J. Physiol. (1975), 253, pp. 527-545 With 6 text-figures Printed in Great Britain
527
MILK SECRETION IN THE RABBIT: CHANGES DURING LACTATION AND THE MECHANISM OF ION TRANSPORT
BY M. PEAKER AND JANET C. TAYLOR* From the Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge CB2 4A T
(Received 13 May 1975) SUMMARY
1. Changes in the yield and composition of milk and in the permeability of the mammary epithelium to labelled disaccharides and ions have been studied during lactation in rabbits of the Dutch breed. 2. Milk yield increased to reach a peak on day 20 of lactation and then declined, but by 30-32 days the yield was still relatively high. Milk [protein] and [fat] increased in late lactation; [Na] and [Cl] decreased from early to established lactation (11-14 days) and then increased, whereas milk [K] and [lactose] showed an inverse pattern to that displayed by [Na] and [Cl]. 3. At all stages of lactation [Na] and [Cl] were both inversely related to milk [lactose] while [K] showed a positive correlation. 4. Labelled lactose and sucrose were found to cross the mammary epithelium at all stages but in increased amounts during late lactation. Sucrose entry from blood into milk was positively correlated with milk [Na], and inversely correlated with [K] and [lactose]. 5. The entry of 24Na and 36C1 into milk from blood paralleled the changes in milk [Na] and [Cl]. 6. Intracellular ionic composition determined in vitro was similar for [Na] and [K] in both established (11-14 days) and late (25-28 days) lactation, but [Cl] was higher in late lactation. 7. Intracellular potentials recorded in vivo were -31 mV (mean) and -36 mV in established and late lactation respectively. Transepithelial p.d. was close to zero at both stages. 8. It is suggested that ions and lactose and other small molecules can cross the mammary epithelium by a paracellular, as well as by a transcellular route, throughout lactation and that the paracellular pathway is increased in late lactation. *
M.R.C. Scholar.
528 M. PEAKER AND JANET C. TA YLOR 9. The site of the proposed paracellular pathway and the implications of such a pathway's presence on ion transport are discussed, and a scheme is suggested to account for the ionic composition of milk in this species. INTRODUCTION
From studies on the aqueous phase of milk and the electrochemical gradients between milk and extracellular fluid, Linzell & Peaker (1971 a, b) proposed a scheme to account for the ionic composition of milk and ion transport in the guinea-pig and goat in full lactation. It was suggested that Na and K are distributed passively across the apical (luminal) membrane of the secretary or alveolar cell according to the electrical potential gradient. Thus the concentrations of these ions are lower in milk than in intracellular fluid but the ratio between them is similar in both compartments. The high K, [K], and low Na concentration, [Na], of intracellular fluid, and therefore milk, is believed to be maintained by a Na+-K+ exchanging pump on the basal (i.e. basolateral) membrane. The situation for Cl is different since both the potential and concentration gradients tend to drive Cl from the cell into milk. However, the concentration of C], [Cl], is lower in milk than in the cell and it was suggested that this ion might be actively transported into the cell across both the apical and basal membranes (see also Linzell & Peaker, 1975). Although the scheme could account for the formation of the aqueous phase of milk in normal full lactation it was obvious that there were cases in which it could not solely apply, for example, during late pregnancy or oxytocin treatment in the goat, when milk [Na] and [Cl] are raised, and [K] and [lactose] are lowered. Evidence has been obtained that in both these conditions, in contrast to the usual situation, labelled sucrose passes from blood into milk, and labelled lactose from milk into blood even though the secretary cells remain impermeable to these disaccharides. Thus it was argued that the passage of sucrose into milk represents a paracellular pathway, probably through 'leaky' tight junctions and that in these circumstances Na and Cl pass into milk, and K and lactose in the reverse direction, down their respective concentration gradients (Linzell & Peaker, 1971 d, 1974) (Fig. 1). Moreover, in late pregnancy in the goat the increased passage of Na and Cl into milk, determined by isotopic flux measurements, could be entirely accounted for by paracellular movements, estimated by [14C]sucrose entry, as opposed to transcellular movements (Linzell & Peaker, 1974). Linzell & Peaker (1971d) showed that in day-to-day changes in milk composition during full lactation in the goat, [lactose] vs. both [Na] and [K] were inverse correlations, whereas [lactose] vs. [Cl] was not significant;
MILK SECRETION IN THE RABBIT 529 these relationships were compatible with the scheme proposed for ion movements during lactation. During oxytocin treatment and in late pregnancy the correlation between [lactose] and [K] was positive and in these cases [lactose] was significantly and inversely correlated with [Cl]. This different pattern is consistent with the hypothesis that in these conditions there is an additional, paracellular pathway across the mammary secretary epithelium which permits Na and Cl to enter milk, and K and lactose to leave (Linzell & Peaker, 1971 d, 1974). Extracellular Basal membrane fluid
Apical (luminal) membrane Milk
Paracellular pathway
Fig. 1. Suggested scheme for ion and lactose movments between extracellular fluid and milk in different physiological conditions. The cellular transport mechanisms including those into and out of the Golgi vesicles (which fuse with, and probably become part of, the apical membrane) are those suggested by Linzell & Peaker (1971 a, b).
