- Email: [email protected]
Sphingomyelinase Activity of LDL
Nairn AC, Picciotto MR: 1994. Calcium/ calmodulin-dependent protein kinases. Semin Cancer Biol 5:295–303. Peracchia C, Sotkis A, Wang XG, et al.: 2000. Calmodulin directly gates gap junction channels. J Biol Chem 275:26,220–26,224. Persechini A, Cronk B: 1999. The relationship between the free concentrations of Ca21 and Ca21-calmodulin in intact cells. J Biol Chem 274:6827–6830. Persechini A, Lynch JA, Romoser VA: 1997. Novel fluorescent indicator proteins for monitoring free intracellular Ca21. Cell Calcium 22:209–216. Persechini A, Stemmer PM, Ohashi I: 1996. Localization of unique functional determinants in the calmodulin lobes to individual EF hands. J Biol Chem 271:32,217–32,225. Rao A, Luo C, Hogan PG: 1997. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15:707–747. Romoser VA, Hinkle PM, Persechini A: 1997. Detection in living cells of Ca21-dependent changes in the fluorescence of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 272:13,270–13,274. Sase K, Michel T: 1997. Expression and regulation of endothelial nitric oxide synthase. Trends Cardiovasc Med 7:28–37. Schumacher MA, Rivard AF, Bachinger HP, Adelman JP: 2001. Structure of the gating domain of a Ca21-activated K1 channel complexed with Ca21/calmodulin. Nature 410:1120–1124. Shoshan-Barmatz V, Ashley RH: 1998. The structure, function, and cellular regulation of ryanodine-sensitive Ca21 release channels. Int Rev Cytol 183:185–270. Takuwa N, Zhou W, Takuwa Y: 1995. Calcium, calmodulin and cell cycle progression. Cell Signal 7:93–104. Tansey MG, Luby-Phelps K, Kamm KE, Stull JT: 1994. Ca21-dependent phosphorylation of myosin light chain kinase decreases the Ca21 sensitivity of light chain phosphorylation within smooth muscle cells. J Biol Chem 269:9912–9920. Teruel MN, Chen W, Persechini A, Meyer T: 2000. Differential codes for free Ca 21calmodulin signals in nucleus and cytosol. Curr Biol 10:86–94. Wang DX, Tolbert LM, Carlson KW, Sadee W: 2000. Nuclear Ca21/calmodulin translocation activated by m-opioid (OP3) receptor. J Neurochem 74:1418–1425. Zuhlke RD, Pitt GS, Deisseroth K, et al.: 1999. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399:159–162. PII S1050-1738(01)00144-X
TCM Vol. 12, No. 1, 2002
TCM
Sphingomyelinase Activity of LDL: A Link between Atherosclerosis, Ceramide, and Apoptosis? Paavo K.J. Kinnunen* and Juha M. Holopainen
Atherosclerosis is characterized by the accumulation in the arterial intima of mainly low-density lipoprotein (LDL)-derived lipids, together with apolipoprotein B-100 (apoB-100), the protein moiety of LDL. Recent studies indicate aggregation of LDL within the arterial wall to represent a critical step in the initiation of this disease. Aggregation of LDL has been further proposed to involve ceramide, the levels of which are elevated in atherosclerotic plaques as well as in LDL isolated from these lesions. Biophysical studies have shown ceramide to have a pronounced tendency for self-aggregation, presumably driven by intermolecular hydrogen bonding. Importantly, the segregated ceramide-enriched lipid phases have high melting temperatures and are in a gel state at 378C. The plasma levels of sphingomyelin, which upon enzymatic hydrolysis by sphingomyelinase (SMase) yields ceramide, have been shown to correlate with the severity of coronary heart disease. The formation of ceramide from sphingomyelin could thus represent a critical step in atherosclerosis. We recently showed that LDL itself possesses SMase activity. Moreover, sequence analogy with bacterial enzymes suggests that this activity may be intrinsic to apoB-100. Possible physiological role of this activity is uncertain, yet could be involved in nonreceptormediated endocytotic entry of LDL into cells. Importantly, it also opens a possible mechanistic link between elevated plasma levels of LDL, apoptosis (programmed cell death), and atherosclerosis. (Trends Cardiovasc Med 2002;12:37–42). © 2002, Elsevier Science Inc.
Low-density lipoprotein (LDL) is the major carrier of cholesterol in the circulation, and provides cholesterol to the Paavo K.J. Kinnunen and Juha M. Holopainen are from the Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, University of Helsinki, Finland. * Address correspondence to: P.K.J. Kinnunen, Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, FIN-00014 University of Helsinki, P.O. Box 63 (Biomedicum, Haartmaninkatu 8), Helsinki, Finland. Tel.: 1358 9 191 25400; fax: 1358 9 191 25444; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
peripheral tissues by LDL receptormediated endocytosis (Brown and Goldstein 1986). The surface of the roughly spherical LDL particle is comprised of a single copy of the apolipoprotein B-100 (apoB-100), together with approximately 500 molecules of phosphatidylcholine, 200 molecules of sphingomyelin, and 400 molecules of unesterified cholesterol, constituing a surface film surrounding a core of cholesteryl esters and triacylglycerols. Yet, LDL is heterogeneous in size, composition, and conformation of apoB100 (Austin et al. 1988). ApoB-100 is one of the largest proteins known and appears to be multifunctional, from pro-
37
viding the recognition site on the LDL particle for its receptor, to diverse catalytic activities. Parthasarathy et al. demonstrated that apoB-100 possesses phospholipase A2 activity, which is inactivated upon oxidation (Parthasarathy and Barnett 1990). Later studies revealed both phospholipase A1 and A2 activities to be present (Reisfeld et al. 1993). In addition, platelet-activating factor acetylhydrolase activity was demonstrated to be associated with LDL (Stafforini et al. 1987). Interestingly, apoB-100 shows sequence homology to Src-1 domains, and has been shown to undergo autophosphorylation of tyrosine residues (Guevara et al. 1995). We have recently shown SMase activity to be associated with LDL, with sequence comparison to bacterial SMases suggesting that this activity could be an intrinsic property of apoB100 (Holopainen et al. 2000c). Aggregation of LDL has been proposed to represent an essential and central process in atherogenesis (Ross 1993), and LDL extracted from atherosclerotic lesions is aggregated or is prone to aggregate. Aggregation of LDL can be achieved by binding of these lipoproteins to proteoglycans, certain acidic phospholipids, vortexing (see Lauraeus et al. 1998, for a brief review), or by modifying the LDL surface by phospholipases A2, C, or SMase (Xu and Tabas 1991). In contrast to unaggregated LDL, aggregated LDL in atherosclerotic lesions is enriched in ceramide (Schissel et al. 1996). The involvement of ceramide is of particular interest, as this lipid has been established as a central messenger in apoptosis (for example, Mathias et al. 1998), albeit the mechanism(s) of action still remain(s) elusive. Taken together, altered sphingolipid metabolism could represent a risk factor for the development of atherosclerosis and the involvement of SMase and ceramide may play a central role in the development of this disease, providing also a link to apoptosis.
