Noradrenaline release and uptake in isolated small dense cored vesicles from rat seminal ducts

Noradrenaline (NA) release and uptake was investigated in an improved preparation of isolated small dense cored vesicles from rat seminal ducts. The vesicle preparation exhibited an Mg²⁺-ATP-dependent uptake of NA. Half maximal uptake after 20 min was seen at 22 μM. Exchangeability of NA between medium and vesicles was 100%. The kinetics of exchange suggested that mos NA is stored in a single pool. The t1/2 for release of NA was 43 min in the presence of Mg²⁺-ATP, as compared to 15 min in the absence of Mg²⁺-ATP. Addition of reserpine (20 μM) did not significantly alter the NA release. The kinetic properties of this preparation was compared to those of earlier reported noradrenergic vesicle preparations from different tissues.     

Granulated vesicles in sympathetic nerve endings in the pineal gland: Observations on the effects of pharmacologic agents by electron microscopy

The effects of pharmacologic agents upon granulated vesicles in the sympathetic nerve endings of the pineal gland were studied electron microscopically. The most common granulated vesicle measures 400 Å and has an eccentric granule (Type IA); a few vesicles are as large as 1000 Å (Type IB). The vesicles also contain fine grains which may be the subunits of which the granules are composed. Reserpine causes many vesicles to become elliptical, raising the possibility of an effect upon the vesicle membrane. Depletion of granules by reserpine is confirmed and fine grains are also depleted. p-Chlorphenylalanine, which reduces serotonin concentration, causes a reduction in number of granules but leaves fine grains. p-Chlorphenylalanine followed by pargyline, induces a reduction in number of granules but a considerable number are present suggesting persistence or re-accumulation of norepinephrine or other catecholamines. Oxypertine and desmethylimipramine (DMI) deplete granulated vesicles but fine grains remain. Another kind of granulated vesicle has a different ultrastructure than the Type IA and IB and measures 1000 Å (Type II). This kind of granulated vesicle is not depleted by reserpine, p-chlorphenylalanine, oxypertine or desmethylimipramine suggesting that it contains neither catecholamines nor serotonin.     

Tyramine‐induced endogenous noradrenaline efflux from in situ cardiac sympathetic nerve ending in cats

With the use of dialysis technique, the effects of tyramine on in situ cardiac sympathetic nerve endings were examined in anaesthetized cats. Dialysis probes were implanted in the left ventricular myocardium, and the concentration of dialysate noradrenaline (NA) served as an indicator of NA output at the cardiac sympathetic nerve ending. Locally applied tyramine (600 μM ) increased dialysate NA levels from 17 ± 1 (pg mL^?1) to 3466 ± 209 (pg mL^?1). Pretreatment with reserpine (vesicle transport NA blocker 1 μM ) did not affect tyramine‐induced NA efflux. The tyramine‐induced NA efflux was augmented by pretreatment with pargyline (1 m M ) but suppressed by pargyline (10 m M ). Pretreatment with α‐methyl‐tyrosine suppressed NA efflux evoked by tyramine. These pretreatments did not affect the time course of NA efflux but only altered peak height of NA efflux. The efflux of NA evoked by tyramine was not associated with any reduction of dihydroxyphenylglycol (DHPG). In contrast, in the pretreatment with reserpine, the efflux of NA was associated with a reduction of DHPG. This result suggests that NA graduation between axoplasm and stored vesicle contributes to maintaining the axoplasmic NA level during carrier‐mediated outward NA transport. The tyramine‐induced NA efflux provides a close reflection of the NA content at the nerve ending. With the use of dialysis, this experimental model is suitable for studying the mechanism of sympathomimetic amine‐induced neurotransmitter efflux.     

