containing (mM) 0.5 P,, 135 NaCI, 4 KCI, 1 MgSO,, 2 CaCI,, 10 glucose and 10 Tris-BES, pH 7.4. When a Na+-free medium was required, choline' was used as a ...
26s Biochernical Society Trcinsdctions ( 1 995)23 The regulation of anion transport in lactating rat mammary tissue
J.M. SHILLINGFORD, D.B. SHENNAN and R.B. BEECHEY Hannah Research Institute, Ayr, Scotland, KA6 5HL and * The Epithelial Function and Development Group, Institute of Biological Sciences, The University of Wales, Aberystwyth, Dyfed, SY23 3DD. Mammary gland metabolism is markedly influenced by the cessation of suckling. This can be attributed to two separate signalling systems. Firstly, a lack of suckling leads to a reduction in the circulating levels of hormones which are required to maintain lactation. Secondly, the resulting milk stasis also appears to affect the metabolic activity of the mammary gland. The latter effect appears to be mediated, via a negative feedback system, by a chemical signal which is secreted into milk [ I ] . This form of intramammary control may allow the rate of milk secretion to be regulated by milk removal. We have designed experiments to test if mammary tissue phosphate and sulphate transport systems are regulated by suckling (i.e. by the endocrine system) and/or milk stasis. A recent study has shown that inorganic phosphate (P,) transport in lactating rat mammary tissue occurs by at least two pathways 121. The predominant route is a (Na+-P,) cotransport pathway which allows P, to be accumulated intracellularly above unity and thus may help generate a large transepithelial Pi gradient. The other pathway is a Pi self-exchange system which can be inhibited by DIDS (4,4'diisothiocyanatostilbene-2,2'disulphonic acid) and appears not to transport anions such as CI- and SO: [2]. In contrast, sulphate transport in the mammary gland is mediated by a DIDS-sensitive anion-exchange system which has a broad substrate specificity [3,4]. Lactating Wistar rats, 10-15 days post partum, were used throughout this study. The transport of nP and '5S0,2- by isolated mammary tissue fragments was assayed at 37°C using standard methods 151. P, uptake was assayed using an incubation medium containing (mM) 0.5 P,, 135 NaCI, 4 KCI, 1 MgSO,, 2 CaCI,, 10 glucose and 10 Tris-BES, pH 7.4. When a Na+-free medium was required, choline' was used as a replacement. To quantitate the Na+gradient driven component we first measured I2P efflux from preloaded tissue slices into a buffer containing Na' followed by one containing choline as the principal cation. P, and SO:. efflux via the exchange systems were measured hy first following efflux from mammary tissue suspended in a solution containing (mM) 150 Na gluconate, 5 K gluconate, 10 glucose and 10 Tris-BES, pH 7.4, prior to transferring the tissue to a similar buffer supplemented with 50mM P, or 50mM CI- a appropriate (Na gluconate was reduced to mantain osmolarity). P, and sulphate efflux rate constants were calculated from the slope of plots of In (N,/N,) where N, = the amount of isotope remaining in the tissue at time t and N, = the amount of isotope associated with the tissue at the start of the efflux incubation. Statistical significance was assessed using unpaired or paired Student's t tests as appropriate. Results were considered significant if P