plaques and calcified plaques in patients who died of atherosclerosis. Interestingly, there is a positive correlation between the plasma concentration of sphingomyelin, a precursor for ceramide, and the severity of coronary heart disease (Jiang et al. 2000). A variety of cell types present in atherosclerotic lesions secrete SMase (Marathe et al. 1998, Schissel et al. 1996). Assuming LDL aggregation to be a prerequisite for the progression of atherosclerosis and as SMase can induce this process, it seems feasible to suggest a role for both SMase and ceramide in the pathophysiology of atherosclerosis. LDL treated with SMase can induce macrophage foam cell formation in vitro (Schissel et al. 1996, Xu and Tabas 1991). The ceramide content of aggregated LDL in lesions was 10–50-fold higher than that of plasma LDL (Schissel et al. 1996). However, nonaggregated lesional LDL was not enriched in ceramide, suggesting that aggregation and the formation of this lipid could be interrelated. Yet, the possibility cannot be excluded that this ceramide could originate from cells, being transferred to LDL by plasma lipid transfer proteins. • SMase Activity Associated with LDL In the course of our studies on the properties and metabolism of LDL we came to the conclusion that it would be worthwhile to explore if SMase was associated with LDL. This turned out to be the case, and the formation of ceramide was evident upon incubation of sphingomyelincontaining liposomes with human plasma LDL (Holopainen et al. 2000c). Instead, VLDL, HDL2, and HDL3 were devoid of SMase activity. The absence of SMase from VLDL would be in keeping with lipolysis-induced changes in the conformation of apoB-100 to be required, as previously demonstrated for the lack of recognition of apoB-100 in VLDL by the LDL receptor (Catapano et al. 1979). Ox-
idized LDL also lacked SMase activity. Oxidation of LDL induces the formation of a number of proteolytic fragments from the single polypeptide chain constituting apoB-100 (Fong et al. 1987), which could be sufficient to disrupt the conformation of this protein necessary for the proper orientation of the residues responsible for catalytic activity. The activity of SMase in LDL from different donors exhibited considerable variation but did not correlate with age, sex, or body mass index. Possible variation in the oxidation state could explain these differences in enzyme activity. Modifications such as methylation and acetylation of lipoproteins could also play a role in atherosclerosis (Steinberg 1993, Wiklund et al. 1991). The influence of these modifications on SMase activity associated with LDL remains to be investigated. Finally, genetic polymorphism of apoB-100 could also be involved. Although a major fraction of LDL is produced from VLDL, studies have shown that liver can produce LDL also directly (for example, Teng et al. 1986). The latter lipoproteins are somewhat smaller and denser than those derived from VLDL. In addition, lipoprotein(a) could also be present in the LDL fraction. The possibility of the above two lipoprotein species possessing SMase activity remains to be studied. The 3D structures for LDL and SMases are not available at present. Moreover, prediction of the 3D structure of apoB100 remains ambiguous. To identify putative region of LDL responsible for the contained SMase activity we compared the amino acid sequence of human apoB100 with those of Bacillus cereus, Leptospira interrogans, and Staphylococcus aureus SMase (Figure 1). Three homologous regions were found in apoB-100 and B. cereus SMase consisting of 24, 39, and 43 amino acids and having 38, 36, and 28% identical amino acids, respectively (Holopainen et al. 2000c). Comparison of apoB-100 with three dif-
• Sphingolipids in the Development of Atherosclerosis Oxidized LDL, but not native LDL, induces apoptosis of arterial smooth muscle cells via the activation of a neutral SMase (Chatterjee 1999). Furthermore, increased activity of this SMase correlates with elevated levels of ceramide and apoptosis in postmortem samples of
38
Figure 1. Illustration of the five distinct domains in the sequence of apoB-100, including the LDL receptor-binding domain as well as the the region containing the putative catalytic site of SMase.
TCM Vol. 12, No. 1, 2002
ferent isoforms of B. cereus SMase revealed that out of the total 305 amino acids of this enzyme 65 (21%) align with the sequence of apoB-100. Alignment of human apoB-100 with B. cereus, L. interrogans, and S. aureus sequences gives 30 amino acids that are common to all. Previous sequence studies have shown deoxyribonuclease I (DNase I) to share common mechanistic characteristics with SMase and identified tentatively in the sequence of SMase eight amino acids, which could be important in substrate recognition (Matsuo et al. 1996, Weston et al. 1992). Interestingly, out of these functionally important amino acids six are conserved, and one is weakly similar also in the alignment of B. cereus SMase and apoB-100. Finally, while sphingomyelin is present in LDL ceramide is almost absent, and has been shown to be present in large amounts only in aggregated LDL (Schissel et al. 1996). The putative active site in apoB-100 assumed to be responsible for the SMase activity of LDL should, therefore, be oriented outwards from the lipoprotein surface, prohibiting access to the sphingomyelin contained in this lipoprotein. Despite the above proteomic approach supporting the notion that the SMase activity of LDL would be intrinsic to apoB-100, other possibilities must also be considered. Accordingly, this enzyme could be secreted by cells in the arterial walls and become subsequently associated with LDL. However, as this activity was not detected in the other lipoproteins the interaction between the secreted SMase and lipoprotein surface should be specific for LDL. Lack of ceramide in plasma LDL further requires that the LDL-bound SMase should bind in a manner not enabling access of this enzyme to its substrate sphingomyelin in the lipoprotein surface film. These issues need to be addressed experimentally. • Possible Physiological Role for the SMase Activity of LDL Using microinjection techniques and giant unilamellar vesicles (GUVs) as biomembrane models, we have recently shown that the formation of ceramide results in a vectorial formation of vesicles (Holopainen et al. 2000a). More specifically, exposure of the outer surface of GUVs to SMase results in a rapid lateral clustering of the reaction prodTCM Vol. 12, No. 1, 2002
uct, ceramide, within the bilayer. The driving force is likely to be efficient intermolecular hydrogen bonding between the ceramide headgroups. Subsequently, the ceramide-enriched membrane domains detach as “endocytotic” vesicles inside the giant liposome. Yet, if SMase is injected into the liposome, thus catalyzing ceramide formation in the inner leaflet of the bilayer, the opposite takes place, resulting in budding of small vesicles outside the giant liposome. Interestingly, in ATP-depleted macrophages and fibroblasts treatment with exogenous SMase results within 10 min in the budding of numerous vesicles from the plasma membrane into the cytoplasm of these cells (Zha et al. 1998). These vesicles lack any observable protein coating and are relatively large, with a diameter of approx. 0.4 mm. Our data thus show that except no proteins other than SMase are necessary for this endocytosis. In line with the above, exogenously incorporated N-hexanoyl-sphingosine (C6-ceramide) induces time- and dose-dependent formation of vesicles (diameter 2–10 mm) in the interior of fibroblasts (Li et al. 1999). Finally, it is important to note that in contrast to the segregation of ceramide, sphingomyelin is miscible in fluid phosphatidylcholine bilayers. This difference is due to the large, hydrated phosphocholine headgroup of sphingomyelin. In subsequent experiments we demonstrated that “endocytotic” vesicles were also induced in giant liposomes by LDL (Holopainen et al. 2000c). This suggests that LDL may enter cells in an ATP- and receptor-independent pathway, owing to “autocytosis” driven by its SMase activity. Evidence for nonreceptor-mediated uptake by rat luteal cells of LDL cholesterol without concomitant apoB-100 ingestion has been presented (Reaven et al. 1986). Recently, aggregated LDL was shown to enter the cells by surfaceconnected compartments by a mechanism not involving the LDL receptor (Zhang et al. 1997). It is tempting to speculate that the above processes would be mediated by the SMase activity present in apoB-100. LDL binds to the proteoglycans on the endothelial wall (Hurt-Camejo et al. 1997). Accordingly, after binding of LDL to the cell surface the contained SMase would have access to its substrate, sphingomyelin. The ceramide formed would first segregate locally into microdomains. Subsequently, because of the
