Polarized distribution of oxalate transport systems in LLC-PK1 cells, a line of renal epithelial cells.

Although oxalate is a major component of kidney stones, the factors affecting renal oxalate handling are poorly understood. This uncertainty stems in part from complexities inherent to available preparations; thus the present studies examined oxalate handling in a simpler model system, LLC-PK1 cells, an epithelial cell line of porcine origin. Initial studies on monolayers in dishes demonstrated that these cells accumulate oxalate via a process or processes sensitive to the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Subsequent studies using LLC-PK1 monolayers on membrane filters examined the characteristics and distribution of these transporter(s). At the apical surface, DIDS-sensitive uptake was sensitive to [Cl-] but not [SO4(2-)] or [HCO3-] and was unaffected by alterations in pH or membrane potential. At the basolateral surface, oxalate uptake was [Cl-] insensitive but markedly affected by variation in pH, [SO4(2-)], or [HCO3-]. Uptake at the two membrane surfaces was also differentially affected by transport inhibitors and organic acids. Thus LLC-PK1 cells appear to express unique transporters at each membrane surface: oxalate/Cl- exchange at the apical surface and oxalate/SO4(2-) (or HCO3-) exchange at the basolateral surface.

A liposome based platelet substitute, the plateletsome, with hemostatic efficacy.

The complexity of platelet mediated hemostasis has hindered development of a platelet substitute for transfusion therapy. In the current study, the hemostatic efficacy of a liposome based modality, the plateletsome, is demonstrated. A deoxycholate extract of a platelet membrane fraction, with a minimum of 15 proteins including GPIb, GPIIb-IIIa and GPIV/III, was incorporated into sphingomyelin: phosphatidylcholine: monosialylganglioside or egg phosphatide small unilamellar vesicles by reverse-phase/sonication and French press extrusion. These plateletsomes decreased bleeding by 67% in the tail bleeding time in rats made thrombocytopenic (platelets < 30,000/microliters) with external irradiation (7-9Gy) by Cesium source. Efficacy was also demonstrated in the thrombocytopathic, Fawn-Hooded rat, but to a lesser extent than in the thrombocytopenic animals. Direct plateletsome infusion to the tail wound was more effective than systemic administration for all effective preparations. On post-mortem examination, no pathologic thrombi were detected by gross and histopathologic examination of the lungs, livers, kidneys, or spleens of thrombocytopenic or normal animals after plateletsome infusion. No evidence of intravascular coagulation, monitored by levels of circulating fibrinogen and platelet counts, was observed when plateletsomes were administered intravenously to rabbits. No deleterious effect, either inhibition or hyperaggregability, on platelet aggregation studies in vitro was observed. While further refinements are clearly required, this study indicates that liposomes bearing specific platelet proteins may provide a basis for a clinically applicable platelet substitute.

Effect of calcium channel blockers on platelet GPIIb-IIIa as a calcium channel in liposomes: comparison with effects on the intact platelet.

The platelet membrane glycoprotein IIb-IIIa complex is essential for platelet aggregation and functions as a fibrinogen receptor on the activated platelet. When incorporated into phospholipid vesicles, this glycoprotein complex can function as an apparent calcium channel which facilitates the transit of calcium across a phospholipid barrier. In order to further evaluate this calcium channel, the effect of calcium channel blockers of the dihydropyridine (nifedipine and nicardipine), arylalkylamine (verapamil) and benzothiazepine (diltiazem) classes were evaluated on GPIIb-IIIa liposomes with encapsulated fura-2 (a fluorescent calcium indicator). Nicardipine, verapamil, and nifedipine significantly inhibited calcium influx into GPIIb-IIIa liposomes; however, this required 190 microM, 400 microM, and 140 microM drug, respectively. These concentrations are 10-1,000 fold greater than those clinically obtainable. In contrast, diltiazem at concentrations greater than 220 microM and amiloride at concentrations greater than 800 microM showed no inhibitory effects. When aspirinized platelets were activated with 30 micrograms/ml bovine fibrillar collagen, both nicardipine and diltiazem produced a decrease in both the initial rise and maximum cytoplasmic calcium concentration. Parallel experiments were performed to assess the effects of verapamil, nicardipine, and diltiazem on platelet aggregation in platelet rich plasma. Nicardipine, 190-380 microM, induced a prolongation of the lag phase, but no effect on the final degree of platelet aggregation to collagen. Similar inhibition of platelet aggregation was seen with diltiazem and verapamil although the effect of diltiazem was less pronounced particularly at higher concentrations of collagen. No effect was seen on aggregation with 32 microM ADP which is release independent, or on the primary wave of low dose ADP induced platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand inhibition of the platelet glycoprotein IIb-IIIa complex function as a calcium channel in liposomes.

