The transport of phosphate in the lactating mammary gland
Jonathan Shillingford
Laboratory of Genetics and Physiology,
National Institute of Diabetes, Digestive and Kidney Diseases,
National Institutes of Health,
Bethesda,
MD 20892
Tel.: 301-435-6634
Fax: 301-480-7312
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Foreword
The aim of this mini-review is to examine the available literature with regard the transport of inorganic phosphate (Pi) in the mammary gland. The experimental data obtained and discussed by the author was carried out under the supervision of Prof. Brian Beechey and Dr. David Shennan whose assistance and advice is gratefully acknowledged.
Current understanding of Pi transport in the mammary gland
In most species, perhaps with the exception of humans, a large trans-epithelial Pi-gradient exists between blood and milk. For example, in both the rat and the sheep the plasma concentration of Pi is between 1 and 2 mM (see Berndt and Knox, 1992) compared to a value of 11 - 12 mM in milk (Holt and Jenness, 1984). Experiments carried out by Neville and Peaker (1979) seem to suggest that Pi is not able to pass from milk to blood via passive movement across the apical membrane. This was demonstrated by injecting radioactive Pi into the mammary gland of the goat via the teat. It was assumed that if Pi is able to cross the apical membrane then the amount of radioactivity would be reduced. However, 98 % of the original dose was recovered 3 h after injection. Although this suggests Pi does not cross the apical membrane via a passive mechanism it does not rule out the possibility that an active transport mechanism may be responsible for transporting Pi into milk.
Much of the work concerning the transport of Pi into milk is based on inference rather than actual experimental data. Several reviews have suggested that Pi is secreted into milk via exocytosis of Golgi-derived vesicles (see Neville et al., 1983; Leong et al., 1990; Abeijon et al., 1997). Indeed it would appear that Pi is produced as a by-product of lactose synthesis (Kuhn and White, 1975; 1977) and possibly also casein phosphorylation (Farrell et al., 1992). However, whether Pi is transported out of the Golgi apparatus, as has been suggested, or is a method whereby Pi is concentrated prior to secretion remains uncertain.
The nature of mammary Pi transport
Irrespective of how Pi is secreted/transported into milk, Pi first has to be transported into the mammary secretory cell via the basolateral membrane. Until recently, nothing was known about the basolateral entry mechanism.
It has long been established that the transport of Pi across the apical membrane of both the intestinal and renal cell occurs via sodium-phosphate (Na-Pi) symport (for reviews see Biber and Murer, 1994; Murer et al., 1994). Thus the energy of the inwardly-directed sodium-gradient is utilized to drive the unfavourable transport of Pi into the cell.
Pi transport has also been shown to occur via anion-exchange. The blood-brain barrier exhibits Pi/bicarbonate exchange (Dallaire and Beliveau, 1992), bacteria possess a number of Pi/sugar-phosphate exchange systems (for review see Maloney et al., 1990), and the dicarboxylate transporter of mitochondria is capable of mediating Pi/dicarboxylate exchange (Johnson and Chappell, 1973)
Since nothing was known about mammary gland Pi transport, either symport, anion-exchange or both of these mechanisms may be responsible for the transport of Pi as suggested by Shennan (1990).
Initial experiments were designed to examine whether Pi transport could be mediated via a Na-Pi symport mechanism. This was achieved with the use of isolated mammary tissue explants and an in situ perfusion technique; currently these techniques are the best available to examine mammary transport mechanisms.
When the uptake of Pi into rat mammary tissue explants was determined in the presence of extracellular sodium, Pi uptake increased steadily over the time-course. In contrast, Pi uptake determined in the absence of sodium (choline replacement) remained low and there was no evidence of an increase in Pi uptake over time (Slide 1).
Further experiments performed at 4ºC established that Pi uptake in the presence of extracellular sodium was totally abolished whereas Pi uptake in a choline medium remained unchanged. Taken together, these results suggest that Pi transport in the mammary gland can occur via a Na-dependent transport process.
To further assess the Na-dependent nature of mammary Pi transport, a series of efflux experiments were performed. In these experiments, tissue explants were loaded with radioactive Pi and the efflux of Pi followed over time. It can be predicted that if mammary Pi transport proceeds via Na-Pi symport, reversing the intracellular to extracellular sodium-gradient should result in an increase in the efflux of Pi. Indeed transferring tissue explants from a sodium medium to a choline medium resulted in a large increase in the fractional efflux of Pi from the explant into the medium. This increase in efflux was abolished when the experiment was performed at 4°C suggesting that the observed increase in efflux was not due to cell lysis or detachment as a result of changes in medium composition (Slide 2).
Finally, Pi uptake was assessed using a rapid paired-tracer dilution technique in conjunction with the in situ perfused rat mammary gland first described by Mendelson and Scow (1972). Using this technique it was demonstrated that the clearance of Pi (i.e. the volume of perfusate notionally cleared of Pi) was entirely dependent on the presence of sodium in the perfusate; replacing sodium with NMDG completely abolished Pi uptake (Slide 3).