macroscopic physical properties of these domains (for instance, negative spontaneous curvature and high bending rigidity, see Holopainen et al. 2000a), they vesiculate from the bilayer, thus acting as local vehicles for “autocytotic” entry of LDL through the plasma membrane, as illustrated schematically in Figure 2. Based on these findings, we have suggested that in addition to its above putative role in LDL processing SMase could be used as a mechanism of entry of particles, such as microbes into mammalian cells, inducing the pinching of “autocytotic” vesicles off the plasma membrane, with the contained pathogen. This mechanism would be generic. Evidence for the involvement of SMase and phosphatidylcholine-specific phospholipase C (PC-PLC) in the entry of Neisseria gonorrhea, S. aureus, and species of mycobacteria into human cells was recently presented (see Holopainen et al. 2000a, for a brief review). The role of PC-PLC could be associated with breakdown of the surrounding vesicle inside the cell, enabling for an entry of the microbe into the cytoplasm. The above mechanism need not be limited to bacteria, but is equally likely to be responsible for the entry of viruses. In this connection it is of interest that Sindbis virus has been observed to trigger apoptosis in cells, with the formation of ceramide (Jan et al. 2000). More detailed understanding of the role(s) of sphingomyelin and other phospholipid-degrading enzymes in the above processess may thus open novel venues for therapeutic intervention. • Concluding Remarks Atherosclerosis is a multifactorial disorder. This is easily understood in the light of the plethora of processes that have been revealed to be involved in the development of this disease, such as oxidation of LDL, macrophage chemotaxis and foam cell formation, smooth muscle cell migration and alteration, lipoprotein retention and aggregation, and changes in the endothelium. Moreover, high HDL levels are “protective” of atherosclerosis (Nikkilä 1976), although the actual molecular level mechanisms still remain perplexing (Kinnunen 1979). Despite the limitations due to the number of factors being involved, the findings summarized in this brief review enable us to outline the following mech-
39
A key role in the above scheme is attributed to the particular features of ceramide. While the chemical structure of ceramide is simple, it imparts quite remarkable biophysical properties to this lipid. Its small polar headgroup contains both hydrogen bond donors and acceptors, and ceramide thus has a pronounced tendency for lateral segregation in membranes, driven by the formation of intermolecular hydrogen bonded networks (Holopainen et al. 1997, 2000b, Moore et al. 1997). Because of the saturated hydrocarbon chains in a large fraction of ceramide, the segregated ceramideenriched phases have their melting temperatures well above the body temperature and are thus in gel state at 378C (Holopainen et al. 1997, 2000a, 2000c). In the aggregated membranes further rearrangements, such as interdigitation of the chains, may take place, making these structures tightly packed and impermeable (Swartzendruber et al. 1989). In this connection it is relevant to note that ceramide, cholesterol, and fatty acids are essential constituents of the skin, and are currently believed to be responsible for its barrier properties (Elias and Menon 1991). Accordingly, those properties of the above lipids that serve useful purposes in skin may well precipitate pathological consequences when manifested in sites such as vascular endothelium. Figure 2. Formation of ceramide from sphingomyelin in a bilayer, catalyzed by the SMase activity of LDL and resulting in the formation of an “autocytotic” vesicle with the contained lipoprotein. For illustrative purposes the diameter of the lipoprotein particle and the thickness of the bilayer are not drawn to scale. The round symbol notifies the phosphocholine headgroup of either sphingomyelin (black acyl chains) or phosphatidylcholine (magenta acyl chains). The lipid with a black square as a headgroup represents ceramide.
anism for the formation of atherosclerotic plaques. High plasma levels of LDL would first shift the equilibrium in arterial walls towards initial, loose particle aggregation, perhaps promoted by binding of LDL to glycosaminoglycans (HurtCamejo et al. 1997). In these aggregates the SMase of LDL would be able to catalyze the formation of ceramide in the contacting, adjacent lipoproteins, resulting in the segregation of gel-like ceramide-enriched lipid microdomains, which further promote LDL–LDL interactions and result in tightly aggregated LDL, in keeping with the fusion of liposomes being promoted by ceramide (Abraham et al. 1988). Likewise, con-
40
tacts of these aggregated LDL with the vascular endothelial cells would enable catalytic formation of ceramide in these cells, triggering apoptosis, together with further ceramide accumulation (Royer and Foote 1971). The above scheme would be compatible with the correlation between elevated plasma levels of sphingomyelin and the severity of coronary heart disease (Jiang et al. 2000), as well as the measured high amounts of sphingomyelin and ceramide in atherosclerotic plaques (Schissel et al. 1996). Following the aggregation of LDL, other lipids in LDL, cholesterol in particular, would start to contribute to the growth of the plaque.
• Future Directions The processes and behavior of lipid mixtures discussed in this brief review are consequences of their physical properties. Lipids represent the liquid crystalline state of matter, which is characterized by a variety of phases and connecting phase transitions, described by phase diagrams. The above considerations warrant studies to establish the phase diagram of the lipids contained in atheroma. Although phase diagrams for multicomponent systems necessarily become complex, they nevertheless provide powerful “roadmaps,” which could open a rational and detailed understanding for the molecular basis of atherosclerosis, on the level of the different lipids, as well as factors such as acyl chain saturation. Availability of these data would also facilitate the development of novel therapeutic means, potentially enabling one to interfere with and prevent the putative formation of the microcrystalline “seeds” of ceraTCM Vol. 12, No. 1, 2002
mide initiating the growth of atheroma. A key role in the development of atheroma in the scheme outlined here is assigned to the SMase activity associated with LDL. It is essential to establish whether this activity is indeed intrinsic to apoB-100 or due to a tightly associated enzyme released from cells. Moreover, the physiological significance of this LDL-associated SMase remains to be studied, together with evaluating the feasibility of the use of specific inhibitors of this catalytic activity in preventing atherosclerosis.
• Acknowledgments We wish to thank Professor Felix Gõni (University of Bilbao, Basque County, Spain) and Dr. Robert Corkery (Procter and Gamble, Cincinnati, OH) for stimulating discussions. Financial support was obtained from Tekes and Finnish Academy (P.K.J.K.). J.M.H. is supported by grants from Farmos Research Foundation, Paulo Research Foundation, and Emil Aaltonen Foundation.
