Effects of liposome-entrapped D -myo-inositol 1,4,5-trisphosphate and D -myo-inositol 1,3,4,5-tetrakisphosphate in the isolated rat aorta

European Journal of Pharmacology, 250 (1993)493-495

© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

EJP 21393 Short communication

Effects of liposome-entrapped D-myo-inositol 1,4,5-trisphosphate and D-myo-inositol 1,3,4,5-tetrakisphosphate in the isolated rat aorta E u g e n Brailoiu

a

D r a g o m i r N. S e r b a n a L a u r e n t i u M. P o p e s c u b, S e b a s t i a n S l a t i n e a n u a, Catalin M. F i l i p e a n u a and D i m i t r i e D. B r a n i s t e a n u .,a

a Department of Physiology, University of Medicine and Pharmacy 'Gr. T. Popa', 16 Universitatii Street, lasi, Romania, and b Department of Cell Biology, University of Medicine and Pharmacy 'Carol Davila', Bucharest, Romania

Received 24 August 1993,accepted 22 October 1993

This study examined the effects of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)- and o-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4)-loaded liposomes upon the contractile activity of vascular smooth muscle, using the isolated (endothelium removed) rat aortic ring as an in vitro model. While control liposomes had no effect, the administration of Ins(1,4,5)P3-containing liposomes contracted the smooth muscle preparation. Furthermore, a similar effect was seen with the administration of Ins(1,3,4,5)P4-filled liposomes but, in this case, the rings developed a significantly higher level of active tension. Pretreatment of the aortic preparation with heparin-loaded liposomes blocked the contractions induced by both Ins(1,4,5)P 3- and Ins(1,3,4,5)Pa-containing liposomes. Liposome; Smooth muscle, aortic; D-myo-Inositol 1,4,5-trisphosphate; D-myo-Inositol 1,3,4,5-tetrakisphosphate

1. Introduction

2. Materials and methods

The hydrolysis of phosphatidylinositol 4,5-bisphosphate into D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) and diacylglycerol is a key step for the initiation of vascular smooth muscle contraction after agonist-receptor coupling. The Ins(1,4,5)P3-induced Ca 2÷ release from intracellular stores is now considered the main mechanism involved in the early cytosolic Ca 2÷ elevation and an essential step for the development of active tension during agonist-induced smooth muscle contraction (Somlyo et al., 1985). Furthermore, in several tissues an increased concentration of o - m y o - i n o s i t o l 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) has been reported during and after stimulation of the phosphoinositide system (Batty et al., 1985; Lambert et al., 1991). Ins(1,3,4,5)P4 may also be able to induce an increase of cytosolic Ca 2+ levels (Cullen et al., 1989; Ely et al., 1990). Liposomes are potentially useful for the intracellular delivery of drugs. These lipid vesicles can be captured by the arterial wall (Hodis et al., 1990). Our study examined the effects of Ins(1,4,5)P 3 and Ins(1,3,4,5)P4, intracellularly administered via liposomes, upon the contractile activity of the isolated aortic smooth muscle.

2.1. Liposome preparation

* Corresponding author. Fax40-8-l12406.

The liposomes used in this study were prepared from egg phosphatidylcholine (Fluka); 60 mg lipid per ml of solution to be incorporated, according to the method described by Bangham et al. (1965) and modified by us as follows. Diethylether was added to the suspension of multilamellar vesicles in a 1 : 10 volume ratio. The organic solvent was then removed by rotary evaporation under reduced pressure. Control liposomes contained only KC1 (140 mM) solution (pH adjusted to 6.9). The same solution was used to prepare Ins(1,4,5)P 3 (10 -5 M)-, Ins(1,3,4,5)P4 (10 -5 M)and heparin (5 × 10 -5 M)-containing liposomes (the three incorporated compounds were from Sigma). In order to remove the non-incorporated solutes, all liposome batches were either subjected to dialysis in the Krebs-Henseleit solution (24 h, 1/600 v / v ratio, at 4°C, using standard dialysis tubing from Sigma), or passed through a Sephadex G50 (2 × 30 cm, Pharmacia Fine Chemicals) chromatography column (similar results were obtained by using either of these two methods). A spectroscopic method was used in order to calculate the encapsulation efficiency for this type of liposomes. The value found for the adenosine encapsulation efficiency was 10.56% (S.E.M. + 0.71%). However,

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this parameter may be different for the compounds used in organ bath experiments in this study. Electron micrographs of the liposome suspensions were obtained using a Tesla BS 500 electron microscope, after staining the vesicles for 90 s in a phosphotungstic acid solution (1.5%). The diameter of the liposomes varied between 0.1 and 0.8 /xm.

