We have studied the contractility of liver sinusoidal stellate (Ito) cells stimulated with endothelin 1, nitric-oxide donors and eicosanoids. Contraction and relaxation of stellate cells were detected by the use of a silicone-rubber method that revealed the traction forces exerted by these cells. Endothelin 1 was a strong elicitor for stellate-cell contraction. 78, 55, 59 and 56% of stellate cells were contracted 2.5, 5, 10 and 20 min, respectively, after exposure to 10 nM endothelin 1. The effect of endothelin 1 was dose dependent and still detectable at an endothelin 1 concentration of 100 pM. Concomitantly, an endothelin-dependent formation of inositol phosphates was apparent; values of InsP, InsP,, and InsP, were 881 rt 99%, 1965 k 368%, and 791 k 120% of control, respectively, 20 min after addition of 10 nM endothelin 1. In addition, endothelin 1 caused a transient increase of [Ca2+], in stellate cells from a basal value of 121 2 9 nM to maximal 1015 5 86 nM. These endothelin-1 effects were much stronger than those of the thromboxane-A, analogue U46619 and of prostaglandin F2=. In contrast, Iloprost, prostaglandin E,, and sodium nitroprusside promoted stellate-cell relaxation; for example, 82, 83 and 71 % of stellate cells relaxed 5, 10, and 20 min, respectively, after addition of 500 pM sodium nitroprusside to contracted cells. Prostaglandin E, and Iloprost led to elevation of CAMP levels in stellate cells from a basal value of 9.2k0.8 pmoU well to 55.1 2 8.0 and 122.2 2 12.2 pmollwell 10 min after addition of prostaglandin E, (5 pM) and Iloprost (5 pM), respectively, in the presence of 3-isobutyl-1-methylxanthine (0.5 mM). However, sodium nitroprusside was a trigger for cGMP accumulation. Intracellular cGMP increased from a basal value of 0.9 k 0.07 pmol/well to 13.4 k 6.7 pmol/well 10 min after addition of 500 pM sodium nitroprusside into the medium. It is interesting that Iloprost and sodium nitroprusside also induced the disappearance of actin stress fibers in contracted cells; F-actin stress fibers became less numerous and de-aggregated; more than 90% of stellate cells were void of stress fibers after 10 pM Iloprost treatment for 30 min. Thus, endothelin 1, eicosanoids and sodium nitroprusside are able to modulate the contractility of stellate cells. The effect of endothelin 1 and prostaglandin F,, seems to be mediated by inositol-phosphate formation and increase of the intracellular Ca" level, that of Iloprost and prostaglandin E, by CAMP. cGMP may be a second messenger for the action of sodium nitroprusside.In a previous study it has been shown that stellate cells (fat-storing cells, Ito cells, or hepatic perisinusoidal lipocytes) can undergo reversible contraction. Increase or decrease of the traction forces exerted by stimulated stellate cells were revealed by using flexible silicone rubber memCorrespondence to K.

To approach experimentally the problem of contractility, stellate cells from rats were isolated and grown on a flexible silicone rubber substrate. Increases or decreases in the number of wrinkles of the silicone membrane beneath the cells that were easily observable by microscopy was employed as semi-quantitative measure of stellate cell motility. Contraction of stellate cells accompanied by diminution of cell body size was induced by U46619 (a thromboxane A2 analogue) and prostaglandin (PG) F2 alpha. Wrinkle formation became detectable 1.5 min after addition of 2 microM-U46619 and reached its maximum 10-15 min later. The effect of PGF2 alpha was not so striking, but lasted for a longer period of time. On the other hand, dibutyryl cyclic AMP, Iloprost (a PGI2 analogue) and PGE2 led to the disappearance or decrease in the number of wrinkles, indicating relaxation of contracted stellate cells. For instance, after addition of 2 microM-Iloprost, 47, 75 and 82% of contracted stellate cells had relaxed within 5, 10 and 20 min respectively. Moreover, dibutyryl cyclic AMP induced disappearance of alpha-smooth muscle actin stress fibres. This response became recognizable 10 min after addition of dibutyryl cyclic AMP; 40 min later, 97% of stellate cells were devoid of stress fibres. Thus stellate cells are able to undergo reversible contraction in primary culture, and the contraction of these cells may be mediated by eicosanoids that can be produced within the liver.