References Abraham W, Wertz PW, Downing DT: 1988. Effect of epidermal acylglucosylceramides and acylceramides on the morphology of liposomes prepared from stratum corneum lipids. Biochim Biophys Acta 939:403–408. Austin MA, Breslow JL, Hennekens CH, et al.: 1988. Low-density lipoprotein subclass patterns and risk of myocardial infarction. JAMA 260:1917–1921. Brown MS, Goldstein JL: 1986. A receptormediated pathway for cholesterol homeostasis. Science 232:34–47. Catapano AL, Gianturco SH, Kinnunen PKJ, et al.: 1979. Suppression of 3-hydroxy-3methylglutaryl-CoA reductase by low density lipoproteins produced in vitro by lipoprotein lipase action on nonsuppressive very low density lipoproteins. J Biol Chem 254:1007–1009. Chatterjee S: 1999. Neutral sphingomyelinase: past, present and future. Chem Phys Lipids 102:79–96. Elias PM, Menon GK: 1991. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv Lipid Res 24:1–26. Fong LG, Parthasarathy S, Witztum JL, Steinberg D: 1987. Nonenzymatic oxida-
TCM Vol. 12, No. 1, 2002
tive cleavage of peptide bonds in apoprotein B-100. J Lipid Res 28:1466–1477. Guevara J Jr. Walch ET, Epstein HF, et al.: 1995. Evidence that apoB-100 of lowdensity lipoproteins is a novel Src-related protein kinase. J Protein Chem 14:627– 631. Holopainen JM, Angelova MI, Kinnunen PKJ: 2000a. Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophys J 78:830–838. Holopainen JM, Lehtonen JYA, Kinnunen PKJ: 1997. Lipid microdomains in dimyristoylphosphatidylcholine-ceramide liposomes. Chem Phys Lipids 88:1–13. Holopainen JM, Lemmich J, Richter F, et al.: 2000b. Dimyristoylphosphatidylcholine/C16: 0-ceramide binary liposomes studied by differential scanning calorimetry and wideand small-angle x-ray scattering. Biophys J 78:2459–2469. Holopainen JM, Penate Medina O, Metso AJ, Kinnunen PKJ: 2000c. Sphingomyelinase activity associated with human plasma low density lipoprotein: possible functional implications. J Biol Chem 275: 16,484–16,489. Hurt-Camejo E, Olsson U, Wiklund O, et al.: 1997. Cellular consequences of the association of apoB lipoproteins with proteoglycans. Potential contribution to atherogenesis. Arterioscler Thromb Vasc Biol 17: 1011–1017. Jan J-T, Chatterjee S, Griffin DE: 2000. Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide. J Virol 74:6425– 6432. Jiang X-c, Paultre F, Pearson TA, et al.: 2000. Plasma sphingomyelin level as a risk factor for coronary artery disease. Arterioscler Thromb Vasc Biol 20:2614–2618. Kinnunen PKJ: 1979. High-density lipoprotein may not be antiatherogenic after all. Lancet ii:34–35. Lauraeus S, Holopainen JM, Taskinen M-R, Kinnunen PKJ: 1998. Aggregation of dimyristoylphosphatidylglycerol liposomes by human plasma low density lipoprotein. Biochim Biophys Acta 1373:147–162. Li R, Blanchette-Mackie EJ, Ladisch S: 1999. Induction of endocytic vesicles by exogenous C6-ceramide. J Biol Chem 274:21,121– 21,127. Marathe S, Schissel SL, Yellin MJ, et al.: 1998. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling. J Biol Chem 273:4081–4088.
Mathias S, Pena LA, Kolesnick RN: 1998. Signal transduction of stress via ceramide. Biochem J 335:465–480. Matsuo Y, Yamada A, Tsukamoto K, et al.: 1996. A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I. Protein Sci 5:2459– 2467. Moore DJ, Rerek ME, Mendelsohn R: 1997. FTIR spectroscopy studies of the conformational order and phase behaviour of ceramides. J Phys Chem B 101:8933– 8940. Nikkilä EA: 1976. Serum high-density-lipoprotein and coronary heart-disease. Lancet 2:320. Parthasarathy S, Barnett J: 1990. Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100. Proc Natl Acad Sci USA 87:9741–9745. Reaven E, Chen YD, Spicher M, et al.: 1986. Uptake of low density lipoproteins by rat tissues. Special emphasis on the luteinized ovary. J Clin Invest 77:1971–1984. Reisfeld N, Lichtenberg D, Dagan A, Yedgar S: 1993. Apolipoprotein B exhibits phospholipase A1 and phospholipase A2 activities. FEBS Lett 315:267–270. Ross R: 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809. Royer M, Foote JL: 1971. The identification of ceramides and glyceryl ethers in unsaponifiable lipid of human aorta. Chem Phys Lipids 7:266–678. Schissel SL, Tweedie-Hardman J, Rapp JH, et al.: 1996. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest 98:1455–1464. Stafforini DM, McIntyre TM, Carter ME, Prescott SM: 1987. Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor. J Biol Chem 262:4215–4222. Steinberg D: 1993. Modified forms of lowdensity lipoprotein and atherosclerosis. J Intern Med 233:227–232. Swartzendruber DC, Wertz PW, Kitko DJ, et al.: 1989. Molecular models of the intercellular lipid lamellae in mammalian stratum corneum. J Invest Dermatol 92:251– 257. Teng B, Sniderman AD, Soutar AK, Thompson GR: 1986. Metabolic basis of hyperapobetalipoproteinemia. Turnover of apolipoprotein B in low density lipoprotein
41
and its precursors and subfractions compared with normal and familial hypercholesterolemia. J Clin Invest 77:663–672. Weston SA, Lahm A, Suck D: 1992. X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 Å resolution. J Mol Biol 226:1237–1256. Wiklund O, Mattsson L, Bjornheden T, et al.: 1991. Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: role of local LDL modification. J Lipid Res 32:55–62. Xu X-X, Tabas I: 1991. Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumu-
lation in macrophages. J Biol Chem 266:24,849–24,858. Zha X, Pierini LM, Leopold PL, et al.: 1998. Sphingomyelinase treatment induces ATPindependent endocytosis. J Cell Biol 140:39–47. Zhang, W-Y, Gaynor PM, Kruth HS: 1997. Aggregated low density lipoprotein induces and enters surface-connected compartments of human monocyte-macrophages. Uptake occurs independently of the low density lipoprotein receptor. J Biol Chem 272:31,700–31,706. PII S1050-1738(01)00143-8
TCM
Voltage Sensing and Activation Gating of HCN Pacemaker Channels Jun Chen, David R. Piper, and Michael C. Sanguinetti*
Activation of pacemaker channels underlie the spontaneous diastolic depolarization of sinoatrial node cells in the heart. Four similar genes encoding these hyperpolarization-activated, cyclic nucleotide-gated channels were recently cloned and subsequently named HCN1-4. Here we review the physiological role of HCN channels and recent findings regarding mechanisms of channel gating. Like all other voltage-gated channels, site-directed mutagenesis analysis indicates that the highly charged S4 transmembrane domain is the voltage sensor. However, unlike most other channels channel, opening occurs in response to membrane hyperpolarization rather than depolarization. (Trends Cardiovasc Med 2002;12:42–45). © 2002, Elsevier Science Inc.