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3. Results

While the administration of control liposomes had no effects, Ins(1,4,5)P3-1oaded liposomes contracted the rat aortic rings to a level representing 45 + 8% (n = 6) of the high K ÷ (40 mM KC1)-induced contraction. Ins(1,3,4,5)P4-filled liposomes also contracted the aortic rings (80 +_ 10% of the mentioned reference level; n = 6). Both types of liposomes were tested either sequentially on the same ring (after washout and relaxation down to baseline tension), or on parallel rings obtained from the same animal (no differences between the results obtained with these two approaches). The above mentioned results correspond to administration of 2 ml liposome suspension to the 8 ml KrebsHenseleit solution in the organ bath ( 1 : 4 v / v ratio). Both contractions, which depended upon intracellular drug delivery from liposomes, reached in 5 rain a stable maximum tension level, maintained for at least 20 min thereafter, and were fully reversible. The reference KCI 40 mM-induced contractions were obtained before the liposome challenges and reproduced after washout and re-equilibration.

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2.2. Tissue preparation To obtain the rat aortic rings, male albino rats (150 g body weight) were decapitated and exsanguinated. The thoracic aorta was rapidly removed and cut into rings, 2 mm wide. The endothelium was rubbed gently with a smooth softwood stick. The rings were then mounted between hooks and their mechanical activity was monitored using an isometric force transducer and a potentiometric pen recorder. The 10-ml organ bath contained Krebs-Henseleit solution (pH 7.4) with the following composition (mM): NaCI, 118; KC1, 4.8; CaC12, 2.5; MgSO 4, 1.6; K H 2 P O 4, 1.2; N a H C O 3, 25; glucose, 5.5. The Krebs-Henseleit solution was kept at 37°C and aerated continuously with 95% 0 2 + 5% CO 2. The resting tension was maintained at 2 g, the preparation being allowed to equilibrate for 2 h before starting the experiment. The lack of a functional endothelium was confirmed in each aortic preparation by its inability to relax in response to 10 .s M carbachol when precontracted by 40 mM K +. The liposome suspension was added to the organ bath in volume ratios between 1/100 and 1/2.

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Fig. 1. Effects of intraliposomal Ins(1,4,5)P 3 (trace b), Ins(1,3,4,5)P4 (trace c) and both (traces b and c; notice the different administration sequences), as compared to the control contraction induced by 40 m M K + (trace a), in de-endothelised rat aortic rings. Percent values of the respective tension levels as compared to the control are shown in the figure (mean _+S.E.M., n = 6).

Administration of higher amounts of liposome suspension did not elicit stronger contractions; the dose described above (intraliposomal concentrations of 10 .5 M lns(1,4,5)P 3 or Ins(1,3,4,5)P4 with an 1:4 v / v administration) has a maximal effect for both Ins(1,3,5)P 3and Ins(1,3,4,5)P4-1oaded liposomes. On the other hand, contractile effects appeared only for volumes of liposome suspension greater than 1.5 ml added to the 8 ml Krebs-Henseleit solution in the organ bath (dilution < 0.16), indicating a rather steep dose-effect curve. No contractile effects were seen with intraliposomal concentrations lower than 5 × 10 -6 M of either of the two phosphoinositols (not shown). Concentrations higher than 10 - 4 M were not affordable due to the price and to the important losses during liposome preparation. Adding Ins(1,4,5)P3-1oaded liposomes to rings precontracted by Ins(1,3,4,5)P4-filled liposomes did not result in any significant additional effect (fig. 1). In contrast, addition of vesicles containing Ins(1,3,4,5)P4 during the contractions induced by Ins(1,4,5)P3-filled liposomes increased the contractile force up to the level elicited by the Ins(1,3,4,5)P4-containing liposomes alone (fig. 1). Pretreatment of aortic preparations with heparinfilled liposomes (5 × 10 5 M intraliposomal heparin, 1:4 v / v administration) blocked the contractions induced by both Ins(1,4,5)P 3- and Ins(1,3,4,5)P4-1oaded liposomes. The effects of intraliposomal heparin upon the already developed contractions were not tested.

4. Discussion

The contraction induced by Ins(1,4,5)P3-1oaded liposomes could be simply explained by the ability of Ins(1,4,5)P 3 to increase the cytosolic Ca z+ level in the aortic smooth muscle (Somlyo et al., 1985). The in-