A piconewton force transducer and its application to measurement of the bending stiffness of phospholipid membranes

The bending stiffness of a phospholipid bilayer (kc) was measured by forming thin bilayer cylinders (tethers) from giant phospholipid vesicles. Based on the balance of forces, the tether force was expeeted to be proportional to the square root of the membrane tension, with a constant of proportionality containingk>c. The membrane tension was controlled via the aspiration pressure in a micropipette used to hold the vesicle. The force on the tether was generated by an electromagnet acting on a paramagnetic bead attached to the vesicle surface. The magnitude of the force was determined from measurements on the magnet current which was adjusted to maintain the position of the bead. Measurements were performed on vesicles composed of stearoyl-oleoyl-phosphatidylcholine plus 5% (by mole) biotinylated phosphatidylethanolamine to mediate adhesion to streptavidin-coated beads. From each vesicle, tethers were formed repeatedly at different values of the membrane tension. The expected relationship between membrane tension and tether force was observed. The mean value ofkc for 10 different vesicles was 1.17×10⁻¹⁹ J (SD=0.08×10⁻¹⁹ J). The precision of these data demonstrates the reliability of this approach, which avoids uncertainties of interpretation and measurement that may be associated with other methods for determiningkc.     

Synthesis of Noradrenaline from 3,4-Dihydroxyphenylalanine (DOPA) and Dopamine in Adrenergic Nerves of Mouse Atrium — Effect of Reserpine,Monoamine Oxidase and Tyrosine Hydroxylase Inhibition

The in vitro synthesis of noradrenaline (NA) from 3,4-dihydroxyphenylalanine (DOPA) and dopamine (DA) in the mouse atrium has been investigated, using isotope, subcellular distribution and histochemical techniques. Incubation of atrial tissue in 5 times 10^-6 M ¹⁴C-DOPA resulted in a formation of ¹⁴C-NA and ¹⁴C-DA at a rate of about 0.13 μg/g/hr, and 0.14 μg/g/hr respectively, whereas the same medium concentration of ³H-DA gave a formation of ³H-NA of about 0.25 μg/g/hr. The synthesis of NA and DA was found to be almost exclusively associated with the presence of adrenergic nerves, since it was considerably reduced after pretreatment with 6-OH-DA. Pretreatment with reserpine decreased formation of radioactive NA and DA which could be counteracted by nialamide. The effect of reserpine may be explained by inhibition of DA uptake in the amine storage granules. The antagonistic effect of nialamide is certainly due to inhibition of DA catabolism. Tyrosine hydroxylase inhibition produced by H44/68, which depleted the endogenous NA store to about 40% of normal, increased the formation of radioactive NA both from ³H-DA and ¹⁴C-DOPA. This may in part be explained by decreased competition of the endogenous precursor DA for DA-β-hydroxylase but also in part by increased storage sites available for newly formed NA.     

Inhibition by Reserpine,Guanethidine and Imipramine of the Uptake of 5-Hydroxytryptamine by Rat Peritoneal Mast Cells in Vitro

The effect of reserpine, guanethidine and imipramine on the uptake of tritiated 5-hydroxytryptamine by rat peritoneal mast cells was studied in vitro. The K_m value for uptake of 5-hydroxytryptamine was 3 × 10^-7 M in this study. Kinetic analysis of the data obtained suggests that the drugs inhibit the uptake in a competitive manner, in the order of decreasing potency: reserpine, imipramine, guanethidine. For reserpine the Dixon plot resulted in two curves, each composed of 2 linear components suggesting inhibition of uptake of 5-hydroxytryptamine at 2 different levels or by 2 different mechanisms with different K₁ values. It is suggested that the higher K₁ value, representing an immediate effect of reserpine, stands for inhibition of uptake at the cell membrane. The lower K₁ value possibly represents inhibition at the level of the membranes surrounding the intracellular storage organelles.     