Jun Chen, David R. Piper, and Michael C. Sanguinetti are from the Department of Medicine, Division of Cardiology and Eccles Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah. * Address correspondence to: M. Sanguinetti, Department of Medicine, Division of Cardiology and Eccles Program in Human Molecular Biology and Genetics, University of Utah, 15 N 2030 E, Room 4220, Salt Lake City, UT 84112-5330, USA. Tel.: (11) 801585-6336; fax: (11) 801-585-3501; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
42
• Physiological Roles and Molecular Basis of Pacemaker Channels The pacemaker current is a hyperpolarization-activated, cation-selective, inward current that modulates the firing rate of cardiac and neuronal pacemaker cells. This current is oftentimes abbreviated as Ih (“hyperpolarization”), If (“funny”), or Iq (“queer”). These currents contribute to normal pacemaking in the sinoatrial node and atrioventricular node of the atria and Purkinje fibers in the ventricle (DiFrancesco 1993, 1995), and to abnor-
mal automatic activity of cardiac myocytes under pathological conditions (Opthof 1998). Ih also mediates repetitive firing in neurons and oscillatory behavior in neuronal networks. In addition, they act to set the resting potential of certain excitatory cells, and may function in synaptic plasticity and activation of sperm (Pape 1996). cAMP causes cardiac pacemaker cells to increase their intrinsic rate of firing, an effect caused by a positive shift in the voltage dependence of Ih activation (Bois et al. 1997). Acetylcholine decreases the amplitude of If, and together with activation of IKACh, slows the pacing rate of nodal cells (Accili et al. 1998, DiFrancesco 1995). If is conducted by both Na1 and K1, and therefore, has a reversal potential around 220 mV (Ho et al. 1994). Block of If by Cs1 and certain drugs does not eliminate pacemaking (DiFrancesco 1995), emphasizing the important role of other currents, such as the rapid delayed rectifier K1 current, IKr , in this activity (Figure 1). The channels that conduct the pacemaker current were recently cloned and shown to share structural features with voltage-gated K1 channels. These features include six transmembrane domains, a GYG K1 channel signature sequence in the pore loop, and a highly positively charged S4 domain that is the putative voltage sensor (Gauss et al. 1998, Ludwig et al. 1998, Santoro et al. 1997, 1998). Pacemaker channels are encoded by the HCN (Hyperpolarization-activated, Cyclic Nucleotide-gated) gene family, and are most homologous to the eag family of K1 channels (for example, erg, eag, elk) and the KAT1 family of plant K1 channels (Biel et al. 1999). All three channel families (HCN, eag, KAT1) contain a consensus cyclic nucleotide binding (CNB) domain in the C-terminus. All known members of the HCN channel family (HCN1-4) are expressed in the brain (Santoro et al. 1997, 1998), whereas only HCN1, HCN2, and HCN4 are expressed in the heart. Based on in situ hybridization, the expression of HCN4 .. HCN2 . HCN1 in the sinoatrial node (Moosmang et al. 2001). HCN2 activates at more negative potentials than does If recorded from atrial pacemaker cells. Thus, it is possible that HCN4 homomultimers or HCN2/HCN4 heteromultimers (Ludwig et al. 1999), form the If channels in these cells. Based on RNAse TCM Vol. 12, No. 1, 2002
TCM Vol. 12, No. 1, 2002
TCM
Sphingomyelinase Activity of LDL: A Link between Atherosclerosis, Ceramide, and Apoptosis? Paavo K.J. Kinnunen* and Juha M. Holopainen
Atherosclerosis is characterized by the accumulation in the arterial intima of mainly low-density lipoprotein (LDL)-derived lipids, together with apolipoprotein B-100 (apoB-100), the protein moiety of LDL. Recent studies indicate aggregation of LDL within the arterial wall to represent a critical step in the initiation of this disease. Aggregation of LDL has been further proposed to involve ceramide, the levels of which are elevated in atherosclerotic plaques as well as in LDL isolated from these lesions. Biophysical studies have shown ceramide to have a pronounced tendency for self-aggregation, presumably driven by intermolecular hydrogen bonding. Importantly, the segregated ceramide-enriched lipid phases have high melting temperatures and are in a gel state at 378C. The plasma levels of sphingomyelin, which upon enzymatic hydrolysis by sphingomyelinase (SMase) yields ceramide, have been shown to correlate with the severity of coronary heart disease. The formation of ceramide from sphingomyelin could thus represent a critical step in atherosclerosis. We recently showed that LDL itself possesses SMase activity. Moreover, sequence analogy with bacterial enzymes suggests that this activity may be intrinsic to apoB-100. Possible physiological role of this activity is uncertain, yet could be involved in nonreceptormediated endocytotic entry of LDL into cells. Importantly, it also opens a possible mechanistic link between elevated plasma levels of LDL, apoptosis (programmed cell death), and atherosclerosis. (Trends Cardiovasc Med 2002;12:37–42). © 2002, Elsevier Science Inc.
Low-density lipoprotein (LDL) is the major carrier of cholesterol in the circulation, and provides cholesterol to the Paavo K.J. Kinnunen and Juha M. Holopainen are from the Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, University of Helsinki, Finland. * Address correspondence to: P.K.J. Kinnunen, Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, FIN-00014 University of Helsinki, P.O. Box 63 (Biomedicum, Haartmaninkatu 8), Helsinki, Finland. Tel.: 1358 9 191 25400; fax: 1358 9 191 25444; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
peripheral tissues by LDL receptormediated endocytosis (Brown and Goldstein 1986). The surface of the roughly spherical LDL particle is comprised of a single copy of the apolipoprotein B-100 (apoB-100), together with approximately 500 molecules of phosphatidylcholine, 200 molecules of sphingomyelin, and 400 molecules of unesterified cholesterol, constituing a surface film surrounding a core of cholesteryl esters and triacylglycerols. Yet, LDL is heterogeneous in size, composition, and conformation of apoB100 (Austin et al. 1988). ApoB-100 is one of the largest proteins known and appears to be multifunctional, from pro-
37
viding the recognition site on the LDL particle for its receptor, to diverse catalytic activities. Parthasarathy et al. demonstrated that apoB-100 possesses phospholipase A2 activity, which is inactivated upon oxidation (Parthasarathy and Barnett 1990). Later studies revealed both phospholipase A1 and A2 activities to be present (Reisfeld et al. 1993). In addition, platelet-activating factor acetylhydrolase activity was demonstrated to be associated with LDL (Stafforini et al. 1987). Interestingly, apoB-100 shows sequence homology to Src-1 domains, and has been shown to undergo autophosphorylation of tyrosine residues (Guevara et al. 1995). We have recently shown SMase activity to be associated with LDL, with sequence comparison to bacterial SMases suggesting that this activity could be an intrinsic property of apoB100 (Holopainen et al. 2000c). Aggregation of LDL has been proposed to represent an essential and central process in atherogenesis (Ross 1993), and LDL extracted from atherosclerotic lesions is aggregated or is prone to aggregate. Aggregation of LDL can be achieved by binding of these lipoproteins to proteoglycans, certain acidic phospholipids, vortexing (see Lauraeus et al. 1998, for a brief review), or by modifying the LDL surface by phospholipases A2, C, or SMase (Xu and Tabas 1991). In contrast to unaggregated LDL, aggregated LDL in atherosclerotic lesions is enriched in ceramide (Schissel et al. 1996). The involvement of ceramide is of particular interest, as this lipid has been established as a central messenger in apoptosis (for example, Mathias et al. 1998), albeit the mechanism(s) of action still remain(s) elusive. Taken together, altered sphingolipid metabolism could represent a risk factor for the development of atherosclerosis and the involvement of SMase and ceramide may play a central role in the development of this disease, providing also a link to apoptosis.