The rabbit is an interesting species in that the composition of the aqueous phase of milk differs from that of other eutherian mammals for which adequate data exist (Linzell & Peaker, 1971 a) and changes markedly during the relatively short period of lactation (Cowie, 1969; Gachev, 1965, 1971; Linzell & Peaker, 1971 a); moreover in this species suckling occurs
530 M. PEAKER AND JANET C. TAYLOR only once a day (Cross & Harris, 1952; Zarrow, Denenberg & Anderson, 1965). In essence, the concentration of lactose in milk is relatively low in established lactation, but while milk yield remains high later in lactation, [Na] and [Cl] increase while [K] and [lactose] decrease, i.e. a similar situation to that in late pregnancy or during oxytocin treatment in goats. Therefore it seemed desirable to examine, using in vivo methods similar to those employed in the goat, the permeability of the mammary secretary epithelium in the rabbit to disaccharides and ions at different stages of lactation in order to determine the pathways of ion movements and to assess the relative importance of transcellular and paracellular routes. METHODS Animals. Female domestic rabbits of a Dutch strain in their second to fourth lactation, with four to seven (mean six) young, were used. They were housed individually; water, hay and oxoid diet SG 1 were freely available. Pregnant rabbits were examined daily. They usually littered in the night and the first day the young were seen was called day 1 of lactation. Determination of milk yield. The females were kept separated from their young except for a short period each morning. The young were disturbed to encourage urination and then weighed. The mother was then allowed access. When suckling ceased, after approximately 15 min, the young were reweighed and milk yield taken as the weight difference (see Cowie, 1969). Yields prior to day 5 were not recorded as interference with the young or mother at this stage often led to neglect and death of the litter. Milk composition. Individual glands were hand-milked at the time the young would have been allowed access, by the method of Goode & Taylor (1974). In most experiments (see Results) the samples from individual glands were pooled for
analysis. Isotope infusions. The experiments were done under light halothane (Fluothane, I.C.I.; in 02) anaesthesia on rabbits separated from their young for 24 hr. The marginal vein of one ear and the central artery of the other ear were cannulated by the technique described by Peaker, Phillips & Wright (1970). An arterial blood sample and a pooled milk sample from the glands on the right-hand side were taken if the animal had previously been given isotope, in order to determine residual levels. A mixture of isotopes (30 ml. sterile 0-154 M-NaCl containing: 50 ,uc, 3HOH; 7-5 ,tc, 3601; 10 tsc, [14C]sucrose and 7-5 /tIc, 24Na) was then infused i.v. at 1 ml./min for 30 min. Arterial blood samples (2 ml.) were taken at 5 min intervals throughout and a pooled milk sample from the glands on the left-hand side was taken from 20 to 30 min.
[14C]lactose movements. Under light halothane anaesthesia [14C]lactose (20 me/mmole) was injected into five ducts of each of four glands (the pair of central glands on each side). The animals were then placed in a metabolism cage for the collection of urine. Blood samples were taken at intervals and after 24 hr individual milk samples were taken from the injected glands and a pooled sample from the others. Milk yield was then determined as described above. Intracellular composition. Intracellular ionic composition in vitro was determined using [14C]sucrose as an extracellular space marker as described by Linzell & Peaker (1971 b). The medium in which the slices were incubated for 1 hr was the
MILK SECRETION IN THE RABBIT
531
bicarbonate-CO2 buffered solution of Krebs & Henseleit (1932) containing 5 mm glucose and 2 mm sodium acetate. Potential differences. Intracellular and blood-milk p.d.s were recorded using the methods described by Evans, Linzell & Peaker (1971).