Zinc inhibition of glucose uptake in brush border membrane vesicles from pig small intestine

The effect of zinc on sodium coupled glucose uptake was studied in pig intestinal brush border membrane vesicles. In this system zinc inhibited glucose uptake and appeared to have a K _i of 0.25 mM. When tested by spectrophotometry, electron microscopy and protein determination following centrifugation, no evidence of significant vesicle aggregation was found with 0.5 mM zinc treatment. Zinc inhibition of glucose uptake persisted when the vesicle membrane potential was clamped with identical KCl concentrations inside and outside the vesicles in the presence of valinomycin. Variation of the glucose and sodium concentrations gave results indicating that zinc reduces glucose affinity for the carrier but not sodium binding to the transporter. The glucose inhibitory effect was not due to a rapid dissipation of the sodium gradient as zinc failed to affect sodium uptake in the absence of glucose. Zinc also failed to inhibit glucose efflux from vesicles under isotopic exchange conditions, when glucose and sodium concentrations were identical inside and outside vesicles. The t1/2 of glucose inhibition by zinc was relatively long, i.e. 6 min. We conclude that zinc acts as an inhibitor of glucose transport by interacting with the sodium-glucose co-transporter. The long zinc incubation time required to achieve maximal inhibition of glucose transport suggests that this interaction takes place within vesicles.     

Chloride transport in human placental microvillus membrane vesicles

³⁶Cl uptake by microvillus brush border membrane vesicles prepared from normal term human placenta has been studied using an ion exchange column assay and a novel method to correct for vesicle recovery. Uptake of this anion, which was largely into an osmotically active space, was time dependent with a half-time of approximately 4 min at 4°C. DIDS at a maximal inhibitory concentration inhibited³⁶Cl uptake by approximately 40%; furosemide (10^–4 M) showed a similar degree of inhibition and the effects of the two drugs appeared not to be additive. Anions including Cl, Br, I, NO₃, salicylate and SCN reduced³⁶Cl uptake, the latter two being the most potent; gluconate was without effect. Inhibition by maximal inhibitory concentrations of DIDS and the anions SCN and salicylate was not additive. Neither the DIDS-sensitive nor the SCN-sensitive component of³⁶Cl uptake was influenced by membrane potential; in contrast the inhibitor-insensitive component was increased by an inside positive membrane potential. These findings suggest that under the conditions employed an electroneutral anion exchange system is responsible for approximately half of the chloride flux across this plasma membrane.     

Characterization of ouabain-induced noradrenaline and acetylcholine release from in situ cardiac autonomic nerve endings

Aim: Although ouabain modulates autonomic nerve ending function, it is uncertain whether ouabain‐induced releasing mechanism differs between in vivo sympathetic and parasympathetic nerve endings. Using cardiac dialysis, we examined how ouabain induces neurotransmitter release from autonomic nerve ending. Methods: Dialysis probe was implanted in left ventricle, and dialysate noradrenaline (NA) or acetylcholine (ACh) levels in the anaesthetized cats were measured as indices of neurotransmitter release from post‐ganglionic autonomic nerve endings. Results: Locally applied ouabain (100 μm ) increased in dialysate NA or ACh levels. The ouabain‐induced increases in NA levels remained unaffected by cardiac sympathetic denervation and tetrodotoxin (Na⁺ channel blocker, TTX), but the ouabain‐induced increases in ACh levels were attenuated by TTX. The ouabain‐induced increases in NA levels were suppressed by pretreatment with desipramine (NA transport blocker) and augmented by reserpine (vesicle NA transport blocker). In contrast, the ouabain‐induced increases in ACh levels remained unaffected by pretreatment with hemicholinium‐3 (choline transport blocker) but suppressed by vesamicol (vesicle ACh transport blocker). The ouabain‐induced increases in NA levels were suppressed by pretreatment with ω‐conotoxin GVIA (N‐type Ca²⁺ channel blocker), verapamil (L‐type Ca²⁺ channel blocker) and TMB‐8 (intracellular Ca²⁺ antagonist). The ouabain‐induced increases in ACh levels were suppressed by pretreatment with ω‐conotoxin MVIIC (P/Q‐type Ca²⁺ channel blocker), and TMB‐8. Conclusions: Ouabain‐induced NA release is attributable to the mechanisms of regional exocytosis and/or carrier‐mediated outward transport of NA, from stored NA vesicle and/or axoplasma, respectively, while the ouabain‐induced ACh release is attributable to the mechanism of exocytosis, which is triggered by regional depolarization. At both sympathetic and parasympathetic nerve endings, the regional exocytosis is because of opening of calcium channels and intracellular calcium mobilization.     