plaques and calcified plaques in patients who died of atherosclerosis. Interestingly, there is a positive correlation between the plasma concentration of sphingomyelin, a precursor for ceramide, and the severity of coronary heart disease (Jiang et al. 2000). A variety of cell types present in atherosclerotic lesions secrete SMase (Marathe et al. 1998, Schissel et al. 1996). Assuming LDL aggregation to be a prerequisite for the progression of atherosclerosis and as SMase can induce this process, it seems feasible to suggest a role for both SMase and ceramide in the pathophysiology of atherosclerosis. LDL treated with SMase can induce macrophage foam cell formation in vitro (Schissel et al. 1996, Xu and Tabas 1991). The ceramide content of aggregated LDL in lesions was 10–50-fold higher than that of plasma LDL (Schissel et al. 1996). However, nonaggregated lesional LDL was not enriched in ceramide, suggesting that aggregation and the formation of this lipid could be interrelated. Yet, the possibility cannot be excluded that this ceramide could originate from cells, being transferred to LDL by plasma lipid transfer proteins. • SMase Activity Associated with LDL In the course of our studies on the properties and metabolism of LDL we came to the conclusion that it would be worthwhile to explore if SMase was associated with LDL. This turned out to be the case, and the formation of ceramide was evident upon incubation of sphingomyelincontaining liposomes with human plasma LDL (Holopainen et al. 2000c). Instead, VLDL, HDL2, and HDL3 were devoid of SMase activity. The absence of SMase from VLDL would be in keeping with lipolysis-induced changes in the conformation of apoB-100 to be required, as previously demonstrated for the lack of recognition of apoB-100 in VLDL by the LDL receptor (Catapano et al. 1979). Ox-
idized LDL also lacked SMase activity. Oxidation of LDL induces the formation of a number of proteolytic fragments from the single polypeptide chain constituting apoB-100 (Fong et al. 1987), which could be sufficient to disrupt the conformation of this protein necessary for the proper orientation of the residues responsible for catalytic activity. The activity of SMase in LDL from different donors exhibited considerable variation but did not correlate with age, sex, or body mass index. Possible variation in the oxidation state could explain these differences in enzyme activity. Modifications such as methylation and acetylation of lipoproteins could also play a role in atherosclerosis (Steinberg 1993, Wiklund et al. 1991). The influence of these modifications on SMase activity associated with LDL remains to be investigated. Finally, genetic polymorphism of apoB-100 could also be involved. Although a major fraction of LDL is produced from VLDL, studies have shown that liver can produce LDL also directly (for example, Teng et al. 1986). The latter lipoproteins are somewhat smaller and denser than those derived from VLDL. In addition, lipoprotein(a) could also be present in the LDL fraction. The possibility of the above two lipoprotein species possessing SMase activity remains to be studied. The 3D structures for LDL and SMases are not available at present. Moreover, prediction of the 3D structure of apoB100 remains ambiguous. To identify putative region of LDL responsible for the contained SMase activity we compared the amino acid sequence of human apoB100 with those of Bacillus cereus, Leptospira interrogans, and Staphylococcus aureus SMase (Figure 1). Three homologous regions were found in apoB-100 and B. cereus SMase consisting of 24, 39, and 43 amino acids and having 38, 36, and 28% identical amino acids, respectively (Holopainen et al. 2000c). Comparison of apoB-100 with three dif-
• Sphingolipids in the Development of Atherosclerosis Oxidized LDL, but not native LDL, induces apoptosis of arterial smooth muscle cells via the activation of a neutral SMase (Chatterjee 1999). Furthermore, increased activity of this SMase correlates with elevated levels of ceramide and apoptosis in postmortem samples of
38
Figure 1. Illustration of the five distinct domains in the sequence of apoB-100, including the LDL receptor-binding domain as well as the the region containing the putative catalytic site of SMase.
TCM Vol. 12, No. 1, 2002
ferent isoforms of B. cereus SMase revealed that out of the total 305 amino acids of this enzyme 65 (21%) align with the sequence of apoB-100. Alignment of human apoB-100 with B. cereus, L. interrogans, and S. aureus sequences gives 30 amino acids that are common to all. Previous sequence studies have shown deoxyribonuclease I (DNase I) to share common mechanistic characteristics with SMase and identified tentatively in the sequence of SMase eight amino acids, which could be important in substrate recognition (Matsuo et al. 1996, Weston et al. 1992). Interestingly, out of these functionally important amino acids six are conserved, and one is weakly similar also in the alignment of B. cereus SMase and apoB-100. Finally, while sphingomyelin is present in LDL ceramide is almost absent, and has been shown to be present in large amounts only in aggregated LDL (Schissel et al. 1996). The putative active site in apoB-100 assumed to be responsible for the SMase activity of LDL should, therefore, be oriented outwards from the lipoprotein surface, prohibiting access to the sphingomyelin contained in this lipoprotein. Despite the above proteomic approach supporting the notion that the SMase activity of LDL would be intrinsic to apoB-100, other possibilities must also be considered. Accordingly, this enzyme could be secreted by cells in the arterial walls and become subsequently associated with LDL. However, as this activity was not detected in the other lipoproteins the interaction between the secreted SMase and lipoprotein surface should be specific for LDL. Lack of ceramide in plasma LDL further requires that the LDL-bound SMase should bind in a manner not enabling access of this enzyme to its substrate sphingomyelin in the lipoprotein surface film. These issues need to be addressed experimentally. • Possible Physiological Role for the SMase Activity of LDL Using microinjection techniques and giant unilamellar vesicles (GUVs) as biomembrane models, we have recently shown that the formation of ceramide results in a vectorial formation of vesicles (Holopainen et al. 2000a). More specifically, exposure of the outer surface of GUVs to SMase results in a rapid lateral clustering of the reaction prodTCM Vol. 12, No. 1, 2002
uct, ceramide, within the bilayer. The driving force is likely to be efficient intermolecular hydrogen bonding between the ceramide headgroups. Subsequently, the ceramide-enriched membrane domains detach as “endocytotic” vesicles inside the giant liposome. Yet, if SMase is injected into the liposome, thus catalyzing ceramide formation in the inner leaflet of the bilayer, the opposite takes place, resulting in budding of small vesicles outside the giant liposome. Interestingly, in ATP-depleted macrophages and fibroblasts treatment with exogenous SMase results within 10 min in the budding of numerous vesicles from the plasma membrane into the cytoplasm of these cells (Zha et al. 1998). These vesicles lack any observable protein coating and are relatively large, with a diameter of approx. 0.4 mm. Our data thus show that except no proteins other than SMase are necessary for this endocytosis. In line with the above, exogenously incorporated N-hexanoyl-sphingosine (C6-ceramide) induces time- and dose-dependent formation of vesicles (diameter 2–10 mm) in the interior of fibroblasts (Li et al. 1999). Finally, it is important to note that in contrast to the segregation of ceramide, sphingomyelin is miscible in fluid phosphatidylcholine bilayers. This difference is due to the large, hydrated phosphocholine headgroup of sphingomyelin. In subsequent experiments we demonstrated that “endocytotic” vesicles were also induced in giant liposomes by LDL (Holopainen et al. 2000c). This suggests that LDL may enter cells in an ATP- and receptor-independent pathway, owing to “autocytosis” driven by its SMase activity. Evidence for nonreceptor-mediated uptake by rat luteal cells of LDL cholesterol without concomitant apoB-100 ingestion has been presented (Reaven et al. 1986). Recently, aggregated LDL was shown to enter the cells by surfaceconnected compartments by a mechanism not involving the LDL receptor (Zhang et al. 1997). It is tempting to speculate that the above processes would be mediated by the SMase activity present in apoB-100. LDL binds to the proteoglycans on the endothelial wall (Hurt-Camejo et al. 1997). Accordingly, after binding of LDL to the cell surface the contained SMase would have access to its substrate, sphingomyelin. The ceramide formed would first segregate locally into microdomains. Subsequently, because of the
macroscopic physical properties of these domains (for instance, negative spontaneous curvature and high bending rigidity, see Holopainen et al. 2000a), they vesiculate from the bilayer, thus acting as local vehicles for “autocytotic” entry of LDL through the plasma membrane, as illustrated schematically in Figure 2. Based on these findings, we have suggested that in addition to its above putative role in LDL processing SMase could be used as a mechanism of entry of particles, such as microbes into mammalian cells, inducing the pinching of “autocytotic” vesicles off the plasma membrane, with the contained pathogen. This mechanism would be generic. Evidence for the involvement of SMase and phosphatidylcholine-specific phospholipase C (PC-PLC) in the entry of Neisseria gonorrhea, S. aureus, and species of mycobacteria into human cells was recently presented (see Holopainen et al. 2000a, for a brief review). The role of PC-PLC could be associated with breakdown of the surrounding vesicle inside the cell, enabling for an entry of the microbe into the cytoplasm. The above mechanism need not be limited to bacteria, but is equally likely to be responsible for the entry of viruses. In this connection it is of interest that Sindbis virus has been observed to trigger apoptosis in cells, with the formation of ceramide (Jan et al. 2000). More detailed understanding of the role(s) of sphingomyelin and other phospholipid-degrading enzymes in the above processess may thus open novel venues for therapeutic intervention. • Concluding Remarks Atherosclerosis is a multifactorial disorder. This is easily understood in the light of the plethora of processes that have been revealed to be involved in the development of this disease, such as oxidation of LDL, macrophage chemotaxis and foam cell formation, smooth muscle cell migration and alteration, lipoprotein retention and aggregation, and changes in the endothelium. Moreover, high HDL levels are “protective” of atherosclerosis (Nikkilä 1976), although the actual molecular level mechanisms still remain perplexing (Kinnunen 1979). Despite the limitations due to the number of factors being involved, the findings summarized in this brief review enable us to outline the following mech-
39
A key role in the above scheme is attributed to the particular features of ceramide. While the chemical structure of ceramide is simple, it imparts quite remarkable biophysical properties to this lipid. Its small polar headgroup contains both hydrogen bond donors and acceptors, and ceramide thus has a pronounced tendency for lateral segregation in membranes, driven by the formation of intermolecular hydrogen bonded networks (Holopainen et al. 1997, 2000b, Moore et al. 1997). Because of the saturated hydrocarbon chains in a large fraction of ceramide, the segregated ceramideenriched phases have their melting temperatures well above the body temperature and are thus in gel state at 378C (Holopainen et al. 1997, 2000a, 2000c). In the aggregated membranes further rearrangements, such as interdigitation of the chains, may take place, making these structures tightly packed and impermeable (Swartzendruber et al. 1989). In this connection it is relevant to note that ceramide, cholesterol, and fatty acids are essential constituents of the skin, and are currently believed to be responsible for its barrier properties (Elias and Menon 1991). Accordingly, those properties of the above lipids that serve useful purposes in skin may well precipitate pathological consequences when manifested in sites such as vascular endothelium. Figure 2. Formation of ceramide from sphingomyelin in a bilayer, catalyzed by the SMase activity of LDL and resulting in the formation of an “autocytotic” vesicle with the contained lipoprotein. For illustrative purposes the diameter of the lipoprotein particle and the thickness of the bilayer are not drawn to scale. The round symbol notifies the phosphocholine headgroup of either sphingomyelin (black acyl chains) or phosphatidylcholine (magenta acyl chains). The lipid with a black square as a headgroup represents ceramide.
