Inhibition of the hyperglycemic effect of cAMP by intravenous injection to mice of carbonic anhydrase entrapped in liposomes.

Mice treated with acetazolamide or cAMP received intravenously liposomes containing carbonic anhydrase. The hyperglycemic effect of both these substances was suppressed by carbonic anhydrase entrapped in liposomes. The later also abolished the glycogenolytic action of cAMP on liver.     

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).     

Role of Surface Glycolipids - Natural or Synthetic Origin on the Biodistribution of Liposomes

Abstract

Various sugar residues were incorporated on the surface of liposomes and its effect on the biodistribution and therapeutic importance were discussed here. Galactosylated liposomes were preferentially taken up by liver parenchymal cells whereas mannosylated liposomes were mainly localized in non-parenchyma1 cells. On the other hand, incorporation of dextran on the surface results in increased circulatory half-life of liposomes. The potential application of such liposomes as a carrier of drugs in the diseased condition is also discussed.     

Effect of lysophosphatidylcholine on the radiation-induced lipid peroxidation in liposomes

The effect of lysophosphatidylcholine (LPC) on the lipid peroxidation process (LPO) in liposomes of rat liver phosphatidylcholine initiated by irradiation of 137Cs source was studied. The formation of diene conjugates (DC) is shown to increase dramatically on incorporation of more than 10% LPC into liposomes. The dependence of DC proportion on the irradiation dose is practically linear in the range of 0 to 5 kGy. The DC concentration in the liposomes without LPC grows at least to dose of 3.3 kGy and is unchanged on further irradiation. The malonic dialdehide accumulation follows the similar dependence. The LPC effect is neutralized by the incorporation of cholesterol into liposomes. The product of free radical fragmentation of LPC, palmitoxyacetone, practically has no influence on the DC concentration. The reasons of LPC effect on the irradiation initiated LPO in liposomes is discussed.     

Effects of radioprotectors on the cAMP and cGMP systems

Summary The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-aminothiazoline, and S-S-3-oxapentane-1,5-diisothiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected cAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fall by 1–3 min after the administration of drug and 1.4-fold rise after 15–20 min); serotonin and dopamine caused a slow rise in the cGMP level; the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin.The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system. The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist.     

Agents that elevate cAMP levels in platelets decrease thrombin binding

The effect of high intracellular levels of cAMP on the ability of rabbit and human platelets to bind and respond to thrombin was examined. Control rabbit platelets differed from human platelets in two interesting respects: they showed thrombin-dependent up-regulation of thrombin binding, but also a 3- to 5-fold lower thrombin-binding capacity. Nevertheless, treatment with prostaglandin E1 + theophylline or with forskolin decreased thrombin binding to both rabbit and human platelets by 60 to 70%. This effect was associated with a marked increase in the level of cAMP and seemed to depend on a decrease in number rather than affinity of thrombin-binding sites. Changes in thrombin binding correlated closely with changes in thrombin-stimulated incorporation of 32Pi into phosphatidic acid and a 40-kDa protein. However, regardless of the amount of thrombin that bound to treated platelets, thrombin-stimulated phosphorylation of a 20-kDa protein and serotonin secretion were severely inhibited. Thus, increased levels of platelet cAMP are associated with a reduced ability to bind and respond to thrombin. However, thrombin binding to platelets correlates more closely with some responses than others, presumably because cAMP inhibits additional platelet reactions.     

Intrahepatic Distribution of Long-Circulating Liposomes Containing Polyethylene Glycol) Distearoyl Phosphatidylethanolamine