Calcium binding activity by chick intestinal brush-border membrane vesicles

Uptake of Ca²⁺ by vesicle preparation of chick intestinal brush-border membranes was rapid and extensive. With tracer quantities of Ca²⁺ uptake was complete in 10 min whereas with 2.0 mM Ca²⁺ maximum uptake by the vesicles occurred after one hour incubation. The maximum concentration of Ca²⁺ found in the vesicles was four times greater than the external Ca²⁺ concentration showing that the majority of the Ca²⁺ was membrane bound. The Ca²⁺ taken up by the vesicles was probably bound to the vesicle's interior since it was not replaced by exposure of loaded vesicles to La³⁺ (5 mM). The uptake of Ca²⁺ by the vesicles at different Ca²⁺ concentrations was analyzed and a high affinity Ca²⁺ binding site was found with an association constant for Ca²⁺ of 5×10^?5 M. More of these sites were found in the duodenum than the ileum and vitamin D increases the number of these sites.     

Chloride transport by self-exchange and by KCI salt diffusion in gramicidin-treated human red blood cells

The permeability of gramicidin-treated human red blood cell membranes to K⁺ and CI^- has been measured at normal ionic strength (1) by tracer exchange at steady-state distribution of salt, and (2) by net transport of salt in the presence of a salt concentration gradient. Under both conditions KCI was the only inorganic salt in cells and medium. In the studies of self-exchanges the electrical driving force on the ions was zero. Calculation of permeability coefficients from net salt transport was simplified because the experiment was designed as a special case of the Nernst-Planck diffusion regime, i.e. the single salt case. Gramicidin altered the cell membranes from being anion to become cation selective. Gramicidin increased the potassium exchange without affecting, the chloride exchange measurably. The chloride exchange showed saturation kinetics as does chloride exchange in normal cells. The net transport of KCI in the presence of a constant concentration gradient increased to a constant value with increasing gramicidin concentration. At high gramicidin concentrations (0°C, pH 7.2) the “chloride permeability coefficient” calculated from tracer exchange (1.9×10^-6 cm/s) was 290 times the chloride permeability coefficient calculated from net salt transport (0.65×10^-8 cm/s). The latter value corresponds to a chloride conductance of 4.2×10^-6 ohm^-1 cm^-2. The chloride permeability coefficient was 2.1×10^-8 cm/s at 25°C (pH 6.8) indicating a value of 3 for the Q₂₅. It appears that normal red cells are anion selective in the sense that anion permeability exceeds cation permeability with a factor of more than a hundred between 0°C and body temperature. The anion exchange, i.e. the Hamburger shift, is a tightly coupled transport process which is several orders of magnitude faster than anion transport by salt diffusion.     

Activity Inhibition of Cytolytic Lymphocytes by Omeprazole

This study examined the in vitro effect of omeprazole (OM) on various types of murine cytocidal lymphocytes. The results show that OM caused a strong inhibition of basal natural killer (NK) activity in spleen cells (SC) from untreated CD2F1 mice; in peritoneal exudate cells and SC activated in vivo by injection of maleic anhydride divinyl ether 1,2-copolymer (MVE-2) or inactivated Candida albicans (CA); in lymphokine-activated killer (LAK) activity generated in vitro from splenocytes cultured with rhIL-2 and in allo-specific cytotoxic lymphocyte-mediated lysis generated in vitro. A significant inhibition of cytotoxic activity of all types of effector cells after 30 min incubation was already induced by OM at 1 × 10^?3 m concentration, after 1 h incubation at 5 × 10^?4 m and after 4 h incubation at 1 × 10^?4 m OM. Complete inhibition of lytic activity was obtained after 4 h incubation of effector cells with 1 × 10^?3 m OM. No inhibitory effect was observed at 5 × 10^?5 m OM concentration. Indomethacin did not abrogate the OM inhibitory effect on NK/LAK activity, suggesting that prostaglandins are not involved in the process leading to suppression of cytocidal activity. When effector cells were incubated with OM in presence of rhIL-2 (500 U/ml), the cytokine failed to antagonize the inhibitory effect of the drug. On the contrary, if OM pretreated cells were incubated with rhIL-2 for a further 18 h after drug removal, this cytokine was able to restore NK activity, but only when NK inhibition was incomplete. These results demonstrate for the first time that in vitro OM causes a rapid, strong effect on various types of cytotoxic lymphocytes ranging from cytotoxicity inhibition to irreversible cell damage.     

