Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in light-scattering induced by gluconeogenic hormones, butyrate and ionophore A23187.

1. The effects of a variety of hormones on the PPi content and light-scattering of isolated rat liver cells was studied. 2. The basal PPi content was about 130 pmol/mg of cell protein, and increased after hormone addition, in parallel with a decrease in light-scattering which we have observed previously [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. 3. The mean increases in PPi content with the agonists shown (as pmol/mg of protein) were: 0.1 microM-glucagon, 25; 20 microM-phenylephrine, 30; 25 nM-vasopressin, 127; glucagon + phenylephrine, 115; glucagon + vasopressin, 382; 100 microM-ADP, 50; 15 microM-A23187, 72; 1 mM-butyrate, 80. 4. In the absence of extracellular Ca2+, vasopressin had little effect on either the PPi content or the light-scattering of hepatocytes. 5. The magnitude of the increase in PPi content correlated with that of the decrease in light-scattering irrespective of the stimulating agent, provided that the PPi did not exceed 300 pmol/mg of protein. Above this value little additional change in light-scattering was observed. 6. Subcellular fractionation showed that over 90% of the cellular PPi was intramitochondrial in both control and stimulated cells. 7. The data support the conclusions of previous experiments using isolated liver mitochondria [Davidson & Halestrap (1987) Biochem. J. 246, 715-723] that hormones increase the mitochondrial matrix volume through a Ca2+-induced rise in matrix [PPi]. 8. It is further proposed that this increase in mitochondrial [PPi] allows entry of ADP into the mitochondria in exchange for PPi and is therefore responsible for the increase in total mitochondrial adenine nucleotides observed after hormone treatment.     

ANGULAR LIGHT-SCATTERING STUDIES ON ISOLATED MITOCHONDRIA

Angular light-scattering studies have been carried out on suspensions of isolated rat liver mitochondria. The angular scatter pattern has a large forward component, typical of large particles. Changes in dissymmetry and in the intensity of light scattered at 90° have been correlated with changes in optical density during the course of mitochondrial swelling and contraction. Such changes can be measured at mitochondrial concentrations much below those required for optical density measurements. Changes in mitochondrial geometry caused by factors "leaking" from mitochondria, not detectable by optical density measurements, have been demonstrated by measuring changes in dissymmetry. Angular light-scattering measurements therefore offer the advantages of increased sensitivity and of added indices of changes in mitochondrial conformation.     

Effective use of renal cortical slices in transport and metabolic studies

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97-98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO2 decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.     

Bacteriorhodopsin induces a light-scattering change in Halobacterium halobium

When suspensions of Halobacterium halobium are exposed to bright light, the light-scattering properties of the bacteria change. This light-scattering response can produce a transmission decrease of about 1% throughout the red and near-infrared region. The action spectrum for the light-scattering response appropriately matches the absorption spectrum of bacteriorhodopsin. The response is eliminated by cyanide p-trifluoro-methoxyphenylhydrazone, a proton ionophore, and by triphenylmethylphosphonium, a membrane permanent cation. A mild hypertonic shock induces a similar light-scattering change, suggesting that bright light causes the bacteria to shrink about 1% in volume, thereby producing the light-scattering response.     

Is the lipolytic response in porcine adipose tissue slices equivalent to the lipolytic response in isolated adipocytes?

• 1.1. In most cases, when isolated adipocytes and adipose tissue slices from the same animal were stimulated with various lipolytic agents (adrenergie agonists, theophylline, adenosine deaminase), the qualitative response was similar.
• 2.2. There were, however, numerous exceptions; e.g. quinterenol did not affect isolated adipocytes whereas it was a partial agonist for adipose slices from the same animal.
• 3.3. The adipocytes present in slices were larger than those isolated from slices by collagenase digestion.
• 4.4. Isolated adipocytes were not more sensitive than tissue slices to stimulation by lipolytic agents.

     

Effective use of renal cortical slices in transport and metabolic studies

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97–98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO₂ decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.     

The molecular weight of Artemia ribosomes, as determined from their refractive-index increment and light-scattering intensity

Cytoplasmic ribosomes were isolated from the cryptobiotic embryos of the brine shrimp Artemia salina. Measurements of their refractive-index increments and light-scattering intensities give a value for their molecular weight of (3.4±0.2)×10⁶.     

