Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases.

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.

Muscle and motor-skill dysfunction in a K+ channel-deficient mouse are not due to altered muscle excitability or fiber type but depend on the genetic background.

The voltage-gated K+ channel Kv3.1 is expressed in skeletal muscle and in GABAergic interneurons in the central nervous system. Hence, the absence of Kv3.1 K+ channels may lead to a phenotype of myogenic or neurogenic origin, or both. Kv3.1-deficient (Kv3.1-/-) 129/Sv mice display altered contractile properties of their skeletal muscles and show poor performance on a rotating rod. In contrast, Kv3.1-/- mice on the (129/Sv x C57BL/6)F1 background display normal muscle properties and perform like wild-type mice. The correlation of poor performance on the rotating rod with altered muscle properties supports the notion that the skeletal muscle dysfunction in Kv3.1-/- 129/Sv mice may be responsible for the impaired motor skills on the rotating rod. Surprisingly, we did not find major differences between wild-type and Kv3.1-/- 129/Sv skeletal muscles in either the resting or action potential, the delayed-rectifier potassium conductance (gK) or the distribution of fast and slow muscle fibers. These findings suggest that the Kv3.1 K+ channel may not play a major role in the intrinsic excitability of skeletal muscle fibers although its absence leads to slower contraction and relaxation and to smaller forces in muscles of 129/Sv Kv3.1-/- mice.

Regional modulation of cyclic nucleotides by endothelin-1 in rat pulmonary arteries: direct activation of G(i)2-protein in the main pulmonary artery.

The ability of endothelin-1 (ET-1) to modulate the cyclic nucleotides, guanosine 3' 5' cyclic monophosphate (cyclic GMP) and adenosine 3' 5' cyclic monophosphate (cyclic AMP) was assessed in the main elastic pulmonary elastic artery (4 - 5 mm i.d.) and the small muscular pulmonary arteries (150 - 200 micrometer i.d.) of the rat. ET-1 caused an increase in cyclic GMP in the larger vessels but had no effect in the smaller arteries. The increase in cyclic GMP was not dependent on an intact endothelium and was inhibited by the ET(A)-receptor antagonist FR139137 (1 microM). ET-1 caused a decrease in cyclic AMP in the main pulmonary arteries, an effect that was partially blocked by FR139317 but not influenced by the ET(B)-receptor antagonist BQ-788 (1 microM) or removal of the vascular endothelium. In contrast, ET-1 caused an increase in cyclic AMP in the small vessels, an effect that was blocked by BQ-788 but unaffected by FR139317. In the main pulmonary arteries, ET-1 caused enhanced incorporation of radiolabelled ADP-ribose by cholera toxin into G(i)2 in the main pulmonary artery, an indicator of its receptor-mediated activation. In summary, we have shown that in the small muscular pulmonary artery of the rat, (where ET(B) mediated vasoconstriction prevails), there is an ET(B)-mediated increase in cyclic AMP with no net effect on cyclic GMP levels. In the large arteries, (where vasoconstriction is mediated via the ET(A) receptor), there is an ET(A)-mediated increase in cyclic GMP (endothelium independent) and an ET(A)-mediated (endothelium independent) decrease in cyclic AMP.

Endothelin-1 modulation of cAMP in rat pulmonary arteries: effect of chronic hypoxia.

The effect of hypoxic shock on the ability of endothelin (ET) receptors to interact with the cAMP second messenger system was assessed in rat pulmonary arteries. Whole pieces of tissue were dissected from the pulmonary arterial system of control and hypoxic (10% O2, 14 days) rats, incubated, where appropriate, with ET-1 (0.1 microM), and the levels of intracellular cAMP measured. Maintenance of rats under hypoxic conditions significantly reduced the basal cAMP levels in all of the arterial branches with the exception of the pulmonary resistance vessels, in which no change was observed. Incubation of the main and first branch extralobar pulmonary arteries from control rats with ET-1 resulted in a consistent decrease in the levels of intracellular cAMP. The ETA receptor antagonist FR139317 partially blocked this ET-1-mediated inhibition of cAMP accumulation in the main extralobar artery. In contrast, ET-1 caused a threefold increase in the levels of this cyclic nucleotide in the pulmonary resistance vessels from the normoxic rat. No ET-1-mediated reduction in intracellular cAMP levels was observed in any of the vessels isolated from hypoxic animals. All vessels showed ligand-activated increases in cAMP production. These results suggest differential modulation of cAMP in the different pulmonary arteries, either by direct activation through Gi and Gs or indirectly via a uncharacterized cross-talk mechanism.