Abstract

We investigated the intrahepatic distribution in rats of liposomes of 85 or 130 nm diameter, which were sterically stabilized with a polyethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) so as to increase their circulation time in blood. Various times after intravenous injection of radiolabeled ([³H-]cholesterylether) liposomes, parenchymal and non-parenchymal cells of the liver were isolated and their radioactivity content was determined. Control liposomes of 85 nm without PEG-PE distributed in an approximately 80:20 ratio to hepatocytes (H) and macrophages (M), respectively; the 130-nm control liposomes showed a 50:50 H/M distribution. Incorporation of PEG-PE reduced the rate of total liver uptake about 4-fold for liposomes of either size and shifted the H/M ratio to 60:40 for the smaller vesicles and to 40:60 for the larger ones. For both liposome sizes, PEG-PE apparently causes a shift in intrahepatic distribution in favor of the macrophages. It is concluded that PEG-PE has a stronger inhibitory effect on liposome uptake by hepatocytes than on uptake by macrophages. Attempts to shift liposome uptake more in favor of hepatocytes, by incorporation of lactosylceramide, failed. This compound, although causing an increase in hepatic uptake, particularly for the 130-nm liposomes, shifted the H/M ratio further towards the macrophages. We conclude that the galactose moiety of the glycolipid is sufficiently exposed on the surface of (PEG-PE)-containing liposomes to allow interaction with the galactose-binding lectin at the surface of the liver macrophage and that the extent of exposure is dependent on vesicle size.     

The effect of blood on the uptake of liposomal lipid by perfused rat liver

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with phosphatidylinositol (PI) and stearylamine. The absorption of the liposomes by perfused rat liver has been studied as a function of blood level (0-7% haematocrit). It has been found that the rate constant for uptake of liposomes (perfusion constant, kp) is markedly reduced by addition of blood to the perfusate particularly in the haematocrit range 0-3%. The perfusion constant is dependent on the liposome composition and decreases with incorporation of PI and increases with incorporation of stearylamine into DPPC liposomes, but is independent of the initial size of the liposomes in the range of weight-average diameter from 40-400 nm. The possible effects of blood components on the liposomes and their subsequent absorption by the liver are discussed.     

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.     

Alteration of the acetylcholine response by intra- and extracellular serotonin application in intracellularly perfused neurons ofLymnaea stagnalis

1. The effect of serotonin on the acetylcholine (ACh) response has been studied by means of voltage clamp and intracellular perfusion in unidentified isolated neurons from parietal and visceral ganglia of Lymnaea stagnalis. 2. In most cells studied serotonin added to the internal or external solution decreases the response to ACh. 3. In other neurons serotonin added to the intracellular solution increases the response to ACh; when it is added extracellularly it produces the opposite effect on the same cells. 4. The decreasing effect of serotonin on ACh currents is mimicked by cyproheptadine, an antagonist of serotonin receptors, and by the intracellular application of cyclic AMP (cAMP) forskolin. 5. The enhancing effect of intracellularly applied serotonin on ACh currents is blocked by cyproheptadine and is not obtained by the intracellular administration of cAMP and forskolin. In some cells the enhancing effect of serotonin appears after forskolin. 6. The results suggest a modulating effect of serotonin on cholinergic synaptic transmission in the nervous system of mollusks. The possible existence of intracellular serotonin receptors is discussed.     

Serotonin increases the cAMP concentration and the phosphoenolpyruvate carboxykinase mRNA in rat kidney, small intestine, and liver.

Within 60 min of the administration of serotonin to fasted-refed rats, there was a 5-, 16-, and 20-fold stimulation of the mRNA coding for the cytosolic form of P-enolpyruvate carboxykinase in the kidney, small intestine and liver, respectively. This stimulation was 5-, 1.3-, and 2-fold higher than noted in the same tissue after 24 h of starvation. Dose- and time-response curves to serotonin in the three tissues were similar. The level of PEPCK mRNA in the liver was significantly elevated within 30 min of serotonin administration, whereas 60 min was required in the small intestine and the kidney. The direct effect of serotonin on PEPCK mRNA was also assessed in hepatocytes maintained in primary culture. Serotonin (10(-8) M to 10(-4) M) caused a dose-dependent increase in the level of PEPCK mRNA and a transient increase in cAMP concentration. Within the first min of serotonin (10(-6) M) addition to cells, cAMP concentration increased 4-fold and returned after 10 min to basal level. Therefore, these results provide functional evidence of serotonin action in the rat peripheric tissues and suggest that cAMP is involved in its intracellular signalling.     

Fast, efficient reconstitution of the cyclooxygenases into proteoliposomes

To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes.     

