Abstract
The effect of the calcium channel blocker verapamil on structure, formation and secretion of very-low-density lipoproteins (VLDL) from rat hepatocytes in suspension was examined. After 30 min incubation at a verapamil dose of 200 ΜM neither free fatty acid (FFA) uptake nor triglyceride (TG) and phospholipid (PL) secretion into the incubation medium were significantly changed. After 90 min incubation the TG secretion was inhibited by about 60%, whereas the PL output was only insignificantly lowered, indicating the secretion of abnormally composed lipoprotein particles. Morphologically, after 30 min incubation the hepatocytes had lost their microvillous border and exhibited a 2–3-fold increase in volume density and average size of the lysosomes. In the Golgi-containing regions an accumulation of smooth-surfaced microvesicles was regularly evident. The configuration of the Golgi complexes was normal. After 90 min incubation the lysosomes showed a further significant elevation in volume and size. The Golgi complexes exhibited only minor changes, but their content in VLDL particles was reduced per Μm2 Golgi complex by about 75%. Commonly, the VLDL were larger and more heterogenous in size. The diameter of those VLDL secreted into the incubation medium ranged from 31 to 84 nm, thus surpassing the control values by 2–3 times. The secretion of large-sized VLDL was regularly associated with the intracytoplasmic appearance of dilated smooth-surfaced vesicles filled with size-modified VLDL. These vesicles were concentrated within Golgicontaining areas from where they were widely dispersed towards the cell periphery. In summary, our results are strongly indicative that size modification and secretion inhibition of the VLDL caused by verapamil primarily takes place in the endoplasmic reticulum.
Similar content being viewed by others
References
Claude A (1970) Growth and differentiation of cytoplasmic membranes in the course of lipoprotein granule synthesis in the hepatic cell. I. Elaboration of elements of the Golgi complex. J Cell Biol 47: 745–766
Dargel R, Zimmermann T, Müller D, Franke H, Kretzschmar M, (1989) Lipid peroxidation in experimental liver cirrhosis. Adv Biosci 76: 269–274
Erdreich A, Rahamimoff (1986) Studies on the mechanism of the inhibitory action of verapamil on calcium transport. In: Rahamimoff R, Katz B (eds) Calcium, neuronal function and transmitter release. Martinus Nijhoff Publishing, Boston, pp 547–555
Erdreich A, Rahamimoff H (1987) The possible involvement of the phospholipid phase of membranes in mediating the effects of verapamil on calcium transport. Biochem Pharmacol 36: 1775–1780
Folch J, Lees M, Sloane-Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226: 497–509
Franke H (1990) Review: substructural alterations in liver parenchymal cells induced by xenobiotics. Exp Pathol 39: 139–155
Franke H, Poli G, Zimmermann T, Dianzani MU, Dargel R (1988) Short-term effects of carbon tetrachloride on the lipoprotein secretion in isolated rat hepatocytes. Virchows Arch B (Cell Pathol) 54: 357–363
Goldstein JL, Brunschede GY, Brown MS (1975) Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339. J Biol Chem 250: 7854–7862
Hay R, Flemming R, O'Connel W, Kirschner J, Oppliger W (1988) Apolipoproteins of the orotic acid fatty liver: Implications for the biogenesis of plasma lipoproteins. J Lipid Res 29: 901–906
Hornick CA, Jones AL, Renaud G, Hradek G, Havel RJ (1984) Effect of chloroquine on low-density lipoprotein catabolic pathway in rat hepatocytes. Am Physiol Soc 193: 187–194
Hummel L, Zimmermann T, Wagner H (1978) Quantitative evaluation of the fetal acid synthesis in the rat. Acta Med Germ 37: 229–232
Kleinig H, Sitte P (1986) Endocytose, coated vesicles, lysosomes. In: Kleinig H, Sitte P (eds) Zellbiologie. Gustav Fischer Verlag, Jena. pp 95–106
Matsuzawa Y, Hostetler KY (1980) Inhibition of lysosomal phospholipase A and phospholipase C by chloroquine and 4,4-bis (diethylaminoethoxy) a, Β-diethyldiphenylethane. J Biol Chem 255: 5190–5194
Merry S, Flangigan P, Schlick E, Freshney RI, Kaye SB (1989) Inherent adriamycin resistance in a murine tumour line: circumvention with verapamil and norverapamil. Br J Cancer 59: 895–897
Navasa M, Bosch J, Reichen J, Bru C, Mastai R, Zysset T, Silva G, Chesta J, Rodes J (1988) Effects of verapamil on hepatic and systemic hemodynamics and liver function in patients with cirrhosis and portal hypertension. J Hepatol 8: 850–854
Nossen JO, Rustan AC, Drevon CA (1987) Calcium-antagonists inhibit secretion of very-low-density lipoprotein from cultured rat hepatocytes. Biochem J 247: 433–439
Olubadewo JO, Cook GA, Heimberg M (1988) Effects of 8-N, N-dethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8)HCl and verapamil on the metabolism of free fatty acids by hepatocytes. Biochem Pharmacol 37: 1463–1471
Reijngoud DJ, Oud PS, Kas J, Tager JM (1976) Relationship between medium pH and that of the lysosomal matrix as studied by two independent methods. Biochim Biophys Acta 448: 290–302
Rustan AC, Nossen JO, Tefre T, Drevon CA (1987) Inhibition of very-low-density lipoprotein secretion by chloroquine, verapamil and monensin takes place in the Golgi complex. Biochim Biophys Acta 930: 311–319
Seglen PO, Reith A (1976) Ammonia inhibition of protein degradation in isolated hepatocytes. Exp Cell Res 100: 276–280
Seglen PO (1976) Preparation of isolated rat liver cells. In: Prescott DM (ed). Methods in cell biology. Vol 8. Academic Press, New York, pp 29–83
Sewell RB, Barham SS, Larusso NF (1983) Effect of chloroquine on the form and function of hepatocyte lysosomes. Morphologic modifications and physiologic alterations related to the biliary excretion of lipids and proteins. Gastroenterology 85: 1146–1153
Stein Y, Ebin B, Bar-On H, Stein O (1977) Chloroquine-induced interferences wiht degradation of serum lipoproteins in rat liver studies in vivo and vitro. Biochim Biophys Acta 484: 286–297
Sturgess JM, De la Iglesia FA (1972) Morphometry of the Golgi apparatus in developing liver. J Cell Biol 55: 524–530
Weibel ER (1979) Point counting methods. In: Weibel ER (ed) Stereological methods. Vol I. Practical methods for biological morphometry. Academic Press, New York, pp 101–161
Winkler L, Schlag B, Leutert F, Dargel R (1981) Quantitative Lipoproteinanalytik mit der Ultrazentrifuge VAC 601 unter Verwendung eines Winkelrotors und eines Fraktionsschneidegerätes. Z Med Lab Diagn 22: 216–225
Zimmermann T, Franke H, Dargel R (1987) Isolation and characterization of parenchymal cells from normal and cirrhotic rat liver. Cell Biochem Funct 5: 47–54
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Franke, H., Müller, D., Schlag, B. et al. Biochemical and ultrastructural studies on the effect of verapamil on formation and secretion of lipoproteins in rat hepatocyte suspensions. Arch Toxicol 64, 656–662 (1990). https://doi.org/10.1007/BF01974694
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01974694