Structure and function of ryanodine receptors.

Membrane depolarization, neurotransmitters, and hormones evoke a release of Ca2+ from intracellular Ca(2+)-storing organelles like the endoplasmic reticulum and, in muscle, the sarcoplasmic reticulum (SR). In turn, the released Ca2+ serves to trigger a variety of cellular responses. The presence of Ca2+ pumps to replenish intracellular stores was described more than 20 years ago. The presence of Ca2+ channels, like the ryanodine receptor, which suddenly release the organelle-stored Ca2+, is a more recent finding. This review describes the progress made in the last five years on the structure, function, and regulation of the ryanodine receptor. Numerous reports have described the response of ryanodine receptors to cellular ions and metabolites, kinases and other proteins, and pharmacological agents. In many cases, comparative measurements have been made using Ca2+ fluxes in SR vesicles, single-channel recordings in planar bilayers, and radioligand binding assays using [3H]ryanodine. These techniques have helped to relate the activity of single ryanodine receptors to global changes in the SR Ca2+ permeability. Molecular information on functional domains within the primary structure of the ryanodine receptor is also available. There are at least three ryanodine receptor isoforms in various tissues. Some cells, such as amphibian muscle cells, express more than a single isoform. The diversity of ligands known to modulate gating and the diversity of tissues known to express the protein suggest that the ryanodine receptor has the potential to participate in many types of cell stimulus-Ca(2+)-release coupling mechanisms.

Functional expression of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum in COS-1 cells.

A full-length cDNA encoding the ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was transiently expressed in COS-1 cells. Immunoblotting studies showed that the expressed ryanodine receptor and the native ryanodine receptor of rabbit skeletal muscle were indistinguishable in molecular size and immunoreactivity. Scatchard analysis of [3H]ryanodine binding to transfected COS-1 cell microsomes resulted in a Bmax of 0.22 pmol/mg of protein and a Kd of 16.2 nM. Expressed ryanodine receptors were solubilized in CHAPS and were shown to cosediment with native ryanodine receptors in a sucrose density gradient. Thus, the expressed receptor, like the native receptor, is assembled as a large oligomeric complex. Single-channel recordings in planar lipid bilayers were used to investigate the functional properties of the sucrose gradient-purified complex. The expressed ryanodine receptor formed a large conductance channel activated by ATP and Ca2+ and inhibited by Mg2+ and ruthenium red. Ryanodine reduced the conductance and increased the mean open time in a manner consistent with that of native channels. These results demonstrated that functional binding sites for the physiological ligands (Ca2+, Mg2+, and ATP) and pharmacological ligands (ruthenium red and ryanodine) controlling gating of the Ca2+ release channel are encoded in the ryanodine receptor cDNA and are faithfully expressed in COS-1 cells. Ryanodine receptors expressed in COS-1 cells displayed several conductance states > or = 1 nS not present in native channels. Such anomalous conductance states of the expressed channel might be referable to lack of muscle-specific posttranslational processing or to the need for components not present in COS-1 cells, which may be required to stabilize the channel structure.

Inhibitors of adenosine catabolism improve recovery of dog myocardium after ischemia.

The effects of inhibitors of adenosine catabolism on contractile function and metabolites were assessed during 15 minutes of ischemia followed by 30 minutes of reperfusion in the open-chest dog heart. As compared to sham treatment, pretreatment with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and dipyridamole (DP) protected contractile function during ischemia, and improved recovery of high energy phosphate content and contractile function during reperfusion following ischemia. Testing EHNA and DP in a free-radical generating system indicated both compounds have some scavenging ability, suggesting the effect of EHNA + DP may not be on adenosine nucleotide metabolism alone. Comparison of end diastolic segment lengths to contractile function indicated the results were not affected by changes in preload resulting from peripheral vasodilation.

Ultrastructure, peroxisomes and lipid peroxidation in reperfused myocardium.

Reperfusion injury was studied in dog myocardium using a transmission electron microscope and 3,3'-diaminobenzidine (DAB) to locate areas of peroxidatic activity. Dark electron dense DAB reaction product was observed in peroxisomes and damaged mitochondria. These results suggest attack by reactive oxygen species on mitochondrial membranes, which might result in the formation of lipid peroxides and prostaglandin-like compounds. It is suggested that the release of lipid peroxide or prostaglandins from the injured cells may contribute to reactive hyperaemia, ventricular fibrillation and angina.