anism for the formation of atherosclerotic plaques. High plasma levels of LDL would first shift the equilibrium in arterial walls towards initial, loose particle aggregation, perhaps promoted by binding of LDL to glycosaminoglycans (HurtCamejo et al. 1997). In these aggregates the SMase of LDL would be able to catalyze the formation of ceramide in the contacting, adjacent lipoproteins, resulting in the segregation of gel-like ceramide-enriched lipid microdomains, which further promote LDL–LDL interactions and result in tightly aggregated LDL, in keeping with the fusion of liposomes being promoted by ceramide (Abraham et al. 1988). Likewise, con-
40
tacts of these aggregated LDL with the vascular endothelial cells would enable catalytic formation of ceramide in these cells, triggering apoptosis, together with further ceramide accumulation (Royer and Foote 1971). The above scheme would be compatible with the correlation between elevated plasma levels of sphingomyelin and the severity of coronary heart disease (Jiang et al. 2000), as well as the measured high amounts of sphingomyelin and ceramide in atherosclerotic plaques (Schissel et al. 1996). Following the aggregation of LDL, other lipids in LDL, cholesterol in particular, would start to contribute to the growth of the plaque.
• Future Directions The processes and behavior of lipid mixtures discussed in this brief review are consequences of their physical properties. Lipids represent the liquid crystalline state of matter, which is characterized by a variety of phases and connecting phase transitions, described by phase diagrams. The above considerations warrant studies to establish the phase diagram of the lipids contained in atheroma. Although phase diagrams for multicomponent systems necessarily become complex, they nevertheless provide powerful “roadmaps,” which could open a rational and detailed understanding for the molecular basis of atherosclerosis, on the level of the different lipids, as well as factors such as acyl chain saturation. Availability of these data would also facilitate the development of novel therapeutic means, potentially enabling one to interfere with and prevent the putative formation of the microcrystalline “seeds” of ceraTCM Vol. 12, No. 1, 2002
mide initiating the growth of atheroma. A key role in the development of atheroma in the scheme outlined here is assigned to the SMase activity associated with LDL. It is essential to establish whether this activity is indeed intrinsic to apoB-100 or due to a tightly associated enzyme released from cells. Moreover, the physiological significance of this LDL-associated SMase remains to be studied, together with evaluating the feasibility of the use of specific inhibitors of this catalytic activity in preventing atherosclerosis.
• Acknowledgments We wish to thank Professor Felix Gõni (University of Bilbao, Basque County, Spain) and Dr. Robert Corkery (Procter and Gamble, Cincinnati, OH) for stimulating discussions. Financial support was obtained from Tekes and Finnish Academy (P.K.J.K.). J.M.H. is supported by grants from Farmos Research Foundation, Paulo Research Foundation, and Emil Aaltonen Foundation.
References Abraham W, Wertz PW, Downing DT: 1988. Effect of epidermal acylglucosylceramides and acylceramides on the morphology of liposomes prepared from stratum corneum lipids. Biochim Biophys Acta 939:403–408. Austin MA, Breslow JL, Hennekens CH, et al.: 1988. Low-density lipoprotein subclass patterns and risk of myocardial infarction. JAMA 260:1917–1921. Brown MS, Goldstein JL: 1986. A receptormediated pathway for cholesterol homeostasis. Science 232:34–47. Catapano AL, Gianturco SH, Kinnunen PKJ, et al.: 1979. Suppression of 3-hydroxy-3methylglutaryl-CoA reductase by low density lipoproteins produced in vitro by lipoprotein lipase action on nonsuppressive very low density lipoproteins. J Biol Chem 254:1007–1009. Chatterjee S: 1999. Neutral sphingomyelinase: past, present and future. Chem Phys Lipids 102:79–96. Elias PM, Menon GK: 1991. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv Lipid Res 24:1–26. Fong LG, Parthasarathy S, Witztum JL, Steinberg D: 1987. Nonenzymatic oxida-
TCM Vol. 12, No. 1, 2002
tive cleavage of peptide bonds in apoprotein B-100. J Lipid Res 28:1466–1477. Guevara J Jr. Walch ET, Epstein HF, et al.: 1995. Evidence that apoB-100 of lowdensity lipoproteins is a novel Src-related protein kinase. J Protein Chem 14:627– 631. Holopainen JM, Angelova MI, Kinnunen PKJ: 2000a. Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophys J 78:830–838. Holopainen JM, Lehtonen JYA, Kinnunen PKJ: 1997. Lipid microdomains in dimyristoylphosphatidylcholine-ceramide liposomes. Chem Phys Lipids 88:1–13. Holopainen JM, Lemmich J, Richter F, et al.: 2000b. Dimyristoylphosphatidylcholine/C16: 0-ceramide binary liposomes studied by differential scanning calorimetry and wideand small-angle x-ray scattering. Biophys J 78:2459–2469. Holopainen JM, Penate Medina O, Metso AJ, Kinnunen PKJ: 2000c. Sphingomyelinase activity associated with human plasma low density lipoprotein: possible functional implications. J Biol Chem 275: 16,484–16,489. Hurt-Camejo E, Olsson U, Wiklund O, et al.: 1997. Cellular consequences of the association of apoB lipoproteins with proteoglycans. Potential contribution to atherogenesis. Arterioscler Thromb Vasc Biol 17: 1011–1017. Jan J-T, Chatterjee S, Griffin DE: 2000. Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide. J Virol 74:6425– 6432. Jiang X-c, Paultre F, Pearson TA, et al.: 2000. Plasma sphingomyelin level as a risk factor for coronary artery disease. Arterioscler Thromb Vasc Biol 20:2614–2618. Kinnunen PKJ: 1979. High-density lipoprotein may not be antiatherogenic after all. Lancet ii:34–35. Lauraeus S, Holopainen JM, Taskinen M-R, Kinnunen PKJ: 1998. Aggregation of dimyristoylphosphatidylglycerol liposomes by human plasma low density lipoprotein. Biochim Biophys Acta 1373:147–162. Li R, Blanchette-Mackie EJ, Ladisch S: 1999. Induction of endocytic vesicles by exogenous C6-ceramide. J Biol Chem 274:21,121– 21,127. Marathe S, Schissel SL, Yellin MJ, et al.: 1998. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling. J Biol Chem 273:4081–4088.