Platelet glycoproteins IIb and IIIa function as a fibrinogen receptor on the activated platelet. We have shown that these glycoproteins can be incorporated onto the surface of phosphatidylcholine vesicles with retention of fibrinogen and antibody binding properties and can permit Ca2+ transit across the phospholipid bilayer. In the current study we demonstrate that this apparent Ca2+ channel function is specifically inhibited by the synthetic analogue of the fibrinogen gamma COOH-terminal peptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (His-12-Val), but not by the adhesive protein sequence Arg-Gly-Asp-Ser (RGDS). Prior incubation of IIb-IIIa liposomes with RGDS prevented Ca2+ transit inhibition by 25 microM His-12-Val, analogous to RGDS inhibition of His-12-Val binding to platelets. His-12-Val inhibited a minor component of transmembrane Ca2+ influx into ADP and thrombin-activated human platelets but had no effect on steady-state platelet 45Ca flux. These data indicate that ligand binding may exert a regulatory influence on transmembrane Ca2+ influx into activated platelets. The difference in inhibitory potency of the peptides studied may be related to differences in conformational changes in the glycoprotein IIb-IIIa complex induced by His-12-Val and RGDS, steric considerations, or differences in interactions with glycoprotein IIb Ca2+ binding domains.

Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes.

Human platelet membrane glycoproteins IIb and IIIa (GPIIb and IIIa) were incorporated into phospholipid vesicles by the reverse-phase technique to assess the ability of GPIIb and IIIa to function as a Ca2+ channel. Movement of Ca2+ across the lipid bilayer was quantitated by injection of proteoliposomes with encapsulated Fura-2 into Ca2+ buffers and measurement of Fura-2 fluorescence as an indicator of Ca2+ influx. Reciprocally, to assess the function of proteins in an inside-out orientation, Ca2+-loaded vesicles were injected into Ca2+-free buffer and Ca2+ efflux monitored by a calcium electrode. Incorporation of the IIb-IIIa complex produced significant facilitation of Ca2+ movement across the lipid bilayer. No net transmembrane Ca2+ movement was seen with dissociated IIb and IIIa. Movement of Ca2+ was proportional to the transmembrane Ca2+ gradient. Ca2+ movement into the vesicles was inversely proportional to extravesicular NaCl from 25 to 150 mmol/L, analogous to several studies in the intact platelet. Adenosine triphosphate had no effect on Ca2+ movement into or out of the vesicles. Specific inhibition of a Ca2+ shift into the vesicles was seen with M148, a monoclonal antibody to IIb/IIIa, while no inhibition was observed with a panel of other anti-IIb/IIIa monoclonal antibodies. This suggests that a specific site on the complex or orientation of the complex is essential for calcium channel function. These data demonstrate that the GPIIb/IIIa complex can serve as a passive Ca2+ channel across a phospholipid bilayer and has the potential to play a role in Ca2+ flux across the platelet plasma membrane.

A major metabolite of delta 1-tetrahydrocannabinol reduces its cataleptic effect in mice.

The results described here demonstrate that THC-induced catalepsy in mice can be substantially inhibited by the prior administration of delta 1-THC-7-oic acid, the major metabolite of THC in most species including humans. This raises the possibility that the intensity and duration of action of THC may depend to a large degree on the levels of this metabolite at the sites of action.

Prostaglandins and cannabis XV. Comparison of enantiomeric cannabinoids in stimulating prostaglandin synthesis in fibroblasts.

Stereospecificity has been reported for a number of actions of the cannabinoids in a variety of systems. In the present report, we have shown that this effect can also be demonstrated when human lung fibroblasts in monolayer culture are stimulated by cannabinoids to produce prostaglandin E2 (PGE2). Three enantiomeric pairs of cannabinoids, (+) and (-)-delta 1-tetrahydrocannabinol (THC), (+) and (-)-delta 6-THC and (+) and (-)-delta 6-dimethylheptyl (DMH) THC were tested. In each case the (-) isomer was significantly more potent in agreement with the findings of others using different systems. Interestingly, very little stereospecificity was found in fibroblasts when the release of arachidonic acid, the precursor of PGE2, was monitored. This suggests that cannabinoids may act at several sites within the cell some of which show comparatively greater stereoselectivity for these agonists.

Effects of cannabinoids on the activities of mouse brain lipases.

