Human, rat and chicken small intestinal Na+ - Cl- -creatine transporter: functional, molecular characterization and localization

Abstract

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [(14)C] creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na(+)- and Cl(-)-dependent, with a probable stoichiometry of 2 Na(+): 1 Cl(-): 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a K(m) for creatine of 29 microM. [(14)C] creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, beta-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na(+)- and Cl(-)-dependent, apical creatine uptake.

Figure 1. Time course of creatine uptake into either chicken enterocytes or rat ileum

[¹⁴C]Creatine uptake was measured in the presence (•, ▴, ○) and absence (□) of extracellular NaCl as a function of time. When required, NaCl was isosmotically substituted by mannitol. At the time indicated by the arrow either 1 mm guanidinopropionic acid (GPA) (▴) or 1 mm unlabelled creatine (○) was added. The dashed line represents the uptake value expected at equilibrium. Inset shows the linearity of uptake below 15 min in the presence of NaCl. Values are means ± s.e.m. of five independent experiments.

Figure 2. Effect of either electrical, Na⁺ or Cl⁻ gradient on 30 min of intestinal creatine uptake

When required, Na⁺ was isosmotically substituted by N-methyl-glucamine, Cl⁻ by gluconate and NaCl by mannitol. Valinomycin (0.02 mm) was added when the external potassium concentration was 40 mm. Other details as in Fig. 1. Values are means ± s.e.m., N =3. *P < 0.001; **P < 0.05, as compared with control conditions, first column.

Figure 3. Na⁺-dependent creatine uptake into chicken enterocytes vs. Na⁺ concentration

Uptake of 1 μm [¹⁴C]creatine was measured for 5 min. The incubation buffer contained (mm): 34 KCl, 1 CaCl₂, 3 K₂HPO₄, 1 MgCl₂, 20 Hepes-Tris, pH 7.4, 20 mannitol, 0.02 valinomycin, 1 mg ml⁻¹ bovine serum albumin and different concentrations of Na⁺, which were made by substitution of NaCl with NMGCl. The Na⁺-dependent component for creatine uptake was calculated by subtracting the uptake measured in the absence of Na⁺ from that measured in its presence. Inset: Hill-type plot of the data, in which the initial velocity (V) was plotted against V/[Na⁺]^1.9, r = 0.96. Values are means ± s.e.m., N =5.

Figure 4. Cl⁻-dependent creatine uptake into chicken enterocytes vs. Cl⁻ concentration

The different concentrations of Cl⁻ were made by substitution of NaCl with sodium gluconate. Other details as in Fig. 3. Inset: Hill-type plot of the data, in which the initial velocity (V) was plotted against V/[Cl⁻]^1.06, r = 0.991. Values are means ± s.e.m., N =5.

Figure 5. Kinetics of creatine uptake into chicken enterocytes

Initial rate (5 min) of chicken enterocyte creatine uptake vs. increasing concentrations of external creatine measured in the presence (•) and absence (▴) of extracellular NaCl. Difference: total uptake minus that measured in NaCl-free conditions. Inset: Eadie-Hofstee plot of the difference data. Values are means ± s.e.m., N =5.

Figure 6. Northern blot analysis of CRT in rat, human and chicken ileum

Equal amounts (10 μg) of poly(A⁺)RNA were loaded onto the gel per lane. The size of the transcripts was determined by ribosomal RNA. The CRT transcripts displayed a motility corresponding to a size of ca 4.4-3 kb for human, 4.2-2.7 kb for rat and 4.4-2.6 kb for chicken ileum.

Figure 7. Expression of CRT mRNA in chicken and rat ileum

Panels are bright-field photomicrographs of rat and chicken small intestine which have been in situ hybridized with either antisense or sense digoxigenin-UTP-labelled riboprobes. The arrows indicate the intestinal crypts. Scale bars: 100 μm for chicken; 50 μm for rat; bars also apply to right panels.

Figure 8. Western blot analysis of heart extracts, intestinal homogenate (H) and apical (BBM) and basolateral (BLM) membranes

A total of 70 μg protein was loaded to each lane. The blots were probed with the polyclonal anti-N-terminal anti-CRT antibody, as described in the Methods section. Histograms represent the relative abundance of CRT protein in homogenate (H), apical (BBM) and basolateral (BLM) membrane. Values are means ± s.e.m., N =3.

Figure 9. Immunolocalization of CRT in chicken, rat and human small intestine

Sections of ileum were immunostained with the polyclonal CRT antibody (A) and with preimmune serum (B), as indicated in the Methods. The arrows indicate the goblet cells. Scale bar represents 100 μm and applies to all panels.