An association between the R990G polymorphism of the CaSR gene, coding for calcium-sensing receptor, and primary hypercalciuria was found in kidney stone formers. To confirm this relationship, we investigated hypercalciuric women without stones and studied the effect of CaSR gene in human embryonic kidney cells (HEK-293). We genotyped for CaSR A986S, R990G, and Q1011E polymorphisms, 119 normocalciuric and 124 hypercalciuric women with negative history of kidney stones. Homozygous (n=2) or heterozygous (n=21) women for the 990G allele considered as one group had an increased risk to be hypercalciuric (odds ratio=5.2; P=0.001) and higher calcium excretion (P=0.005) in comparison with homozygous women for the 990R allele (n=220). HEK-293 cells were transfected with the variant allele at the three CaSR gene polymorphisms and with the most common allele with no variants. The transient increment of intracellular calcium caused by the stepwise increase of extracellular calcium was evaluated in stable transfected cells loaded with fura-2 AM. The extracellular calcium concentration producing the half-maximal intracellular calcium response was lower in HEK-293 cells transfected with the 990G allele than in those transfected with the wild-type allele (P=0.0001). Our findings indicate that R990G polymorphism results in a gain-of-function of the calcium-sensing receptor and increased susceptibility to primary hypercalciuria.

Casein phosphopeptides L L-CN(1^25)4P and K K s1 -CN(59^79)5P, from L L-and K K s1 -casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-GluGlu, were chemically synthesized and administered to HT-29 cells di¡erentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca 2+ ] i due to in£ux of extracellular Ca 2+ . The response was quantitatively higher with L L-CN(12 5)4P than K K s1 -CN(59^79)5P. The synthetic peptide corresponding to the 'acidic motif' was ine¡ective and the dephosphorylated form of L L-CN(1^25)4P almost inactive. The lack of the N-terminally located ¢ve amino acids, or sequence modi¢-cations within the N-terminal segment of L L-CN(1^25)4P, caused a total loss of activity, whereas the lack of the C-terminal segment preserved activity. In conclusion, the in£ux of calcium into HT-29 cells caused by L L-CN(1^25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region.

We investigated the direct effects of casein phosphopeptides (CPP), which are formed by the proteolytic degradation of alpha- and beta-caseins, on calcium uptake by human HT-29 intestinal tumor cells, which undergo an enterocytically oriented differentiation in culture. A commercial preparation containing a mixture of purified CPP and an individual CPP of 25 amino acids, both containing the characteristic Ca(2+) binding motif, ser(P)-ser(P)-ser(P)-glu-glu, were employed. The study was performed at the single-cell level and on a cell population and measured the changes in cytosolic calcium concentration before and after CPP addition. In the presence of 2 mmol/L extracellular calcium, both CPP preparations induced a transient rise of free intracellular calcium ions, which did not influence ATP-induced release of calcium from intracellular stores, and which disappeared completely in the absence of extracellular calcium. Pretreatment of these cells with thapsigargin, which completely empties the intracellular calcium stores, did not abolish the cell responses to CPP. Repetitive stimulation of HT-29 cells with CPP always elicited a transient calcium rise, suggesting a lack of desensitization. The CPP-stimulated cytosolic calcium rise was dependent on CPP dose, in a seemingly nonsaturating mode, and on cell numbers. All of this is consistent with the hypothesis that CPP do not influence membrane-bound receptors or ion channels, but may act as calcium ionophores or calcium carriers across the membrane. The reported findings provide a new basis on which to assess the possibility that CPP enhance calcium absorption and bioavailability in animals.

The thermotropic behavior of palmitoylsphingomyelin vesicles containing GM1 ganglioside and cholesterol has been investigated by high-sensitivity differential scanning calorimetry. The thermograms exhibited by binary palmitoylsphingomyelin/GM1 mixtures are resolvable into two components. The relative contribution of the minor component, undetectable in the absence of ganglioside, to the total enthalpy and its transition temperature (>40 degrees C) increase with the concentration of the glycolipid embedded in the vesicles. These data suggest the occurrence of lateral phase separation and that more ordered, higher melting GM1 ganglioside-enriched domains are present within the sphingomyelin bilayer. Studies on binary sphingomyelin/cholesterol mixtures confirmed the known tendency of the sterol to decrease the total enthalpy of sphingomyelin, forming cholesterol-enriched domains. The thermograms exhibited by ternary sphingomyelin/ganglioside/cholesterol mixtures in variable proportions (up to 20% molar GM1 or Chol) displayed, on increasing the content of either the sterol or the ganglioside, features addressable to sphingomyelin/cholesterol (peaks centered at temperature 40 degrees C), respectively. This trend was confirmed by deconvolution analysis, showing that the thermograms are resolvable into components addressable to GM1-enriched and to cholesterol-enriched domains. Taken all together, the results show that the architectural features of sphingomyelin bilayers are strongly dependent on the presence of GM1 ganglioside and cholesterol, whose presence is leading to the formation of separate, GM1-enriched and cholesterol-enriched distinct domains. Ganglioside-sphingomyelin and sphingomyelin-cholesterol, together with mutual ganglioside-ganglioside, interactions could contribute to maintain a network of bonds extending to proteins, forming specialized membrane domains, such as caveolae, or others, whose experimental clues are the glycolipid-enriched detergent-insoluble fractions that can be isolated from cell membranes.

An intestinal 70/30 Caco2/HT-29 co-culture was set up starting from the parental populations of differentiated cells to mimic the human intestinal epithelium. Co-culture was harvested at confluence 0 (T0) and at 3, 6, 10, and 14 days post confluence after plating (T3, T6, T10, and T14, respectively) for morphological and functional analysis. Transmission electron microscopy revealed different features from T0 to T14: microvilli and a complete junctional apparatus from T6, mucus granules from T3, as also confirmed by PAS/Alcian Blue staining. The specific activity of alkaline phosphatase (ALP), aminopeptidase N (APN), and dipeptidyl peptidase IV (DPPIV) progressively increased after T0, indicating the acquirement of a differentiated and digestive phenotype. Transepithelial electrical resistance (TEER), indicative of the barrier properties of the monolayer, increased from T0 up to T6 reaching values very similar to the human small intestine. The apparent permeability coefficient for Lucifer Yellow (LY), along with morphological analysis, reveals a good status of the tight junctions. At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a versatile and suitable in vitro model of human intestinal epithelium for the presence of more than one prevalent intestinal cell type, by means of a minimum of 6 to a maximum of 14 post-confluence days obtained without the need of particular inducers of subclones and growth support to reach an intestinal differentiated phenotype.