Effect of salts and electron transport on the conformation of isolated chloroplasts. I. Light-scattering and volume changes

Whole chloroplasts isolated from the leaves of spinach (Spinacia oleracea L.) exhibit 2 types of conformational change during electron transport. Amine-uncoupled chloroplasts swell and atebrin-uncoupled chloroplasts shrink. Chloroplasts uncoupled by carbonylcyanide phenylhydrazones and by treatment with ethylenediamine tetraacetic acid do not change their volumes or light-scattering properties during electron transport. Phosphorylating chloroplasts shrink only slightly.The rate and extent of the conformational change parallel the rate of electron transport; both the decrease in turbidity with methylamine and the increase in turbidity with atebrin are rougly proportional to the Hill reaction rate. Consequently the great volume and light-scattering changes which occur in the presence of these uncouplers can be attributed, in part, to the very high rates of uncoupled electron transport. However, for a given rate of electron transport the atebrin-induced scattering increase is very much greater than the increase observed during photophosphorylation.When uncouplers are combined, the carbonylcyanide phenylhydrazone effect (no change) supercedes both the methylamine effect (swelling) and the atebrin effect (shrinking). The methylamine effect supercedes the atebrin (shrinking) and ethylenediamine tetracetic acid (no change) effects. The atebrin effect supercedes the ethylenediamine tetraacetic acid effect. A similar hierarchy of effects is observed with regard to the rate of the uncoupled electron transport.These light-scattering changes of whole chloroplasts reflect similar changes which occur in very small digitonin particles of chloroplasts. Therefore one must look among chloroplast substructures for the basic mechanism of swelling and shrinking.Many salts (including methylamine hydrochloride) cause the chloroplasts to shrink. This phenomenon is not osmotic since comparable osmolarities of sucrose are without effect. Magnesium chloride and calcium chloride are most effective but all salts tested gave major volume decrease when less than 0.05 m. The salt-shrunken chloroplasts show greater light-scattering changes during electron transport than do low-salt chloroplasts.     

Lipolysis in brown adipose tissue of cold- and heat-acclimated hamsters

Rates of release of free fatty acids (FFA) and glycerol to the incubation medium by brown adipose tissue (BAT) slices isolated from heat-acclimated (H), cold-acclimated (C), and control (N) hamsters in the absence or presence of epinephrine (E) were studied. Rates of FFA and glycerol release by tissue slices isolated from H and N animals were similar. In tissue slices isolated from C animals rate of release of FFA and glycerol was three times as high. Addition of E to the incubation medium (200 microgram/ml) had no effect on the rate of FFA and glycerol release of slices from C animals, but tripled the rates of slices from N, resulting in similar values for the two groups. In slices from H animals the rate of release was lower than in the other two groups, increasing only 1.5-fold. Pretreatment of N animals with triiodothyronine (T3; 0.8 microgram/100 g daily for 7 days) doubled the rates of FFA and glycerol release. Addition of E to the medium affected both pretreated and nontreated slices similarly. Two possible mechanisms by which temperature acclimation controls the lipolytic rate of BAT are suggested by 1) the concentration of specific enzymes and 2) cellular metabolites and hormones which activate existing systems. It seems that both operate in temperature-acclimated hamsters.     

Regulation by Ca2+ of membrane elasticity of bovine chromaffin granules

In a range of [Ca2+] similar to cytosolic transient, a drastic reduction from about 20 dyn/cm to almost zero was observed in the membrane elastic modulus of bovine chromaffin granules, isolated in a solution containing 0.3 M sucrose and 5 mM Hepes at pH 7.0, and measured by combination of osmotic swelling and dynamic light-scattering (DLS) methods. This result suggests that the granule membrane becomes extremely flexible as a prelude to exocytosis.     

Effects of calcium ionophores on the transport and distribution of calcium in isolated cells and in liver and kidney slices

Summary The effects of calcium ionophores on cellular calcium metabolism were studied in cultured kidney cells, in cells freshly isolated from rat kidney, and in liver and kidney slices. In isolated cells, these ionophores decreased the total cellular Ca content and the mitochondrial Ca.⁴⁵Ca efflux from prelabelled cells was also stimulated even in the absence of extracellular Ca. In slices, the ionophore A23187 increased the total slice Ca and the uptake of⁴⁵Ca. However, the mitochondria isolated from these slices treated with the ionophore had a lower total Ca and a depressed relative radioactivity. These results suggest that the increased cytosolic Ca produced by Ca ionophores may be due to mobilization of intracellular Ca stores rather than to a net shift of Ca from the extracellular fluids to the cell.     

