-0.04 and 7.67+/-0.05, respectively, at 25 degrees C. The oxygen equilibrium curves were linear when transformed to Hill-plots and Hills n (the haemoglobin subunit co-operativity) ranged between 2.24 and 2.75. The in vitro blood O(2) binding properties corresponded well with in vivo data.     

Stimulation of sperm motility and oxygen consumption of fowl spermatozoa by a low molecular weight fraction of seminal plasma

Washed fowl spermatozoa were incubated in a phosphate buffer containing various concentrations of fowl seminal plasma at 41 degrees C, normal body temperature, and the motility and oxygen consumption of spermatozoa were determined. Immediately after the incubation, spermatozoa showed good motility in the various diluents. However, with concentrations of seminal plasma at or below 20%, spermatozoa quickly became immotile. In contrast, at concentrations higher than 40% seminal plasma, spermatozoa were motile even after 15 min. As the concentration of seminal plasma was increased, oxygen consumption of spermatozoa also increased. A filtrate of the seminal plasma, obtained by passing the fluid through an Amicon YM-2 ultra-filtration membrane (Mr less than 1000), also stimulated the motility and oxygen consumption of spermatozoa. These results suggest that some low molecular weight factor(s) in fowl seminal plasma stimulated motility and oxygen consumption of fowl spermatozoa at 41 degrees C. A physiological role of this factor(s) may be to assist passage of spermatozoa through the vagina after natural mating.     

Iron binding to, and release from, the basolateral membrane of mouse duodenal enterocytes

The basolateral membrane of mouse duodenal enterocytes can be selectively labelled in vitro with 59Fe by incubating intact enterocytes with 59Fe(III)-nitrilotriacetate at 0-4 degrees C. It has been proposed that this labelling represents binding to a site important in the transfer of intracellular Fe to the portal plasma (Snape, S., Simpson, R.J. and Peters, T.J. (1990) Cell Biochem. Funct. 8, 107-115). Studies presented here show binding to intact enterocytes in vitro was complete within 1 h and was proportional to enterocyte protein concentration. Binding to enterocytes isolated from both normal and chronically hypoxic mice showed a hyperbolic dependence on medium Fe(III) concentration, consistent with a single class of binding sites. Neither apparent binding constant nor maximal binding were increased by hypoxic exposure of mice, suggesting that the increased in vivo labelling of this site in hypoxia is not due to an increase in affinity or capacity of this site for iron. Release of iron from intact enterocytes, labelled at 0-4 degrees C, was measured at 37 degrees C and 0-4 degrees C. Release of 59Fe was extensive and more rapid at 37 degrees C with highest release to mouse serum. Iron released to serum was found to be bound to transferrin. Prior dialysis of serum against buffer led to complete failure of enterocytes to release iron. Reconstituting serum by adding back the dialysate restores release to levels seen in fresh serum, suggesting that low molecular weight serum components, notably bicarbonate, mediate iron transfer from the basolateral membrane to serum transferrin. The properties of the basolateral membrane iron binding site described here are consistent with a role in the iron transfer process.     

Elimination of defective alpha-factor pheromone receptors.

This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface. A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins. We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed. When the ste2-3 mutant cells were cultured continuously at 34 degrees C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a half-life of 33 min for wild-type receptor protein (Ste2p). Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene. At 34 degrees C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway. Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover. Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention. In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process. When ste2-3 mutant cells were cultured continuously at 22 degrees C, Ste2-3p accumulated in the plasma membrane. When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life. Thus, structural defects in Ste2-3p synthesized at 34 degrees C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34 degrees C.     

A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis.

Cells infected with a temperature-sensitive mutant (ts-26) of Rauscher murine leukemia virus (R-MuLV) or with wild-type virus were labeled with 35S-methionine, and cell extracts were examined for radioactive polypeptides which could be precipitated by monospecific antisera to viral proteins. When shifted from permissive (31 degrees C) to nonpermissive (39 degrees C) temperature, cells infected with ts-26 rapidly begin to accumulate gPr90enr, the glycoprotein precursor to the membrane envelope glycoprotein gp70 and to the membrane-associated protein p15E. Simultaneously, formation of these mature virion proteins ceases. In addition, lactoperoxidase-catalyzed surface labeling with 125I--iodine indicates that the plasma membrane of cells infected with ts-26 becomes depleted of gp70 antigens at 39 degrees C. Nevertheless, at 39 degrees C these cells release defective MuLVs which lack gp70 and p15E but contain an outer membrane. The released particles also contain an aberrantly processed form of the major virion core protein p30, and many of these virion cores have an unusual immature crescent shape. It has previously been reported that cells infected with the ts-26 mutant of R-MuLV process a 65,000 dalton precursor (Pr65gag) of the virion core proteins more slowly at 39 degrees C than do cells infected with wild-type virus (Stephenson, Tronick and Aaronson, 1975). Although we have confirmed these results, this effect is relatively small and it is known that various alterations of MuLV assembly can lead secondarily to inhibited processing of Pr65gag. We propose that the ts-26 mutant has a primary temperature-sensitive defect in membrane glycoprotein synthesis and that this change causes pleiotropic effects on core morphogenesis.     