Autonomous in vitro growth of myeloid leukemic colony-

Nitric oxide (NO), apart from its properties as a vasodilator, is a cytotoxic agent released from macrophages upon stimulation with immunomodulating agents such as interferon-and endotoxin.In rat Kupffer cells endotoxin causes the release of NO as well as of tumor necrosis factor-a and prostaglandin E 2 (PGE 2 ). This eicosanoid and its second messenger, cyclic AMP, have been shown to increase nitric oxide formation in Kupffer cells treated with endotoxin (Gaillard et al. (1991) Pathobiology 59, 280-283). But not only added PGE 2 but also the prostaglandin produced endogenously upon stimulation with endotoxin increases NO synthesis.Neither tumor necrosis factor-nor interleukin-1/3 stimulate NO synthesis by themselves, but together with PGE 2 they are as effective as lipopolysaccharide plus PGE 2 . To replace PGE 2 in the combination with the cytokines, however, dibutyryl cAMP has to be present in higher concentrations than with LPS. Interleukin-6 alone or in combination with PGE 2 or dibutyryl cAMP is without any effect.Anti-TNF-o! as well as anti-PGE 2 antibodies reduce the release of NO upon stimulation with LPS. Consequently, the effect of LPS on NO production seems to be in part due to the self-stimulating effect of PGE 2 and some cytokines, both produced by Kupffer cells upon LPS stimulation. Regulation der durch Cytokine ausgelösten Synthese von Stickoxid durch Prostaglandin E 2 in Makrophagen der RattenleberZusammenfassung: Neben seiner Eigenschaft als Vasodilatator ist Stickoxid ein cytotoxisches Agens, das von Makrophagen nach Anregung durch Immunmodulatoren, wie Interferon-und Endotoxin gebildet wird.Endotoxin führt in Kupffer-Zellen der Ratte zur Freisetzung sowohl von NO als auch von Tumornekrosefaktor-(TNF-a) und Prostaglandin (PG)E 2 . Es war bereits gezeigt worden, daß dieses Eikosanoid und sein "second messenger", cAMP, die Stickoxidbil-

Macrophages have been described to release nitric oxide (NO) as a cytotoxic radical. This highly unstable substance is as well known as endothelium-derived relaxing factor produced by vascular endothelial cells. Because of its cytotoxic activity the synthesis of NO by rat Kupffer cells, the liver macrophages, upon stimulation with endotoxin (lipopolysaccharide; LPS) and tumor necrosis factor-α (TNF-α) in combination with prostaglandin E₂ (PGE₂) and dibutyryl cAMP (dBcAMP) was studied. Kupffer cells were stimulated after 48 h of primary culture. NO was quantified as NO₂ in the cell medium 24 h after stimulation. LPS stimulated NO generation 5- to 10-fold over the basal level. This increase could be further enhanced by PGE₂ and dBcAMP, especially when added 1 h after LPS. NO generation after stimulation with LPS or LPS + PGE₂ depended on the simultaneous production of PGE₂ by the stimulated Kupffer cells. It could be partly inhibited by anti-PGE₂ antibody or acetylsalicylic acid. While murine TNF-α did not stimulate NO synthesis significantly, added PGE₂ raised NO synthesis about 6-fold. The addition of dBcAMP to TNF-α in the same concentration as with LPS, however, had no effect. Thus, stimulation by LPS + PGE₂ equals that of LPS + dBcAMP whereas TNF-α + PGE₂ does not equal TNF-α + dBcAMP, indicating differences in the mode of action of PGE₂ on LPS- or TNF-α-treated Kupffer cells.