Protein-bound phosphorylserine in acid hydrolysates of brain tissue. The determination of [32P]phosphorylserine by ion-exchange chromatography and electrophoresis

1. Partial acid hydrolysates of proteins derived from cortical slices of guinea-pig brain were divided into two parts and fractionated by ion-exchange chromatography and high-voltage electrophoresis. 2. The apparent yield of protein-bound phosphorylserine by the ion-exchange method was about three times that obtained by electrophoresis. 3. The specific radioactivity of phosphorylserine isolated from (32)P-labelled slices by electrophoresis was twice that isolated by chromatography. 4. The discrepancies were found to be due to the presence of unlabelled phosphates of unknown composition in the ;phosphorylserine' fraction obtained by the ion-exchange method. 5. Electrical stimulation of slices respiring in the presence of [(32)P]phosphate increased the specific radioactivity of the total phosphate in the chromatographic ;phosphorylserine' fraction by 53+/-11%, as compared with only 19+/-5% for the phosphorylserine isolated by electrophoresis.     

Characterization of intracellular accumulation of poly-beta-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 by flow cytometry

Poly-beta-hydroxybutyrate (PHB) accumulates in individual cells of Alcaligenes eutrophus in the form of refractile bodies which alter the light-scattering properties of individual cells. Flow cytometry has been applied to measure the distributions of single-cell light-scattering intensity in Alc. eutrophus populations during batch cultivation of the organism. These measurements clearly identify heterogeneities in the inoculum which influence the lag interval prior to beginning of exponential growth. Light-scattering distributions show greater homogeneity and are extremely similar during balanced, exponential growth. After exhaustion of the nitrogen source and with carbon source still available, significant PHB accumulations occur and the flow cytometry measurements reveal extreme heterogeneity in single-cell light-scattering properties. These measurements clearly demonstrate the potential advantages of single-cell light-scattering measurements by flow cytometry for analysis and control of certain fermentation processes. Single-cell light-scat light-scattering measurements in conjunction with flow sorting instrumentation have been applied to demonstrate enrichment of PHB-producing cells, initially present in a number concentration of 0.01%by a factor of 300 in a single pass. Flow cytometry-cell sorting technology should find significant application in strain improvement and mutant selection.     

Light- and nucleotide-dependent binding of phosphodiesterase to rod disk membranes: correlation with light-scattering changes and vesicle aggregation

Under conditions in which large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent changes in near-infrared transmission and vesicle aggregation and disaggregation occur, we have observed a striking change in the binding of PDE to rod disk membranes. The change in PDE binding is nucleotide and light dependent as are the light-scattering changes. The cGMP- and PDE-dependent light-scattering signal can be produced by a 500-nm light flash which bleaches 1/(1 X 10(7] rhodopsin molecules. Mg ions are an essential cofactor for the nucleotide-dependent PDE binding and light-scattering changes. 3-Isobutyl-1-methylxanthine and other competitive inhibitors of PDE hydrolytic activity support increased PDE binding to the disk membrane, vesicle aggregation, and the light-scattering signal. However, treatments which block GTP-dependent activation of PDE hydrolytic activity (colchicine, GDP, or ethylenediaminetetraacetic acid) also block these phenomena. Thus, GTP-dependent activation of PDE rather than its hydrolytic activity appears to be correlated with the light-scattering signal.     

Polymeric structure of a high-molecular-weight glycoprotein from bovine cervical mucus.

A 16 X 10(6)-Mr glycoprotein isolated from bovine oestrus cervical mucus when reduced under conditions where disulphide-bond cleavage is essentially quantitative produces chains whose Mr from light-scattering and from sedimentation and diffusion data is some 4 X 10(6)-5 X 10(6). Pronase digestion of the chains indicates that glycosylated sequences of Mr 0.3 X 10(6)-0.5 X 10(6) are interspersed with enzyme-susceptible non-glycosylated peptide sequences.     

Myelin changes induced by incubation of brain slices with serum

Forebrain and brain stem slices prepared from adult rats were incubated with pooled normal human serum. Following the incubation, the tissue was homogenized and the fraction floating on 0.32 M sucrose as well as two myelin subfractions (light and heavy) were isolated. Addition of serum into the incubation medium increased generation of the floating fraction by the cerebral slices. Changes in the myelin membrane were also observed. Thus, myelin isolated from forebrain slices revealed pronounced increase in the buoyant density of its particles and loss of basic protein. Furthermore, in spite of the intensive washing employed during the isolation procedure, some serum proteins were found firmly attached to the membraneous fractions. The demonstration of the myelin alterations in the living cerebral tissue exposed to serum during incubation may contribute to understanding the pathogenesis of multiple sclerosis.     

INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL : II. Transport to Condensing Vacuoles and Zymogen Granules

In the previous paper we described an in vitro system of guinea pig pancreatic slices whose secretory proteins can be pulse-labeled with radioactive amino acids. From kinetic experiments performed on smooth and rough microsomes isolated by gradient centrifugation from such slices, we obtained direct evidence that secretory proteins are transported from the cisternae of the rough endoplasmic reticulum to condensing vacuoles of the Golgi complex via small vesicles located in the periphery of the complex. Since condensing vacuoles ultimately become zymogen granules, it was of interest to study this phase of the secretory cycle in pulse-labeled slices. To this intent, a zymogen granule fraction was isolated by differential centrifugation from slices at the end of a 3-min pulse with leucine-¹⁴C and after varying times of incubation in chase medium. At the end of the pulse, few radioactive proteins were found in this fraction; after +17 min in chaser, its proteins were half maximally labeled; they became maximally labeled between +37 and +57 min. Parallel electron microscopic radioautography of intact cells in slices pulse labeled with leucine-³H showed, however, that zymogen granules become labeled, at the earliest, +57 min post-pulse. We assumed that the discrepancy between the two sets of results was due to the presence of rapidly labeled condensing vacuoles in the zymogen granule fraction. To test this assumption, electron microscopic radioautography was performed on sections of zymogen granule pellets isolated from slices pulse labeled with leucine-³H and subsequently incubated in chaser. The results showed that the early labeling of the zymogen granule fractions was, indeed, due to the presence of highly labeled condensing vacuoles among the components of these fractions.     

Studies on the effect of inflammation on the acute phase response using rat liver slices

Liver slices from control and inflamed rats were incubated in McCoy's medium and incorporation of [3H]leucine into liver and medium proteins and into albumin and alpha 1-acid glycoprotein was monitored over 48 hr. The release of the new acute phase reactant, sialyltransferase was also monitored in this system. Earlier observations in which liver slices were incubated for 6 hr showed that increased leucine incorporation into liver and medium proteins and alpha 1-acid glycoprotein, coupled with decreased incorporation into albumin, correlated with the acute phase response of these proteins. Increased incorporation of leucine into these proteins was found following 48 hr incubation in McCoy's medium showing that slices were able to express the changes characteristic of the acute phase response over this longer time period of incubation. Sialyltransferase was released into medium in a linear fashion up to 15 hr and continued to increase for 30 hr in this system; there was a substantial increase in release of enzyme activity from slices from inflamed rats when compared to controls. Monokine-conditioned medium prepared from peritoneal exudate cells isolated from rats at various times after lipopolysaccharide administration was used to induce the acute phase response by intraperitoneal injection. Slices were prepared from these rats and sialyltransferase release from slices was monitored. Monokines prepared from peritoneal exudate cells isolated from rats at about 30 hr were most effective in stimulating sialyltransferase release from liver slices.     

In vitro effects of parathyroid hormone on kidney cortical slices: cAMP responses and concomitant inhibition of the Na+ gradient-dependent uptake of phosphate by brush border membrane vesicles isolated from the renal slices

Mouse renal cortical slices were incubated with parathyroid hormone (30 U/ml) for 2 min. Brush border membrane vesicles isolated from the treated slices had a decreased Na+ gradient-dependent uptake of phosphate. Concomitantly, the hormone elicited the activation of adenylate cyclase, the increase in tissue level of cAMP, and the enhancement of cAMP-dependent protein kinase.     

Changes in mobility of chromaffin granules in actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin.

As a final stage of cell signal transduction, secretory cells release hormones by exocytosis. Before secretory granules contact with the cell membrane for fusion, an actin-network barrier must dissociate as a prelude. To elucidate dynamical behaviors of secretory granules in actin networks, in vitro assembly and disassembly processes of actin networks were examined by means of dynamic light-scattering spectroscopy. We studied actin polymerization in the presence of chromaffin granules isolated from bovine adrenal medullas and found that the entanglement of actin filaments rapidly formed cages that confined granules in them. We also studied the effect of gelsolin, one of actin-severing proteins, on the network of actin filaments preformed in the presence of chromaffin granules. It turned out that the cages that confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. A semiquantitative analysis of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or the translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in the literature, a model is proposed for biophysical situations before, during, and after an exocytotic event.