Analytical method Milk. Na, K, Cl and 'lactose' were determined as described by Fleet, Linzell & Peaker (1972), protein by the orange G dye-binding method of Udy (1956) modified for use in a Technicon autoanalyser, and fat by the method of Fleet & Linzell (1964). Milk water content was determined by drying at 102° C to constant weight with the precautions recommended by Hagemeijer, Rorive & Schoffeniels (1965). It should be noted that the method for lactose determines all reducing sugars. While the results are given as lactose concentrations, other sugars in milk would lead to an error when calculating the osmotic effect of milk sugars (see Discussion). Pkwma and tiasue digest. Na, K and Cl were determined, using standard methods in a Technicon autoanalyser (see Peaker, 1971). Radio-isotopes. In all fluids studied separation of radioactivity from 24Na, 36C1, ['4C]sucrose and 3HOH was achieved using the methods described by Linzell & Peaker (1974). Expression of results. The concentrations of milk components are expressed as m-mole/l. milk water or as weight/unit weight milk water. RESULTS
Milk yield The daily yield, determined from days 5 to 32 of lactation, increased to reach a peak of approximately 135 g/day on day 20; thereafter yield declined but even at 30-32 days was still in the order of 105 g/day (Fig. 2). This time course is similar to that obtained by Cowie (1969) in rabbits of the same breed but which were given oxytocin (0.5 i.u. i.v.) before suckling commenced. Milk composition Validity of pooled samples Since most of the experiments described depend upon a comparison between animals it was necessary to establish that a pooled milk sample is representative and therefore that milk composition is similar in all eight glands. This was done in four rabbits twice during lactation when 2 ml. samples were taken from each gland. Analysis of variance for each milk component showed no statistical significance for differences between glands. Furthermore, serial samples taken from one gland showed no significant changes in composition; similar results were obtained by Cowie (1969). Changes during lactation In these and subsequent studies four stages of lactation were considered: early (days 4-7) (daily yield increasing), established (11-14)
M. PEAKER AND JANET C. TAYLOR (daily yield increasing), peak (18-21) (yield at maximum) and late (25-28) (yield decreasing) (see Fig. 2). In the late stage it was observed that the young were eating a little solid food but were always eager to take milk. Protein and fat. Although there was a tendency for [protein] to decrease between early and peak lactation, the changes were not statistically significant. However, in late lactation [protein] increased significantly to reach approximately 5 g/100 ml. milk water (Fig. 3). [Fat] showed a small but significant increase between early and established lactation (Fig. 3). 532
140 -
120 F
100pk bO 4'I
._b f
80160140
2010'
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0
I
I
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I
I
I
1
-
I
I
I
I
4 6
8 10 12 14 16 18 20 22 24 26 28 30 32 Days of lactation l l I IL I Early Established Peak Late
2
Fig. 2. Changes in milk yield from days 5 to 32 of lactation in the rabbit (mean ± s.E. of mean in six animals). The bars below the time scale show the stages at which milk composition and mammary permeability were studied.
Sodium, potassium, chloride and lactose. Changes in [Na] and [Cl] showed similar pattern, as did those of [K] and [lactose] in the opposite direction. [Na] and [Cl] decreased significantly between early and established lactation but then increased until late lactation. At this time [Na] was at its maximum (mean 112 m-mole/l. milk water), but [Cl] was not increased by a similar proportion and the concentration at this time (62 mM) did not exceed that in early lactation. In contrast, [K] and [lactose] showed a reverse pattern an increase up to established lactation and then a signia
-
MILK SECRETION IN THE RABBIT 533 ficant decrease (Fig. 3). Thus at 11-14 days, the stage we have called established lactation, milk composition was nearest to that of goats and cows.