Mathias S, Pena LA, Kolesnick RN: 1998. Signal transduction of stress via ceramide. Biochem J 335:465–480. Matsuo Y, Yamada A, Tsukamoto K, et al.: 1996. A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I. Protein Sci 5:2459– 2467. Moore DJ, Rerek ME, Mendelsohn R: 1997. FTIR spectroscopy studies of the conformational order and phase behaviour of ceramides. J Phys Chem B 101:8933– 8940. Nikkilä EA: 1976. Serum high-density-lipoprotein and coronary heart-disease. Lancet 2:320. Parthasarathy S, Barnett J: 1990. Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100. Proc Natl Acad Sci USA 87:9741–9745. Reaven E, Chen YD, Spicher M, et al.: 1986. Uptake of low density lipoproteins by rat tissues. Special emphasis on the luteinized ovary. J Clin Invest 77:1971–1984. Reisfeld N, Lichtenberg D, Dagan A, Yedgar S: 1993. Apolipoprotein B exhibits phospholipase A1 and phospholipase A2 activities. FEBS Lett 315:267–270. Ross R: 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809. Royer M, Foote JL: 1971. The identification of ceramides and glyceryl ethers in unsaponifiable lipid of human aorta. Chem Phys Lipids 7:266–678. Schissel SL, Tweedie-Hardman J, Rapp JH, et al.: 1996. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest 98:1455–1464. Stafforini DM, McIntyre TM, Carter ME, Prescott SM: 1987. Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor. J Biol Chem 262:4215–4222. Steinberg D: 1993. Modified forms of lowdensity lipoprotein and atherosclerosis. J Intern Med 233:227–232. Swartzendruber DC, Wertz PW, Kitko DJ, et al.: 1989. Molecular models of the intercellular lipid lamellae in mammalian stratum corneum. J Invest Dermatol 92:251– 257. Teng B, Sniderman AD, Soutar AK, Thompson GR: 1986. Metabolic basis of hyperapobetalipoproteinemia. Turnover of apolipoprotein B in low density lipoprotein
41
and its precursors and subfractions compared with normal and familial hypercholesterolemia. J Clin Invest 77:663–672. Weston SA, Lahm A, Suck D: 1992. X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 Å resolution. J Mol Biol 226:1237–1256. Wiklund O, Mattsson L, Bjornheden T, et al.: 1991. Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: role of local LDL modification. J Lipid Res 32:55–62. Xu X-X, Tabas I: 1991. Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumu-
lation in macrophages. J Biol Chem 266:24,849–24,858. Zha X, Pierini LM, Leopold PL, et al.: 1998. Sphingomyelinase treatment induces ATPindependent endocytosis. J Cell Biol 140:39–47. Zhang, W-Y, Gaynor PM, Kruth HS: 1997. Aggregated low density lipoprotein induces and enters surface-connected compartments of human monocyte-macrophages. Uptake occurs independently of the low density lipoprotein receptor. J Biol Chem 272:31,700–31,706. PII S1050-1738(01)00143-8
TCM
Voltage Sensing and Activation Gating of HCN Pacemaker Channels Jun Chen, David R. Piper, and Michael C. Sanguinetti*
Activation of pacemaker channels underlie the spontaneous diastolic depolarization of sinoatrial node cells in the heart. Four similar genes encoding these hyperpolarization-activated, cyclic nucleotide-gated channels were recently cloned and subsequently named HCN1-4. Here we review the physiological role of HCN channels and recent findings regarding mechanisms of channel gating. Like all other voltage-gated channels, site-directed mutagenesis analysis indicates that the highly charged S4 transmembrane domain is the voltage sensor. However, unlike most other channels channel, opening occurs in response to membrane hyperpolarization rather than depolarization. (Trends Cardiovasc Med 2002;12:42–45). © 2002, Elsevier Science Inc.
Jun Chen, David R. Piper, and Michael C. Sanguinetti are from the Department of Medicine, Division of Cardiology and Eccles Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah. * Address correspondence to: M. Sanguinetti, Department of Medicine, Division of Cardiology and Eccles Program in Human Molecular Biology and Genetics, University of Utah, 15 N 2030 E, Room 4220, Salt Lake City, UT 84112-5330, USA. Tel.: (11) 801585-6336; fax: (11) 801-585-3501; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
42
• Physiological Roles and Molecular Basis of Pacemaker Channels The pacemaker current is a hyperpolarization-activated, cation-selective, inward current that modulates the firing rate of cardiac and neuronal pacemaker cells. This current is oftentimes abbreviated as Ih (“hyperpolarization”), If (“funny”), or Iq (“queer”). These currents contribute to normal pacemaking in the sinoatrial node and atrioventricular node of the atria and Purkinje fibers in the ventricle (DiFrancesco 1993, 1995), and to abnor-
mal automatic activity of cardiac myocytes under pathological conditions (Opthof 1998). Ih also mediates repetitive firing in neurons and oscillatory behavior in neuronal networks. In addition, they act to set the resting potential of certain excitatory cells, and may function in synaptic plasticity and activation of sperm (Pape 1996). cAMP causes cardiac pacemaker cells to increase their intrinsic rate of firing, an effect caused by a positive shift in the voltage dependence of Ih activation (Bois et al. 1997). Acetylcholine decreases the amplitude of If, and together with activation of IKACh, slows the pacing rate of nodal cells (Accili et al. 1998, DiFrancesco 1995). If is conducted by both Na1 and K1, and therefore, has a reversal potential around 220 mV (Ho et al. 1994). Block of If by Cs1 and certain drugs does not eliminate pacemaking (DiFrancesco 1995), emphasizing the important role of other currents, such as the rapid delayed rectifier K1 current, IKr , in this activity (Figure 1). The channels that conduct the pacemaker current were recently cloned and shown to share structural features with voltage-gated K1 channels. These features include six transmembrane domains, a GYG K1 channel signature sequence in the pore loop, and a highly positively charged S4 domain that is the putative voltage sensor (Gauss et al. 1998, Ludwig et al. 1998, Santoro et al. 1997, 1998). Pacemaker channels are encoded by the HCN (Hyperpolarization-activated, Cyclic Nucleotide-gated) gene family, and are most homologous to the eag family of K1 channels (for example, erg, eag, elk) and the KAT1 family of plant K1 channels (Biel et al. 1999). All three channel families (HCN, eag, KAT1) contain a consensus cyclic nucleotide binding (CNB) domain in the C-terminus. All known members of the HCN channel family (HCN1-4) are expressed in the brain (Santoro et al. 1997, 1998), whereas only HCN1, HCN2, and HCN4 are expressed in the heart. Based on in situ hybridization, the expression of HCN4 .. HCN2 . HCN1 in the sinoatrial node (Moosmang et al. 2001). HCN2 activates at more negative potentials than does If recorded from atrial pacemaker cells. Thus, it is possible that HCN4 homomultimers or HCN2/HCN4 heteromultimers (Ludwig et al. 1999), form the If channels in these cells. Based on RNAse TCM Vol. 12, No. 1, 2002