Cannabinoids were found to augment phospholipase activities and modify lipid levels of mouse brain synaptosomes, myelin and mitochondria. Delta-1-tetra-hydrocannabinol (delta 1-THC) and several of its metabolites induced a dose-dependent (0.32-16 microM) stimulation of phospholipase A2 (PLA2) activity resulting in the increased release of free arachidonic acid from exogenous [1-14C]phosphatidylcholine (PC). The potencies of the cannabinoids in modulating PLA2 activity were approximately of the order: 7-OH-delta 1-THC greater than delta 1-THC greater than 7-oxo-delta 1-THC greater than delta 1-THC-7-oic acid = 6 alpha OH-delta 1-THC much greater than 6 beta-OH-delta 1-THC. The hydrolysis of phosphatidylinositol (PI) by synaptosomal phospholipase C (PLC) was enhanced significantly by delta 1-THC and promoted diacylglyceride levels by greater than 100 percent compared to control values. In contrast, arachidonate was the major product resulting from phospholipase activities of a 20,000 g pellet. Synaptosomal diacylglyceride lipase activity was inhibited by delta 1-THC. [1-14C]Arachidonic acid was readily incorporated into subcellular membrane phospholipids and after exposure to cannabinoids led to diminished phosphoglyceride levels and concomitant increases in released neutral lipid products. These data suggest that cannabinoids control phospholipid turnover and metabolism in mouse brain preparations by the activation of phospholipases and, through this mechanism, may exert some of their effects.

Prostaglandins and cannabis--XVI. Antagonism of delta 1-tetrahydrocannabinol action by its metabolites.

Prior exposure of cells in vitro to delta 1-tetrahydrocannabinol-7-oic acid (delta 1-THC-7-oic acid) reduced the degree of stimulation of prostaglandin synthesis incurred by subsequent treatment with delta 1-THC. The site of action of this inhibitory effect seemed to be on cyclooxygenase and not at the earlier step involving the phospholipase-mediated release of arachidonic acid. delta 1-THC-7-oic acid is a major metabolite of delta 1-THC and has no psychoactivity in humans. Our findings raise the possibility, however, that it may influence the in vivo activities of delta 1-THC by antagonizing its stimulatory action on cellular prostaglandin synthesis.

Prostaglandins and cannabis XIV. Tolerance to the stimulatory actions of cannabinoids on arachidonate metabolism.

The stimulation of prostaglandin E2 synthesis by delta 1-tetrahydrocannabinol in cultured cells is rapidly diminished by successive exposures to the drug at 24-hr intervals. Cannabidiol and cannabicyclol, two other constituents of cannabis, also displayed this in vitro tolerance effect. The phenomenon could, in addition, be observed by measuring the release of arachidonic acid from these cells, suggesting that the site of action of the cannabinoids is at one or more of the lipases that are believed to control prostaglandin synthesis under most conditions. Tolerance to cannabinoid action has been reported for a variety of in vivo parameters; thus, this in vitro system exhibits similar behavior and may, therefore, be a good model for studies on the molecular mechanisms involved in tetrahydrocannabinol action.

Prostaglandins and cannabis--XIII. Cannabinoid-induced elevation of lipoxygenase products in mouse peritoneal macrophages.

The phospholipases controlling the release of arachidonic acid in mouse peritoneal macrophages have been shown to be stimulated by the natural psychoactive cannabinoids. A close correlation was observed between the potencies of these substances in elevating arachidonate levels in vitro and the reported activities in a behavioral assay in monkeys and in producing a "high" in humans. The order of activity with the macrophages was delta 1-tetrahydrocannabinol (delta 1-THC) greater than 7-OH-delta 1-THC greater than 6 alpha-OH-delta 1-THC greater than 6 beta-delta 1-THC much greater than delta 6-THC-7-oic acid. It is suggested that this effect, which has now been shown in several diverse cell types, may serve as a model for studying the mechanism of action of THC.

Stimulation of sphingomyelin hydrolysis by cannabidiol in fibroblasts from a Niemann-Pick patient.

The hydrolysis of sphingomyelin in cells derived from a Niemann-Pick patient was studied using both a labelled precursor and measurement of endogenous levels. In vitro exposure of the cells to cannabidiol resulted in a large decrease in both the relative and absolute amounts of this lipid; the drug had a smaller effect on normal fibroblasts. Cannabidiol has been tested in the clinic as an antiepileptic agent with some success; our findings suggest that it may also be useful in relieving the symptoms associated with Niemann-Pick disease.