-P< 0-01O
Na
120 100
80 60
4' 0
E
d* P < 005 lr -P < 0.05
P < 0401 n.s._ -r
40 20 0 P < 0.01
I
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,P
527
MILK SECRETION IN THE RABBIT: CHANGES DURING LACTATION AND THE MECHANISM OF ION TRANSPORT
BY M. PEAKER AND JANET C. TAYLOR* From the Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge CB2 4A T
(Received 13 May 1975) SUMMARY
1. Changes in the yield and composition of milk and in the permeability of the mammary epithelium to labelled disaccharides and ions have been studied during lactation in rabbits of the Dutch breed. 2. Milk yield increased to reach a peak on day 20 of lactation and then declined, but by 30-32 days the yield was still relatively high. Milk [protein] and [fat] increased in late lactation; [Na] and [Cl] decreased from early to established lactation (11-14 days) and then increased, whereas milk [K] and [lactose] showed an inverse pattern to that displayed by [Na] and [Cl]. 3. At all stages of lactation [Na] and [Cl] were both inversely related to milk [lactose] while [K] showed a positive correlation. 4. Labelled lactose and sucrose were found to cross the mammary epithelium at all stages but in increased amounts during late lactation. Sucrose entry from blood into milk was positively correlated with milk [Na], and inversely correlated with [K] and [lactose]. 5. The entry of 24Na and 36C1 into milk from blood paralleled the changes in milk [Na] and [Cl]. 6. Intracellular ionic composition determined in vitro was similar for [Na] and [K] in both established (11-14 days) and late (25-28 days) lactation, but [Cl] was higher in late lactation. 7. Intracellular potentials recorded in vivo were -31 mV (mean) and -36 mV in established and late lactation respectively. Transepithelial p.d. was close to zero at both stages. 8. It is suggested that ions and lactose and other small molecules can cross the mammary epithelium by a paracellular, as well as by a transcellular route, throughout lactation and that the paracellular pathway is increased in late lactation. *
M.R.C. Scholar.
528 M. PEAKER AND JANET C. TA YLOR 9. The site of the proposed paracellular pathway and the implications of such a pathway's presence on ion transport are discussed, and a scheme is suggested to account for the ionic composition of milk in this species. INTRODUCTION
From studies on the aqueous phase of milk and the electrochemical gradients between milk and extracellular fluid, Linzell & Peaker (1971 a, b) proposed a scheme to account for the ionic composition of milk and ion transport in the guinea-pig and goat in full lactation. It was suggested that Na and K are distributed passively across the apical (luminal) membrane of the secretary or alveolar cell according to the electrical potential gradient. Thus the concentrations of these ions are lower in milk than in intracellular fluid but the ratio between them is similar in both compartments. The high K, [K], and low Na concentration, [Na], of intracellular fluid, and therefore milk, is believed to be maintained by a Na+-K+ exchanging pump on the basal (i.e. basolateral) membrane. The situation for Cl is different since both the potential and concentration gradients tend to drive Cl from the cell into milk. However, the concentration of C], [Cl], is lower in milk than in the cell and it was suggested that this ion might be actively transported into the cell across both the apical and basal membranes (see also Linzell & Peaker, 1975). Although the scheme could account for the formation of the aqueous phase of milk in normal full lactation it was obvious that there were cases in which it could not solely apply, for example, during late pregnancy or oxytocin treatment in the goat, when milk [Na] and [Cl] are raised, and [K] and [lactose] are lowered. Evidence has been obtained that in both these conditions, in contrast to the usual situation, labelled sucrose passes from blood into milk, and labelled lactose from milk into blood even though the secretary cells remain impermeable to these disaccharides. Thus it was argued that the passage of sucrose into milk represents a paracellular pathway, probably through 'leaky' tight junctions and that in these circumstances Na and Cl pass into milk, and K and lactose in the reverse direction, down their respective concentration gradients (Linzell & Peaker, 1971 d, 1974) (Fig. 1). Moreover, in late pregnancy in the goat the increased passage of Na and Cl into milk, determined by isotopic flux measurements, could be entirely accounted for by paracellular movements, estimated by [14C]sucrose entry, as opposed to transcellular movements (Linzell & Peaker, 1974). Linzell & Peaker (1971d) showed that in day-to-day changes in milk composition during full lactation in the goat, [lactose] vs. both [Na] and [K] were inverse correlations, whereas [lactose] vs. [Cl] was not significant;
MILK SECRETION IN THE RABBIT 529 these relationships were compatible with the scheme proposed for ion movements during lactation. During oxytocin treatment and in late pregnancy the correlation between [lactose] and [K] was positive and in these cases [lactose] was significantly and inversely correlated with [Cl]. This different pattern is consistent with the hypothesis that in these conditions there is an additional, paracellular pathway across the mammary secretary epithelium which permits Na and Cl to enter milk, and K and lactose to leave (Linzell & Peaker, 1971 d, 1974). Extracellular Basal membrane fluid
Apical (luminal) membrane Milk
Paracellular pathway
Fig. 1. Suggested scheme for ion and lactose movments between extracellular fluid and milk in different physiological conditions. The cellular transport mechanisms including those into and out of the Golgi vesicles (which fuse with, and probably become part of, the apical membrane) are those suggested by Linzell & Peaker (1971 a, b).