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.     

Adenovirus-dependent release of choline from plasma membrane vesicles at an acidic pH is mediated by the penton base protein.

It has been suggested that during receptor-mediated endocytosis of human adenovirus (Ad) type 2 into cells, Ad disrupts the membrane of endocytic vesicles to enter the cytosol. To understand the mechanism of Ad-mediated disruption of the endocytic vesicles, I exposed Ad to plasma membrane vesicles derived from KB cells. Ad caused release of choline from the plasma membrane vesicles preloaded with [3H]choline. The efflux of choline was dependent on (i) the concentration of Ad, with a half-maximal effect at 0.5 microgram/ml; (ii) the pH of the buffer, with the optimum pH of the reaction ranging from 5.5 to 6.0; (iii) the length of the incubation, with a half-maximal release at 2 min; and (iv) the temperature of the incubation, with the optimum temperature being 37 degrees C. The Ad-dependent release of choline was inhibited by anti-penton base, while antihexon did not block the effect. These results suggest roles for a low-pH environment and the penton base protein in the Ad-dependent efflux of choline from plasma membrane vesicles.     

Microwaves and the cell membrane. II. Temperature, plasma, and oxygen mediate microwave-induced membrane permeability in the erythrocyte

Microwaves (2450 MHz) are shown to increase 22Na permeability of rabbit erythrocytes for exposures only within the narrow temperature range of 17.7 to 19.5 degrees C (Tc) which coincides with a nonlinearity in the Arrhenius plot reflecting an apparent membrane phase transition. Significantly, this response is not observed for cholesterol-loaded erythrocyte membranes which exhibit a linear Arrhenius plot and no apparent phase transition at Tc. The permeability increase at Tc is a nonlinear function of absorbed power but is a linear function of the internal electric field strength of the sample and saturates at approximately 400 mW/g and 600 V/m, respectively. The permeability increase was found to be reversible and transient in that immediately following termination of exposure sodium influx is significantly reduced but returns to normal within 60 min. Extracellular factors exert a significant influence on the microwave effect. The presence of plasma markedly potentiates the increase in 22Na permeability at Tc. Oxygen also modulates the microwave effect with relative hypoxia (5 mm Hg) and hyperoxia (760 mm Hg) enhancing the permeability increase. In contrast, the presence of two antioxidants, ascorbic acid or mercaptoethanol, inhibits the effect. These findings raise important questions about the physical and chemical nature of microwave interactions with cell membranes and also shed light on earlier studies reporting either positive or negative effects on membrane permeability.     

Role of seminal plasma in damage to turkey spermatozoa during in vitro storage

In vitro storage of turkey spermatozoa is performed without consideration of the potential role of seminal plasma on sperm functions. We report the effects of seminal plasma on membrane permeability, lipid metabolism, energy status, motility and fertility of turkey spermatozoa stored at 4 or 20 degrees C. Phospholipid content (1077 nmol/10(9) spz versus 1219 nmol/10(9) spz at 48 h) and membrane permeability of spermatozoa were significantly damaged by the presence of seminal plasma after 48 h of storage at 4 degrees C, whereas damage to ATP content and fertility occurred earlier damaged by this presence (fertility after 24h storage 51% with seminal plasma versus 71% without). At 20 degrees C, seminal plasma decreased the phospholipid content of spermatozoa in the first hour of storage (1326 nmol/10(9) spz versus 1636 nmol/10(9) spz). Twenty-four hours later, this effect was masked by intense lipid peroxidation. These results show that seminal plasma is deleterious to storage of turkey spermatozoa at 4 degrees C and is involved in phospholipid metabolism of spermatozoa. Lipid peroxidation could be responsible for the acceleration of the degradation of sperm phospholipids during storage at 20 degrees C. However, lipid peroxidation seems not to be active at 4 degrees C. In this case, we suggest that phospholipase activation may contribute to sperm degradation, especially in the presence of seminal plasma.     

