Effect of a calcium chelator on morphine tolerance development

To assess the effects of calcium ion chelation on narcotic tolerance development, adult Sprague-Dawley rats were implanted with i.c.v. cannulae connected to osmotic minipumps for continuous infusions of saline or EGTA (2 mumol/24 h) during chronic morphinization by s.c. pellet implantation. Additional rats receiving the same morphine pellet treatment but without minipumps served as non-surgical controls. After 64 h the minipumps and pellets were removed and narcotic tolerance was assessed by the tail-flick technique. A positive analgetic response to an 8.50 mg/kg s.c. dose of morphine sulfate was noted in 6/12 no-minipump controls and 4/9 saline-infused controls. In placebo-treated rats caused no alteration of either baseline tail-flick latencies or in analgetic responsiveness to lower doses of morphine. It is concluded that the enhancement of morphine tolerance by EGTA (a calcium-specific chelator) results from a facilitation of certain adaptive changes of calcium ion disposition that are related to the neurochemical mechanisms of narcotic tolerance and dependence development.     

Calcium transport and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in sarcoplasmic reticulum from normal and ethanol-tolerant rats

Sarcoplasmic reticulum (SR) of high purity and functional integrity was isolated from skeletal muscle of normal and ethanol-tolerant rats. Ethanol at low concentrations (0.1 to 0.2 M), added in vitro to isolated SR, resulted in slight inhibition of both calcium loading and calcium-stimulated ATPase rates. Higher concentrations of ethanol resulted in further inhibition of calcium loading, but not of calcium-stimulated ATPase. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isolated SR membranes showed a small decrease in anisotropy with the addition of ethanol in vitro. Sarcoplasmic reticulum from normal and ethanol-tolerant rats did not differ in calcium pump function or in fluorescence polarization of DPH, in the presence or absence of ethanol.     

beta-Adrenergic stimulation of the adenosine 3',5'-monophosphate system regulated by cholinergic stimuli in the prostate

Acetylcholine significantly inhibited isoproterenol-stimulated adenosine 3',5'-monophosphate (cAMP) levels of rat prostatic tissue in a concentration-dependent fashion. Atropine but no hexamethonium reversed the inhibitory action of acetylcholine. Tetracaine and verapamil abolished the inhibitory effect of acetylcholine on isoproterenol-stimulated accumulation of cAMP. Exclusion of calcium also eliminated the effect of acetylcholine. Inhibitory regulation of cAMP levels was reproduced by the divalent cation ionophore A23187. These observations suggest that beta-adrenergic stimulation of the cAMP system of the prostate is regulated by cholinergic stimulation involving a specific muscarinic receptor with calcium-dependent mechanism sensitive to verapamil or tetracaine.     

Effects of sodium-deficient media and of a calcium ionophore (A-23187) on the release of (3H)-noradrenaline, (14C)-α-aminoisobutyrate,and (3H)-γ-aminobutyrate from superfused slices of rat neocortex

The calcium ionophore A-23187, at a concentration of 250 μg/ml, had no significant effects on the efflux of (³H)-noradrenaline or of (¹⁴C)-α-aminoisobutyrate, in superfused slices from rat neocortex. Sodium-deficient solutions (sodium partly substituted by choline), however, were found to increase the efflux of (¹⁴C)-α-aminoisobutyrate and of (³H)-γ-aminobutyrate. The enhanced efflux of both these substances appeared with no latency, was maintained for long periods, and did not change appreciably when calcium was omitted. Media low in sodium enhanced noradrenaline efflux in a more complex manner. There was, initially, a small increase in efflux that was similar both in latency and lack of dependence upon calcium to that of the above amino acids. This was followed by a very large efflux peak of noradrenaline, with a change in the fractional rate constant of up to 0.06 min^?1. This large efflux peak did not occur when no calcium was added to the superfusing fluid. The efflux of noradrenaline occurred when calcium was replaced by strontium or barium ions, but the latter were less effective. Finally, when media free of added calcium were made sodium-deficient, there was a moderate increase in the efflux of noradrenaline that began after a delay of about 30 min.It is suggested that the effects of sodium-deficient media on the efflux of amino acids and transmitters are due to two different mechanisms. The first mechanism involves interference with sodium-dependent active transport processes that carry substances from the extracellular fluid across the plasma membrane; this could account for the increased efflux of α-aminoisobutyrate and of γ-aminobutyrate and for the first, small, increased efflux of noradrenaline, all of which are independent of external calcium concentration. The second mechanism is mediated by an increase in intracellular calcium, induced by low external sodium, which promotes secretion of noradrenaline present in synaptic vesicles. The large efflux of noradrenaline, that is calcium-dependent, might be the result of this mechanism. In addition, solutions low in both sodium and calcium induce non-specific increases in membrane permeability, that lead to the delayed release of noradrenaline.     

