Phosphocreatine interacts with phospholipids, affects membrane properties and exerts membrane-protective effects

Abstract

A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.

Figure 1. Phosphocreatine and creatine are retained by liposomes as determined in a biochemical assay.

LUVs consisting of phosphatidylcholine and cardiolipin (PC/CL) were incubated either with Cr or PCr at different concentrations in TES buffer (10 mM pH 7.0, 50 mM NaCl), centrifuged, the pellet washed twice, and remaining Cr and PCr quantified. Data are given as mean±SD (n = 3).

Figure 2. Direct interaction of phosphocreatine, creatine, their cyclic analogues, glucose and glucose phosphate with liposomes as determined by surface plasmon resonance.

(A) Concentration-dependence of glucose (Glc, white), Cr (dark grey), glucose-6-phosphate (Glc-6-P, light grey), and PCr (black) binding to LUVs consisting of cholesterol, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin (CH/PE/PC/SP). (B, C) Binding of PCr, Cr, their cyclic analogues, as well as glucose and Glc-6-P to LUVs consisting of either (B) CH/PE/PC/SP or (C) PC/CL. Runs were performed in TES buffer (10 mM pH 7.0, 50 mM NaCl). Bound metabolites were quantified at a reporting point 60 s after beginning of dissociation. The given SPR signal corresponds to measured response units (RU) divided by the molecular mass (Da) of the corresponding metabolite. Data in (A) were normalized to the response at 100 mM PCr. All data are given as mean ± SD (n = 4 independent experiments).

Figure 3. Interaction of phosphocreatine and creatine with lipid vesicles as visualized by solid-state ³¹P-NMR spectroscopy.

(A) Simulation of the static ³¹P-NMR spectrum of a spherical liposome. (B), (C) Static spectra of lipid vesicles (CH/PE/PC/SP) incubated with (B) 25 mM Cr or PCr, or (C) 25 mM glucose were compared to spectra of control MLVs without additions. Spectra were recorded at 300 MHz and 303 K, referenced to 85% H₃PO₄ and normalized. Differences between control and treated lipid vesicles observed in (B) but not in (C) are indicated: (*) change in the lineshape in the powder spectrum of Cr and PCr; (**) small resonance peak at ∼0 ppm in the Cr spectrum due to isotropic lipids (micelles or other small aggregates); (#) resonance peak at 1.9 ppm in the PCr spectrum corresponding to inorganic phosphate; (##) resonance peak at −3.2 ppm in the PCr spectrum corresponding to the phosphate moiety of PCr.

Figure 4. Effect of phosphocreatine, creatine and dimethylbiguanidium chloride on the lipid phase transition as determined by differential scanning calorimetry.

Thermograms (left) and quantified phase transition temperature shifts (right) obtained by heating or cooling of a suspension of SOPC in PIPES buffer (20 mM PIPES pH 7.4, 140 mM NaCl, 1 mM EDTA) alone (middle scans in A or B, labeled (−). (A) one heating and one cooling scan shown with added 100 mM PCr, Cr or dimethylbiguanidium chloride (DMBG), or (B) two cycles of heating and cooling shown for each sample. 500 mM PCr Na-salt or Tris-salt (omitting NaCl). Thermograms (left) are from representative runs, histograms (right) represent averaged data from 3 independent experiments (H  =  heating; C  =  cooling).

Figure 5. Phosphocreatine and creatine protect lipid vesicles against melittin-induced permeabilization.

50 µM of CH/POPE/POPC/SP-LUVs entrapping a quenched ANTS/DPX solution were injected into PIPES buffer (20 mM pH 7.4, isomolarity of the entrapped solution was adjusted to be equal to that of the external media with NaCl). The media external to the liposomes contained only phosphate buffer and NaCl (trace 1) or 100 mM DMBG (trace 2), Cr (trace 3), or PCr (trace 4). Leakage was begun with 500 nM melittin. All buffers and entrapped ANTS/DPX solution were adjusted to the same osmolarity and each compound was tested twice. Fluorescence dequenching was followed at 530 nm and normalized to the signal obtained after full permeabilization of LUVs.

Figure 6. Phosphocreatine protects red blood cells against doxorubicin-, saponin- and hypoosmotic stress- induced lysis.

Red blood cells isolated from fresh pig blood were preincubated for 10 min without any additions or in presence of 50 mM PCr, glucose (Glc) or glucose-6-phosphate (Glc-6-P). Hemolysis was induced by (A) 300 µM doxorubicin (DXR) at 37°C, (B) incubation for 10 min at 200 mOsm at room temperature, or (C) incubation with 1–10 µg/ml saponin for 10 min at room temperature. Data in (A) were corrected for absorbance of doxorubicin; hemolysis was evaluated directly (0 h) and 5 h after addition of doxorubicin. Stability of PCr under the given experimental condition was verified by HPLC. Data are given as mean±SD (n = 3). Note: Glc-6-P also exerts a protective effect.

Figure 7. Structure and putative phospholipids interaction mechanism of phosphocreatine and its analogues.

(A) Chemical structures of Cr, PCr (top) as well as the electrostatic potential (red, positive; blue, negative) at the solvent-accessible surface (mesh representation), superimposed on the ball-and-stick representations of Cr and PCr (bottom). (B) Chemical structures of the cyclic analogues cCr and PcCr. (C) Proposed interaction between PCr (bold line representation) and the zwitterionic headgroups of an array of eight dioleoyl phosphatidylcholine molecules (DOPC, thin line representation) with (partial) charges indicated. Structures prepared with WebLabViewer Pro v4.0 (for details see Materials and Methods).