Cysteine string protein expression in mammary epithelial cells.

Cysteine string protein (Csp) is a secretory vesicle protein previously demonstrated to be required for Ca2+-regulated exocytosis in neurons and endocrine cells. It has been suggested to function by regulating voltage-gated Ca2+ channels or, alternatively, to have a more direct effect on the regulated exocytotic machinery. Here we demonstrate the expression of Csp in mammary epithelial cells and in the KIM-2 mammary cell line. In KIM-2 cells, Csp was found to be associated with a population of small vesicles and showed partial co-distribution with the vesicle protein cellubrevin. KIM-2 cells do not express detectable levels of voltage-gated Ca2+ channels, ruling these out as a site of action. Using the release of transfected growth hormone (GH) as an assay of secretion, we found that GH is secreted in an exclusively constitutive manner from KIM-2 cells. Overexpression of Csp1 inhibits regulated exocytosis in other cell types but has no effect on constitutive GH release by KIM-2 cells. These results suggest that Csp does not have a major function in constitutive exocytosis.

Acetate and formate uptake into vesicles isolated from the basolateral region of the plasma membrane of ovine parotid acinar cells.

The transport of acetate and formate into plasma membrane vesicles derived from the basolateral face of the ovine parotid acinar cell has an absolute requirement for an anion to be present within the intravesicular space: bicarbonate, formate, acetate, propionate, and butyrate support the uptake of either acetate or formate. A pH gradient across the vesicle membrane, pHi 7.4, pH0 5.5, enhances the uptake of formate, but not acetate. There is no direct relationship between the rate of exchange and the degree of protonation of formate or acetate in the extravesicular medium. The process is saturable and can be inhibited by a range of functional group reagents. When mannitol is the main external osmoticum, the uptake of acetate and formate is still rapid; thus, no other ions are involved in the process apart from the external formate or acetate and the intravesicular anion. This activity could play a major role in the provision of energy in ruminant tissues.

Phosphate transport via Na+ -Pi cotransport and anion exchange in lactating rat mammary tissue.

Inorganic orthophosphate (Pi) transport, using 32P-labelled orthophosphate as tracer, by lactating rat mammary tissue has been examined using both tissue explants and the intact perfused gland. Pi uptake was predominantly via a Na+ -dependent pathway. Li+, however, unlike choline, was able to partially substitute for Na+. In addition, Pi release from tissue explants preloaded with 32Pi was stimulated by reversing the Na+ gradient. Thus transferring mammary explants from a buffer containing Na+ to one which was Na+ free (choline replacement) doubled the Pi efflux rate constant. The uptake of Pi by tissue explants was saturable with respect to external Pi, having apparent K(m) and V(max) values of 1.13 mM and 3.36 mmol (kg cell water)-1 (15 min)-1, respectively. The stimulation of Pi uptake by tissue explants by external Na+ was also saturable; the K(m) for Na+ was 9.7 mM. These results, taken together, suggest that the Na+ -dependent pathway is a Na+ -Pi cotransport mechanism. The transport of Pi by the perfused lactating rat mammary gland was examined using a rapid, paired-tracer dilution technique. Pi uptake by the perfused gland was found to be Na+ dependent and displayed saturable kinetics. The results suggest that the Na+ -Pi cotransporter is situated at the basolateral aspect of the secretory cells. The release of Pi from preloaded tissue explants was trans-accelerated by external Pi but not by Cl- or SO4(2-). However, external Pi stimulated Pi efflux with low affinity.

Amino acid sequence and the cellular location of the Na(+)-dependent D-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell.

The Na(+)-dependent D-glucose symporter has been shown to be located on the basolateral domain of the plasma membrane of ovine parotid acinar cells. This is in contrast to the apical location of this transporter in the ovine enterocyte. The amino acid sequences of these two proteins have been determined. They are identical. The results indicated that the signals responsible for the differential targeting of these two proteins to the apical and the basal domains of the plasma membrane are not contained within the primary amino acid sequence.

Mechanisms involved in the uptake of D-glucose into the milk producing cells of rat mammary tissue.

There is a rapid stimulation of the rate of efflux of 3-O-methyl D-glucose from intact lactating rat mammary tissue slices in response to a change in the Na(+)-gradient across the basolateral plasma membrane. RNA extracted from lactating mammary glands contains a 4 kb transcript which hybridises with the cDNA for rabbit intestinal SGLT1. A fraction of the mammary gland cells which is enriched in endoplasmic, Golgi and plasma membranes contains a protein that is immunologically similar to the Na(+)-dependent D-glucose symporter present in the plasma membrane of polarised epithelial cells, e.g., rabbit enterocytes, ovine enterocytes and parotid acinar cells.

Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport.

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.

The expression of the Na+/glucose cotransporter (SGLT1) gene in lamb small intestine during postnatal development.

We have shown previously that the activity and abundance of the intestinal Na+/glucose cotransporter (SGLT1) declines dramatically during the postnatal development of lambs, and that it can be restored in the intestine of ruminant sheep by intra-luminal infusion of D-glucose. The work presented in this paper has followed the expression of the SGLT1 gene along the vertical and horizontal axes of the ovine small intestine during early development, using quantitative in situ hybridisation histochemistry. Along the vertical axis, SGLT1 mRNA was first detectable just below the crypt-villus junction and rose rapidly to a peak level approx. 150 microns above this point. After reaching a maximum, the amount of message gradually declined towards the villus tip. This pattern of mRNA accumulation along the crypt-villus axis was similar in all intestinal positions and age groups. Along the length of the small intestine (horizontal axis), a decline in the level of SGLT1 mRNA was observed first in the distal intestine. This decrease in SGLT1 mRNA was significant in the intestine (75% of length) of 5-week-old lambs when compared to tissue taken from 25 and 50% of length (P < 0.01 and P < 0.02, respectively). However, the observed fall in the expression of this gene during weaning did not coincide with the fall in activity and amount of SGLT1. In adult animals, where the activity of SGLT1 is very low, the amount of message was greatly reduced. This work supports the finding that the expression of SGLT1 is primarily controlled at the post-transcriptional level during the postnatal development of ovine intestine.