The rabbit is an interesting species in that the composition of the aqueous phase of milk differs from that of other eutherian mammals for which adequate data exist (Linzell & Peaker, 1971 a) and changes markedly during the relatively short period of lactation (Cowie, 1969; Gachev, 1965, 1971; Linzell & Peaker, 1971 a); moreover in this species suckling occurs
530 M. PEAKER AND JANET C. TAYLOR only once a day (Cross & Harris, 1952; Zarrow, Denenberg & Anderson, 1965). In essence, the concentration of lactose in milk is relatively low in established lactation, but while milk yield remains high later in lactation, [Na] and [Cl] increase while [K] and [lactose] decrease, i.e. a similar situation to that in late pregnancy or during oxytocin treatment in goats. Therefore it seemed desirable to examine, using in vivo methods similar to those employed in the goat, the permeability of the mammary secretary epithelium in the rabbit to disaccharides and ions at different stages of lactation in order to determine the pathways of ion movements and to assess the relative importance of transcellular and paracellular routes. METHODS Animals. Female domestic rabbits of a Dutch strain in their second to fourth lactation, with four to seven (mean six) young, were used. They were housed individually; water, hay and oxoid diet SG 1 were freely available. Pregnant rabbits were examined daily. They usually littered in the night and the first day the young were seen was called day 1 of lactation. Determination of milk yield. The females were kept separated from their young except for a short period each morning. The young were disturbed to encourage urination and then weighed. The mother was then allowed access. When suckling ceased, after approximately 15 min, the young were reweighed and milk yield taken as the weight difference (see Cowie, 1969). Yields prior to day 5 were not recorded as interference with the young or mother at this stage often led to neglect and death of the litter. Milk composition. Individual glands were hand-milked at the time the young would have been allowed access, by the method of Goode & Taylor (1974). In most experiments (see Results) the samples from individual glands were pooled for
analysis. Isotope infusions. The experiments were done under light halothane (Fluothane, I.C.I.; in 02) anaesthesia on rabbits separated from their young for 24 hr. The marginal vein of one ear and the central artery of the other ear were cannulated by the technique described by Peaker, Phillips & Wright (1970). An arterial blood sample and a pooled milk sample from the glands on the right-hand side were taken if the animal had previously been given isotope, in order to determine residual levels. A mixture of isotopes (30 ml. sterile 0-154 M-NaCl containing: 50 ,uc, 3HOH; 7-5 ,tc, 3601; 10 tsc, [14C]sucrose and 7-5 /tIc, 24Na) was then infused i.v. at 1 ml./min for 30 min. Arterial blood samples (2 ml.) were taken at 5 min intervals throughout and a pooled milk sample from the glands on the left-hand side was taken from 20 to 30 min.
[14C]lactose movements. Under light halothane anaesthesia [14C]lactose (20 me/mmole) was injected into five ducts of each of four glands (the pair of central glands on each side). The animals were then placed in a metabolism cage for the collection of urine. Blood samples were taken at intervals and after 24 hr individual milk samples were taken from the injected glands and a pooled sample from the others. Milk yield was then determined as described above. Intracellular composition. Intracellular ionic composition in vitro was determined using [14C]sucrose as an extracellular space marker as described by Linzell & Peaker (1971 b). The medium in which the slices were incubated for 1 hr was the
MILK SECRETION IN THE RABBIT
531
bicarbonate-CO2 buffered solution of Krebs & Henseleit (1932) containing 5 mm glucose and 2 mm sodium acetate. Potential differences. Intracellular and blood-milk p.d.s were recorded using the methods described by Evans, Linzell & Peaker (1971).