Glucagon secretion in the hibernating edible dormouse (Glis glis)

Plasma glucose and glucagon concentrations were measured in edible dormice during the bout of hibernation, arousal and active periods. During lethargy, plasma glucose and glucagon were low, compared to active values and did not fluctuate throughout the phase. During rewarming, plasma glucose regularly increased from 17 degrees to 37 degrees C while plasma glucagon rose after the 17 degrees C stage and reached the higher values at 26 degrees C, then slightly decreased at 37 degrees C. During arousal, plasma levels of free amino acids progressively increased. The effect of temperature and secretagogue (glucose and arginine) on glucagon secretion was studied using perfused pancreas from hibernating edible dormouse. In vitro rewarming of pancreas induced an increase in glucagon secretion. Glucagon secretion was regulated by glucose (inhibitory effect) and by arginine (stimulating effect) up to 25 degrees C. The effect of temperature and glucagon on oxygen uptake of hibernating edible dormouse brown fat was studied using an in vitro technique. Rewarming strongly increased oxygen consumption from 10 to 37 degrees C. Glucagon enhanced oxygen consumption up to 20 degrees C.     

Serum and plasma stimulate prostaglandin production by alveolar macrophages

Fetal bovine serum (FBS) stimulated rabbit alveolar macrophages to synthesize prostaglandins (PG) and release lysosomal enzymes. This stimulatory action was not entirely due to the effect of foreign protein in FBS, since rabbit serum and plasma, both homologous and autologous, also induced release of PGs and lysosomal enzymes. Rabbit serum and plasma are less effective than FBS as a stimulus for PG release, with rabbit serum being more potent than plasma at the same concentration. Bovine serum albumin elicited a dose-dependent increase of arachidonic acid release by macrophages, but not of PG production. Hence, the fatty acid "trapping" effect of albumin in serum and plasma is not responsible for the PG stimulation. The PG stimulating factors were stable at 56 degrees C for 30 min., but lost half the activity after heating at 100 degrees C for 10 min. Gel permeation chromatography of FBS showed several peaks of PG stimulating and arachidonic acid releasing activity. The molecular weight of the major one (150,000 daltons) is similar to that of immunoglobulin G. Rabbit IgG, when added to the macrophage culture, stimulated release of arachidonic acid and PGs. However, the major stimulatory effect in serum or plasma is not all due to IgG, since removal of IgG by a Protein A-agarose column did not remove the stimulatory effect of FBS and rabbit serum. The possibility of other factors, such as complement fragments, is discussed.     

Processing of the insulin-like growth factor-II-mannose 6-phosphate receptor in isolated liver subcellular fractions.

A truncated, soluble form of the insulin-like growth factor-II-mannose 6-phosphate (IGF-II-M6P) receptor has been identified in serum and shown to be released from cultured tissues and cells, liver being the main contributor to serum receptor in adult rats. In the present study, the processing of the IGF-II-M6P receptor has been characterized in isolated liver subcellular fractions using ligand binding, affinity crosslinking, and Western immunoblotting techniques. The receptor in plasma membrane fractions differed from that in Golgi-endosomal fractions by: (i) a lower molecular size upon reducing polyacrylamide gel electrophoresis (245 vs. 255 kDa); (ii) a less tight membrane association as judged upon extractibility by NaCI; and (iii) the inability to recognize antibody anti-22C, directed against the cytoplasmic domain of the receptor. Incubation of cell fractions at 30 degrees C led to a pH- and time-dependent release of the receptor into the medium. The pH optimum for release was 5.5 in the Golgi-endosomal fraction and 7.5 in plasma membrane fractions; at this pH, approximately 2% and 20%-30% of total receptors were released per hour, respectively. Receptor release was inhibited in a dose-dependent manner by aprotinin, benzamidine, and leupeptin in the Golgi-endosomal fraction, and by 1,10 phenanthroline in plasma membrane fractions, although high concentrations were required for inhibition. The receptor released from Golgi-endosomes showed a 5-10 kDa reduction in size and a loss of ability to recognize antibody anti-22C, but that released from plasma membranes showed little or no changes in size. We conclude that soluble, carboxy-terminally truncated forms of the IGF-II-M6P receptor are generated from the intact receptor in isolated Golgi-endosomal and plasma membrane fractions. However, receptor processing in these fractions exhibits different properties, suggesting the involvement of different proteases.