Interaction of bromosulfophthalein with mitochondrial membranes-uptake of bromosulfophthalein and effect on ANS-fluorescence

Bromosulfophthalein uptake by rat liver mitochondria is fast, but reversible and can reach > 150 nmole/mg protein without lysis of mitochondria. There are no sets of characteristic binding sites with definite affinities, but affinity continuously decreases with increased binding. The decrease in binding affinity is ascribed to an increased negative surface charge resulting from insertion of the negatively charged bromosulfophthalein molecule into the hydrophilic-hydrophobic interphase of the membrane and rendering further binding of anionic molecules like bromosulfophthalein itself or ANS more difficult. This has been shown by the decrease of ANS-binding and fluorescence. The increase in surface charge on energization also results in decreased bromosulfophthalein binding. Although the amount of dye free in the matrix space, presumably is very small, the compound is possibly translocated and the amount bound to the inner side of the membrane may vary with the mitochondrial metabolic state.     

Calcium-dependent potassium conductance studied on internally dialysed nerve cells

The Ca-dependent tetraethylammonium-resistant potassium conductance was studied in the membrane of internally dialysed isolated snail neurones. In these conditions the tetraethylammoniumresistant noninactivating outward potassium current decreased when the transmembrane influx of Ca²⁺ was suppressed either by blocking the calcium channels with external Cd²⁺ or internal F^? or by substituting Mg²⁺ for Ca²⁺ in the external solution. Elevation of intracellular Ca²⁺ concentration by dialysing the cell with solution containing Ca-EGTA buffer resulted in an increase of potassium outward current. The effect could be measured with Ca²⁺ concentrations as low as 10^?7 M. Much higher concentrations of Sr²⁺ or Ba²⁺ inside the cell did not potentiate the potassium current. The corresponding ionic channels are shown to be less selective for potassium ions as compared to the Ca-insensitive voltage-dependent channels.A conclusion is made that the changes in Ca²⁺ concentration near the inner surface of the membrane are the key factor which makes these channels ready for activation.     

Production and biomedical applications of virus-like particles derived from polyomaviruses

Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins. Since their first recombinant production 27 years ago these particles have been investigated for a myriad of biomedical applications. These virus-like particles are safe, easy to produce, can be loaded with a broad range of diverse cargos and can be tailored for specific delivery or epitope presentation. We will highlight the structural characteristics of polyomavirus-derived VLPs and give an overview of their applications in diagnostics, vaccine development and gene delivery.     

Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner

BACKGROUND & AIMS: Prevention of bile acid-induced apoptosis is of therapeutic interest and requires the understanding of underlying mechanisms. METHODS: The effect of tauroursodeoxycholate (TUDC) on taurolithocholic acid-3 sulfate (TLCS)-induced apoptosis was studied in cultured rat hepatocytes. RESULTS: TLCS induced activation of caspases 8, 9, and 3 and hepatocyte apoptosis. These effects were abolished by TUDC in a PI 3-kinase-/protein kinase B (PKB)-, p38(MAPK)-, and extracellular signal-regulated kinase-2 (Erk-2)-independent manner. These protein kinases were activated by both TLCS and TUDC, however, with different kinetics. TLCS, but not TUDC, led to a sustained activation of c-Jun N-terminal kinase (JNK) and CD95 trafficking to the plasma membrane; both TLCS effects were prevented by TUDC. Inhibition of JNK1 or protein kinase C prevented TLCS-induced CD95 membrane trafficking and blunted the apoptotic response. The apoptotic potency of other bile acids paralleled their ability to induce sustained JNK activation. CONCLUSIONS: Protection by TUDC against TLCS-induced apoptosis starts upstream of caspase 8 activation and is independent of a PI 3-kinase-dependent survival pathway. JNK activation may be important for bile acid-induced apoptosis by triggering ligand-independent CD95 surface trafficking and activation of apoptosis.