Analytical method Milk. Na, K, Cl and 'lactose' were determined as described by Fleet, Linzell & Peaker (1972), protein by the orange G dye-binding method of Udy (1956) modified for use in a Technicon autoanalyser, and fat by the method of Fleet & Linzell (1964). Milk water content was determined by drying at 102° C to constant weight with the precautions recommended by Hagemeijer, Rorive & Schoffeniels (1965). It should be noted that the method for lactose determines all reducing sugars. While the results are given as lactose concentrations, other sugars in milk would lead to an error when calculating the osmotic effect of milk sugars (see Discussion). Pkwma and tiasue digest. Na, K and Cl were determined, using standard methods in a Technicon autoanalyser (see Peaker, 1971). Radio-isotopes. In all fluids studied separation of radioactivity from 24Na, 36C1, ['4C]sucrose and 3HOH was achieved using the methods described by Linzell & Peaker (1974). Expression of results. The concentrations of milk components are expressed as m-mole/l. milk water or as weight/unit weight milk water. RESULTS
Milk yield The daily yield, determined from days 5 to 32 of lactation, increased to reach a peak of approximately 135 g/day on day 20; thereafter yield declined but even at 30-32 days was still in the order of 105 g/day (Fig. 2). This time course is similar to that obtained by Cowie (1969) in rabbits of the same breed but which were given oxytocin (0.5 i.u. i.v.) before suckling commenced. Milk composition Validity of pooled samples Since most of the experiments described depend upon a comparison between animals it was necessary to establish that a pooled milk sample is representative and therefore that milk composition is similar in all eight glands. This was done in four rabbits twice during lactation when 2 ml. samples were taken from each gland. Analysis of variance for each milk component showed no statistical significance for differences between glands. Furthermore, serial samples taken from one gland showed no significant changes in composition; similar results were obtained by Cowie (1969). Changes during lactation In these and subsequent studies four stages of lactation were considered: early (days 4-7) (daily yield increasing), established (11-14)
M. PEAKER AND JANET C. TAYLOR (daily yield increasing), peak (18-21) (yield at maximum) and late (25-28) (yield decreasing) (see Fig. 2). In the late stage it was observed that the young were eating a little solid food but were always eager to take milk. Protein and fat. Although there was a tendency for [protein] to decrease between early and peak lactation, the changes were not statistically significant. However, in late lactation [protein] increased significantly to reach approximately 5 g/100 ml. milk water (Fig. 3). [Fat] showed a small but significant increase between early and established lactation (Fig. 3). 532
140 -
120 F
100pk bO 4'I
._b f
80160140
2010'
I
0
I
I
I
I
I
I
1
-
I
I
I
I
4 6
8 10 12 14 16 18 20 22 24 26 28 30 32 Days of lactation l l I IL I Early Established Peak Late
2
Fig. 2. Changes in milk yield from days 5 to 32 of lactation in the rabbit (mean ± s.E. of mean in six animals). The bars below the time scale show the stages at which milk composition and mammary permeability were studied.
Sodium, potassium, chloride and lactose. Changes in [Na] and [Cl] showed similar pattern, as did those of [K] and [lactose] in the opposite direction. [Na] and [Cl] decreased significantly between early and established lactation but then increased until late lactation. At this time [Na] was at its maximum (mean 112 m-mole/l. milk water), but [Cl] was not increased by a similar proportion and the concentration at this time (62 mM) did not exceed that in early lactation. In contrast, [K] and [lactose] showed a reverse pattern an increase up to established lactation and then a signia
-
MILK SECRETION IN THE RABBIT 533 ficant decrease (Fig. 3). Thus at 11-14 days, the stage we have called established lactation, milk composition was nearest to that of goats and cows.
-P< 0-01O
Na
120 100
80 60
4' 0
E
d* P < 005 lr -P < 0.05
P < 0401 n.s._ -r
40 20 0 P < 0.01
I
E E
,P