Biochemical consequences of follicle-stimulating hormone binding to testicular macrophages in culture

The present studies demonstrate that testicular macrophages respond to follicle-stimulating hormone (FSH) by: 1) stimulating the rate of incorporation of amino acids into secreted proteins; 2) increasing the rate of incorporation of uridine into RNA; and 3) enhancing the accumulation of intracellular cyclic adenosine monophosphate (cAMP; which was potentiated by the addition of 1 mM 3-isobutyl-1-methylxanthine; MIX). In addition, dibutyryl cAMP (dbcAMP) enhanced the incorporation of amino acids into secreted proteins; however, this cAMP analog had no effect on the incorporation of uridine into RNA. Finally, it was demonstrated that testicular macrophages possess specific receptors with a high affinity for FSH.     

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.     

Permeability properties of liposomes prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin

Liposomes have been prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin.Permeability properties of liposomes thus prepared were studied toward glucose. The glucose permeability of liposomes with saturated lecithins (dipalmitoyllecithin and dimyristoyllecithin) and sphingomyelin appears to be more strongly temperature dependent than that of liposomes with lecithin containing unsaturated fatty acyl chains (egg and rat liver lecithins). The permeability of glucose through vesicles of dipalmitoyllecithin or dimyristoyllecithin was enhanced drastically at their transition temperatures, while the incorporation of about 25 mole% of egg lecithin into liposomes of saturated lecithins suppressed the enhanced permeation rates of glucose above the transition temperatures.The incorporation of small amounts of cholesterol enhanced the temperature-dependent permeability of glucose through the bilayer of saturated lecithins or sphingomyelin. This tendency was best shown in the case of dipalmitoyl-lecithin, in which 20 mole% of cholesterol had the most stimulating effect on the temperature-dependent permeability. The introduction of more than 33 mole% of cholesterol showed, however, reduced effects on the temperature-dependent permeability through liposomes with saturated lecithins or sphingomyelin. It was also shown that cholesterol had a much larger effect on the regulation of the temperature-dependent permeability of liposomes prepared with saturated lecithins or sphingomyelin than on that of liposomes prepared with phospholipids containing unsaturated fatty acids.     

Receptor-mediated uptake of asialoganglioside liposomes: sub-cellular distribution of the liposomal marker in isolated liver cell types

The use of asialo GM1-containing small unilamellar liposome preparations in vivo caused a 2.8-fold increase in the uptake by the liver as compared with the control (neutral) preparations (without asialo GM1). The uptake of negatively charged dicetylphosphate and dipalmitoyl phosphatidic acid-containing small unilamellar liposomes was found to be 1.6-and 1.8-fold respectively higher than that of the neutral preparations. In studies with isolated liver cell types, inhibition of the galactosylated liposome uptake by asialofetuin indicated a possible involvement of hepatic galactose receptors in the recognition of asialo GM1 liposomes by the hepatic parenchymal cells, which in turn were found to be mainly responsible for the enhanced incorporation of these liposomes in the liver. Sub-cellular distribution studies with isolated liver cell types indicated lysosomal localization of the liposomes both in parenchymal and nonparenchymal cells, and it has been proposed that the asialo GM1 liposomes are cointernalized with asialofetuin through a common lysosomal route of ligand internalization.     

Involvement of the membrane fluidity of lactosylceramide-targeted liposomes in their intrahepatic uptake

Incorporation of N-lignoceroyldihydrolactocerebroside (lactosylceramide) enhanced liver uptake of small unilamellar liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate (molar ratio, 4:5:1). The increase in liver uptake was mostly accounted for by an enhanced uptake into the parenchymal cells. The enhancing effects of lactosylceramide on uptake of the liposomes into liver in vivo and into isolated parenchymal cells in vitro were greater with dipalmitoylphosphatidylcholine-based liposomes than with dimyristoylphosphatidylcholine-based ones. In contrast, addition of lactosylceramide had no significant effect on egg phosphatidylcholine vesicle uptake. The stimulated uptake of lactosylceramide liposomes by parenchymal cells was counteracted by added asialofetuin. These observations suggest that transfer of the targeted liposomes via a galactose-specific receptor into parenchymal cells may be controlled by the membrane fluidity of the liposomes.     