Effects of noradrenaline and flow on lactate uptake in the perfused rat hindlimb

Skeletal muscle can release or take up lactate depending on the lactate concentration gradient across the cell membrane. In the perfused rat hindlimb without arterial lactate, both noradrenaline (NA) infusion and increased flow promote lactate release and oxygen consumption (VO ₂). However, it is unclear whether NA or increased flow rate have similar effects on lactate uptake. The present study compares these effects in the rat hindlimb perfused at a basal flow rate of 0.33 mL min^?1 g^?1 and 25 °C in the presence of added arterial lactate. When 10 mmol L^?1 L-(+)-lactate was added to the arterial perfusate, lactate was taken up (16 ± 1.0 μmol g^?1 h^?1, n = 13) by the hindlimb with a 35% higher VO ₂ than that without added lactate. Doubling perfusion flow rate enhanced lactate uptake and VO ₂ by 120% and 40%, respectively. Glucose uptake was also increased (by 253%) with increased flow. Infusion of NA increased perfusion pressure, VO ₂ and glucose uptake similarly to those induced by increased flow rate. However, lactate uptake was inhibited by NA. This inhibition was not altered by the β-adrenergic antagonist propranolol. Vasopressin also showed similar effects to NA to decrease lactate uptake associated with increased VO ₂ and vasoconstriction. These data indicate that in the presence of a high arterial lactate concentration, NA has opposite effects from increased flow rate on skeletal muscle lactate uptake although both have similar effects on lactate release in the absence of arterial lactate. Inhibition of lactate uptake may relate to the vasoconstrictive action of NA.     

p-Aminohippurate/2-oxoglutarate exchange in bovine renal brush-border and basolateral membrane vesicles

The transport of the amphiphilic organic anion, P-aminohippurate (PAH), across the luminal (brush-border) and contraluminal (basolateral) membrane of renal proximal tubule cells was studied with membrane vesicles isolated from bovine kidney cortex. On the basis of the enrichment of specific activities of marker enzymes, leucine aminopeptidase and Na⁺/K⁺-ATPase, brush-border and basolateral membrane vesicles can be obtained from bovine kidneys in reasonably pure form. The uptake of [³H]PAH into both brush-border and basolateral membrane vesicles was trans-stimulated by intravesicular PAH and by 2-oxoglutarate. In the absence of Na⁺, [³H]PAH/2-oxoglutarate exchange was cis-inhibited by unlabelled 2-oxoglutarate in the medium. In the presence of an inward Na⁺ gradient, 10 M 2-oxoglutarate, but no other Krebs cycle derivative, cis-stimulated [³H]PAH uptake, indicating that a Na³-coupled dicarboxylate transporter and PAH/2-oxoglutarate exchanger cooperate in both membranes to enhance [³H]PAH uptake. [³H]PAH uptake showed a non-saturable and a saturable component with similar apparent K _m values in brush-border and basolateral membranes. Although one negatively charged PAH molecule exchanges with one doubly negatively charged 2-oxoglutarate molecule the exchange was electroneutral. Probenecid inhibited [³H]PAH/2-oxoglutarate exchange in brush-border and basolateral membrane vesicles with indistinguishable kinetics. We conclude that similar or identical PAH transporters are located in brush-border and basolateral membranes of bovine kidney proximal tubule cells. This arrangement seems species-specific since a Na⁺ gradient plus 2-oxoglutarate caused concentrative [³H]PAH uptake in brush-border membrane vesicles from bovine, but not from rat kidney.     