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hibitory effect of liposome-entrapped heparin upon this contraction is most probably based upon the ability of heparin to block the Ins(1,4,5)P3-dependent Ca 2+ release from the endoplasmic reticulum (Watras et al., 1989). However, its interaction with G proteins could also be somehow involved (Kobayashi et al., 1988). The contraction elicited by Ins(1,3,4,5)Pa-filled liposomes could hardly be explained by a direct activation of Ca 2÷ release from the sarcoplasmic reticulum, while such an effect has been excluded by others (Watras et al., 1989). Addition of Ins(1,4,5)P3-containing liposomes does not alter the contraction induced by Ins(1,3,4,5)P4-filled liposomes. This suggests that Ins(1,3,4,5)P4-dependent sequestration of Ca 2÷ into an intracellular pool sensitive to Ins(1,4,5)P 3, as shown by Hill et al. (1988) in rat liver cells, does not seem to occur in this model. We could consider the possibility that Ins(1,3,4,5)P4 also acts through its conversion into Ins(1,4,5)P 3 (Cullen et al., 1989). However, this would not account for the observed effects, since at similar concentrations the level of the contractile force developed in the presence of Ins(1,3,4,5)Pa-filled liposomes is higher than that induced by Ins(1,4,5)P3-1oaded vesicles. On the other hand, Ins(1,3,4,5)P4 could compete with type I Ins(1,4,5)P 3 5-phosphatase (Erneux et al., 1989), but it is rather difficult to relate this to our results. Ely et al. (1990) suggested that Ins(1,3,4,5)P4 stimulates Ca 2+ release from bovine adrenal microsomes by a mechanism independent of the Ins(1,4,5)P 3 receptor. The blocking effect of heparin-filled liposomes upon the contraction elicited by both Ins(1,4,5)P 3- and Ins(1,3,4,5)P4-1oaded liposomes could support the involvement of some similar events in the two contractile effects we discuss here. Ins(1,3,4,5)P4 could also increase the cytoplasmic Ca 2+ levels, as shown in other models, via mobilization of Ca 2÷ from intracellular Ins(1,4,5)P 3 independent stores (Ely et al., 1990) or by activation of Ca 2÷ entry from the extracellular space (Irvine and Moor, 1986). Our data show that, in aortic smooth muscle, Ins(1,4,5)P 3 and Ins(1,3,4,5)P4 administered via liposomes induce contractions which can both be prevented by heparin (e.g. heparin also interferes with Ins(1,3,4,5)P4-activated mechanisms). Finally, the results possibly suggest the existence of processes limiting the ability of Ins(1,4,5)P 3, but not that of Ins(1,3,4,5)P4, to release Ca 2+ from intracellular stores.

Acknowledgements We would like to thank Dr. James Watras from the Department of Cardiology, University of Connecticut, Health Center, Farminton, CT, USA, for helpful discussions. We must also thank Mr. Adrian Zosin for his excellent technical assistance.

References Bangham, A.D., M.M. Standish and J.C. Watkins, 1965, Diffusion of univalent ions across the lamellae of swollen phospholipids, J. Mol. Biol. 13, 238. Batty, I.R., S.R. Nahorski and R.F. Irvine, 1985, Rapid formation of inositol 1,3,4,5-tetrakisphosphate following muscarinic receptor stimulation of rat cerebral cortical slices, Biochem. J. 232, 211. Cullen, P.J., R.F. Irvine, B.K. Drobak and A.P. Dawson, 1989, Inositol 1,3,4,5-tetrakisphosphate causes release of Ca e÷ from permeabilized lymphoma L 1210 cells by its conversion into inositol 1,4,5-trisphosphate, Biochem. J. 259, 931. Ely, A.J., L. Hunyady, A.J. Baukal and K.J. Catt, 1990, lnositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor, Biochem. J. 268, 333. Erneux, C., M. Lemos, B. Verjans, P. Vanderhaeghen, A. Delvaux and E. Dumont, 1989, Soluble and particulate Ins(1,4,5)P3/ Ins(l,3,4,5)P4 5-phosphatase in bovine brain, Eur. J. Biochem. 181,317. Hill, D.T., N.M. Dean and A.L. Boynton, 1988, Inositol 1,3,4,5-tetrakisphosphate induces Ca 2+ sequestration in rat liver cells, Science 242, 1176. Hodis, H.N., J.K. Amartey, D.W. Crawford, E. Wickham and D.H. Blankenhorn, 1990, In vivo hypertensive arterial wall uptake of radiolabeled liposomes, Hypertension 15, 600. Irvine, R.F. and R.M. Moor, 1986, Micro-injection of inositol 1,3,4,5 tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca ÷+, Biochem. J. 240, 917. Kobayashi, S., A.V. Somlyo and A.P. Somlyo, 1988, Heparin inhibits the inositol 1,4,5-trisphosphate-dependent, but not the independent, calcium release induced by guanine nucleotide in vascular smooth muscle, Biochem. Biophys. Res. Commun. 153, 625. Lambert, D.J., R.A.J Challiss and S.R. Nahorski, 1991, Accumulation and metabolism of Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 in muscarinic-receptor-stimulated SH-SY5Y neuroblastoma cells, Biochem. J. 273, 791. Somlyo, A.V., M. Bond, A.P. Somlyo and A. Scarpa, 1985, Inositol triphosphate-induced calcium release and contraction in vascular smooth muscle, Proc. Natl. Acad. Sci. USA 82, 5231. Watras, J., D. Benevolensky and C. Childs, 1989, Calcium release from aortic sarcoplasmic reticulum, J. Mol. Cell. Cardiol. 21 (Suppl. 1), 125.