Influence of cyclic AMP on DNA synthesis in slices of regenerating rat liver.

DNA synthesis in slices of regenerating rat liver is inhibited by adenosine cyclic 3',5'-monophosphate [cAMP]. The number of cells synthesizing DNA as assayed by 2-14C-thymidine incorporation is reduced by 65% in the presence of 10(-3) M cAMP. The inhibition of cAMP is not specific; other adenosine compounds, N6,O2,-dibutyryl adenosine 3',5'-monophosphate, 5'AMP and adenosine have the same effect. Moreover, the concentration of cAMP in the cell required for this inhibition is much higher than the normal levels of cAMP in liver cells.     

Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition

We investigated the intrahepatic distribution of small unilamellar liposomes injected intravenously into rats at a dose of 0.10 mmol of lipid per kg body weight. Sonicated liposomes consisting of cholesterol/sphingomyelin (1:1), (A); cholesterol/egg phosphatidylcholine (1:1), (B); cholesterol/sphingomyelin/phosphatidylserine (5:4:1), (C) or cholesterol/egg-phosphatidylcholine/phosphatidylserine (5:4:1), (D) were labeled by encapsulation of [3H]inulin. The observed differences in rate of blood elimination and hepatic accumulation (A much less than B approximately equal to C less than D) confirmed earlier observations and reflected the rates of uptake of the four liposome formulations by isolated liver macrophages in monolayer culture. Fractionation of the liver into a parenchymal and a non-parenchymal cell fraction revealed that 80-90% of the slowly clearing type-A liposomes were taken up by the parenchymal cells while of the more rapidly eliminated type-B liposomes even more than 95% was associated with the parenchymal cells. Incorporation of phosphatidylserine into the sphingomyelin-based liposomes caused a significant increase in hepatocyte uptake but a much more substantial increase in non-parenchymal cell uptake, resulting in a major shift of the intrahepatic distribution towards the non-parenchymal cell fraction. For the phosphatidylcholine-based liposomes incorporation of phosphatidylserine did not increase the already high uptake by the parenchymal cells while uptake by the non-parenchymal cells was only moderately elevated; this resulted in only a small shift in distribution towards the non-parenchymal cells. The phosphatidylserine-induced increase in liposome uptake by non-parenchymal liver cells was paralleled by an increase in uptake by the spleen. Fractionation of the non-parenchymal liver cells in a Kupffer cell fraction and an endothelial cell fraction showed that even for the slowly eliminated liposomes of type A endothelial cells do not participate to a measurable extent in the elimination process, thus excluding involvement of fluid-phase pinocytosis in the uptake process.     

Effects of alkyl glycosides incorporated into liposomes prepared from synthetic amphiphiles on their tissue distribution in Ehrlich solid tumor-bearing mice.

A study of the effects of alkyl glycosides incorporated into synthetic liposomes with respect to their stability, their in vivo distribution in Ehrlich solid tumor-bearing mice and their in vitro interaction with liver cells was undertaken. The synthetic liposomes were prepared from N,N-didodecyl-N alpha-[6-(trimethylammonio)hexanoyl]-L-alaninamide bromide (N+C5Ala2C12) and labeled with 99mTc. n-Dodecyl glucoside (DG) and n-dodecyl sucrose (DS) were used as alkyl glycosides. The stability was hardly changed by incorporation of alkyl glycosides into the liposomes in saline and serum. The uptake of DG- and DS-modified N+C5Ala2C12 liposomes decreased in liver and spleen compared with that of unmodified N+C5Ala2C12 liposomes, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, where the DS-modified N+C5Ala2C12 liposomes had a marked tendency. It was observed with electron micrographs that the size of N+C5Ala2C12 liposomes became small by incorporation of alkyl glycoside. The smaller N+C5Ala2C12 liposomes were found to result in the lower uptake in liver. The interaction of the liposomes with liver cells in vitro indicated that both DG- and DS-modified liposomes had a low affinity for liver cells compared with the unmodified liposomes and the extent of interaction of the DS-modified liposomes was weaker than that of the DG-modified liposomes.