The effects of quinidine on sodium-dependent calcium efflux in isolated rod photoreceptors of the salamander retina

The effect of quinidine on the membrane current generated by the NaCa,K exchange has been investigated in the outer segment of isolated rod photoreceptors from the retina of the larval tiger salamander. The inward exchange current associated with the efflux of Ca²⁺ was selectively recorded by introducing a Ca²⁺ load through the light-sensitive channels, and then shutting these channels with a bright light. Quinidine (20–1000 M) reduced the magnitude of the exchange current and slowed its decay during the removal of a Ca²⁺ load. Quinidine did not alter the form of the relation between the exchange current and the total concentration of exchangeable calcium remaining within the outer segment, [Ca]_T, showing that it does not change the affinity of the exchange mechanism for internal Ca²⁺. The relation between exchange current inhibition and the quinidine concentration could be described by a simple Michaelis relation with a K _i of 287 M and a maximum inhibition of 50%. The incomplete block of the NaCa,K exchange current by quinidine shows that it does not act by simple competition with external Na⁺, and suggests that the inhibition of the exchange by quinidine may be non-specific.     

Cd2+- and K+-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction

Summary Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd²⁺- or K⁺-induced ACh release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd²⁺ in Ca²⁺-free medium for a period of 3 h, (2) 25 or 40mM K⁺ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluoresceinconjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated -bungarotoxin. Unstimulated and K⁺-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd²⁺ in Ca²⁺-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K⁺-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd²⁺ stimulation in Ca²⁺-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd²⁺ stimulation in Ca²⁺-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K⁺ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.     

Subcellular Distribution of 3H-noradrenaline in Adrenergic Nerves of Mouse Atrium — Effect of Reserpine,Monoamine Oxidase and Tyrosine Hydroxylase Inhibition

The subcellular distribution of exogenously administered ³H-noradrenaline in adrenergic nerves of mouse atria after pretreatment with nialamide, reserpine and nialamide, or the methylester of a-methyl-p-tyrosine (H44/68) has been investigated. Parallel fluorescence histochemical studies have been performed. Both reserpine and H44/68 cause a pronounced decrease in the endogenous noradrenaline stores. The subcellular distribution was practically identical in untreated, nialamide-pretreated and H44/68-pretreated animals; approximately 30 %³H-noradrenaline was recovered in the particulate fraction which in all probability contains the amine storage granules. In the reserpine-nialamide pretreated mice, however, most of the ³H-noradrenaline was found in the supernatant iraction, and about 10 % in the particulate fraction. These studies thus confirm earlier investigations that reserpine is a potent inhibitor of the noradrenaline uptake in the amine storage granules, although there is a small reserpineresistant uptake. Furthermore, after tyrosine hydroxylase inhibition both the ‘membrane pump’ and the granular uptake mechanism seem to operate. The morphological studies disclosed differences in the adrenergic nerves when the noradrenaline taken up is mainly granularly or extragranularly stored, since the varicosities are in the former case distinct and the latter case less distinct, while the pre-terminals are more prominent. Methodological studies of the homogenization procedure for subcellular distribution studies have also been performed, the results of which are discussed in view of the experimental data obtained. It is now well established, based on both biochemical and electronmicroscopic evidence. that the peripheral adrenergic transmitter NA^I is mainly stored in special intraneuronal storage granules (Euler and Hillarp 1956. Schümann 1958. Camps and Shideman 1962. Potter and Axelrod 1962, Lundborg 1967, Hokfelt 1969). Exogenously administered NA is rapidly taken up by the axonal membrane of the adrenergic neuron and subsequently incorporated into the storage granules, mainly by means of an ATP-Mg++-dependent uptake mechanism. This latter process can be efficiently-blocked by reserpine (Carlsson et al. 1963, Euler and Lishajko 1963.     