Further experiments using the perfusion technique established that Pi uptake was saturable with respect to increasing concentrations of perfusate Pi. The apparent Km and Vmax values were calculated to be 32 mM and 20 nmoles.min-1.g-1 tissue wet weight, respectively.
All these results taken together strongly suggest that mammary Pi transport can be mediated by a Na-Pi symport mechanism. On the basis of mammary gland architecture it is suggested that this transport process is situated on the basolateral membrane, placing it in an ideal position to transport Pi into the cell.
Molecular identity of mammary Na-Pi symport
The possible molecular identity of the mammary Na-Pi symporter was investigated using the techniques of Northern blotting and RT-PCR. Radiolabelled cDNA derived from NaPi-1 (Werner et al., 1991), NaPi-2 and NaPi-3 (Magagnin et al., 1993) were used to screen rat and sheep mammary total RNA. However, under the conditions used, Northern analyses failed to reveal any related mRNA transcripts. The lack of a Na-Pi transporter of the renal type was supported by RT-PCR experiments using NaPi-specific primers. These experiments failed to result in the amplification of cDNA sequences with any homology to the renal NaPi family or the brain-specific Na-Pi symporter, BNPI (see Ni et al., 1994; 1996). These results suggest that the mammary Na-Pi symporter is probably unrelated to any of the cloned Na-Pi transporters described to date. The apparent lack of a specific molecular probe means that other techniques will need to be used to establish the molecular identity of the mammary Na-Pi symporter. The expression cloning technique first described by Hediger et al. (1985) may prove useful in this endeavour.
These results are further supported by data recently published by The Jackson Laboratory. RT-PCR analysis, using primers derived from a region of a kidney Na-Pi symporter (NaPi-2), failed to result in the amplification of a mRNA transcript in the mammary gland sample.
Evidence for anion-exchange
An anion-exchange mechanism which mediates sulphate/sulphate, sulphate/chloride and sulphate/iodide exchange has previously been described in rat mammary tissue (Shennan, 1989). However, the inability of external Pi to trans-stimulate radioactive sulphate efflux and the inability of external sulphate and chloride to trans-stimulate radioactive Pi efflux (Shillingford et al., 1996) demonstrates that this anion-exchange system does not transport Pi.
Several anion-exchange systems are capable of mediating Pi self-exchange (see Fliege et al., 1978; Rosenburg et al., 1982; Indiveri et al., 1989; Stappen and Kramer, 1994). Therefore the ability of external Pi to trans-stimulate Pi efflux was tested. These experiments show that external Pi was capable of trans-stimulating Pi efflux in a dose-dependent manner. Furthermore, this efflux was inhibited by DIDS, a chemical inhibitor known to inhibit a variety of anion-exchange processes (see Cabantchik and Greger, 1992).
On the basis of further experiments it would seem that this anion-exchange process is distinct from the Pi/bicarbonate exchange described in the blood-brain barrier (Dallaire and Beliveau, 1992) and the Pi/dicarboxylate exchange system of the mitochondria (Johnson and Chappell, 1973).
It is possible that Pi self-exchange and sodium-phosphate occur via the same transport process or two (or more) separate transport processes. There are two pieces of experimental data which suggest they are separate transporters. Firstly, DIDS inhibits Pi-self exchange but has no effect on sodium-phosphate symport. Secondly, efflux via the Pi self-exchange system occurs in the complete absence of sodium (potassium replacement).
The physiological significance of the Pi-self exchange system is not known. One possibility is that it represents a non-physiological mode of a physiologically-significant transport process. Further experiments need to be carried out to investigate this possibility.
The possible role of Na-Pi symport in trans-epithelial Pi movment
A possible scheme that may account for trans-epithelial Pi movement across the mammary secretory cell is presented in Slide 4.
A recent paper (Neckelmann and Orellana, 1998) has investigated the transport of UDP-glucose across the Golgi membrane of cells isolated from pea stems. The experimental data obtained indicate that both UMP and Pi are able to exit the Golgi. It is apparent that whilst the exit of UMP is coupled to the entry of UDP-glucose (see also Capasso and Hirschberg, 1984) the nature of the Pi transporter remains known. However, these results are the first to present data indiciating that a Pi transporter of some description resides in the Golgi membrane. Whether such a transporter also exists in the Golgi apparatus of the mammary gland remains to be established.
The results presented in this mini-review and Shillingford et al. (1996) describe a transporter with properties indicative of the presence of a mammary Na-Pi symporter mechanism. The basolateral location of this transporter places it in an ideal position to drive the transport Pi from the blood into the mammary secretory cell. However, further understanding of the trans-epithelial movement of Pi will require the study of intracellular Pi movement and the movement of Pi into milk via the apical membrane. The role of the Na-Pi symporter in this scheme remains to be investigated.
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Resources
Below is a list of resources the reader may find interesting
- A brief introduction to membrane transport