Enhanced adhesion of endothelial cells onto a polypropylene hollow-fiber membrane by plasma discharge treatment and high inoculum cell density

The effect of plasma discharge treatment of a microporous polypropylene hollow-fiber membrane commonly used for gas exchange in a conventional artificial lung on the adhesion of endothelial cells was investigated with the aim of constructing a hybrid artificial lung bearing endothelial cells on the membrane. The initial adhesion density and growth rate of the cells on the membrane were markedly increased following plasma discharge treatment (13.56 MHz, 30W) of the membrane for 5 to 20 min in the presence of 0.05 mmHg of various gases, such as ammonia, oxygen, and water vapor. Treatment of the membrane with ammonia for 5 min resulted in the highest increase in the cell adhesion density on 5 days from 1.4×10² to 2.0×10³ cells/cm², and the cell density reached 5.0×10³ cells/cm² after cultivation for 14 days. Increasing the inoculum cell concentration from 3.3×10⁵ to 3.2×10⁶ cells/ml resulted in an initial cell adhesion of 0.9×10⁵ cells/cm², even after 1 day. It was observed under a microscope that the cells were distributed uniformly to cover almost all of the surface area of the membrane. After the plasma discharge treatment, the permeability of the membrane to water increased to 9% of that of a polyethylene hollow fiber having pore diameters larger than 0.4 μm.     

Release of [3H]norepinephrine from synaptic vesicles isolated from rat brain after the intracisternal administration of [3H]norepinephrine: influence of nucleotides, ions and drugs, and destabilization of transmitter storage caused by acute or chronic lithium administration

Following the intracisternal administration of [³H]norepinephrine to rats pretreated with a monoamine oxidase inhibitor, synaptic vesicles containing the radio label could be isolated from isotonically prepared microsomal fractions of rat brain. Incorporation of [³H]norepinephrine into the vesicles was reduced by pretreatment of the rats with desmethylimipramine and was also reduced if the rats had not been pretreated with a monoamine oxidase inhibitor. Incorporation of the label was totally eliminated by pretreatment with reserpine. Release in vitro of [³H]norepinephrine from the labeled vesicles was monophasic with a half-time of about 12 min at 30°C. The release was slowed by addition of adenosine 5′-triphosphate plus Mg²⁺ by a mechanism different from that of the vesicular amine uptake system; this was shown by the failure of inhibitors of adenosine triphosphate-Mg²⁺-stimulated uptake (reserpine,N-ethylmaleimide, lithium) to block the effect of adenosine triphosphate plus Mg²⁺ on release. Several other nucleotides also were able to slow the release of [³H]norepinephrine. Unlike adrenomedullary vesicles, rat brain synaptic vesicles did not show enhancement of amine release by chloride in the presence or absence of adenosine triphosphate plus Mg²⁺. The yield of labeled vesicles was substantially reduced if vesicles were prepared by hypotonic lysis of synaptosomes instead of isotonically from the microsomal fraction; the isotonic preparation appears to be superior for studies of vesicle uptake and storage properties.This preparation is readily utilizable for studies of the effects of in vivo administration of drugs thought to act on vesicular storage of catecholamines, a point illustrated by the destabilization of norepinephrine storage caused by acute or chronic lithium administration.     

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes

Aim: We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker‐stained vesicles in cultured rat astrocytes. Methods: Time‐lapse confocal images were acquired, and vesicle mobility was analysed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. Results: At rest, when mobility was monitored for 2 min in bath with Ca²⁺, two vesicle populations were discovered: (1) non‐mobile vesicles (6.1%) with total track length (TL) < 1 μm, averaging at 0.33 ± 0.01 μm (n = 1305) and (2) mobile vesicles (93.9%) with TL > 1 μm, averaging at 3.03 ± 0.01 μm (n = 20 200). ALS IgG (0.1 mg mL^?1) from 12 of 13 patients increased the TL of mobile vesicles by approx. 24% and maximal displacement (MD) by approx. 26% within 4 min, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca²⁺, indicating that ALS IgG vesicle mobility enhancement involves changes in Ca²⁺ homeostasis. To examine whether enhanced mobility relates to elevated Ca²⁺ activity, cells were stimulated by 1 mm ATP, a cytosolic Ca²⁺ increasing agent, in the presence (2 mm ) and in the absence of extracellular Ca²⁺. ATP stimulation triggered an increase in TL by approx. 7% and 12% and a decrease in MD by approx. 11% and 1%, within 4 min respectively. Interestingly, none of the stimuli triggered the release of vesicle cargo. Conclusion: Amyotrophic lateral sclerosis‐IgG‐enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.