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The excretion of prostacyclin (PGI2) in milk and its possible role as a vasodilator in the mammary gland of goats
Camp. Biochem. Physiol. Vol. 93A, No. 2, pp. 477-481, Printed in Great Britain
1989
0
0300-9629/89 S3.00 + 0.00 1989 Pergamon Press plc
THE EXCRETION OF PROSTACYCLIN (PGI,) IN MILK AND ITS POSSIBLE ROLE AS A VASODILATOR IN THE MAMMARY GLAND OF GOATS K.
CHRISTENSEN,*
M.
0.
NIELSEN
and N. JmLwt
The Royal Veterinary and Agricultural University, Department of Animal Physiology and Biochemistry; and tDepartment of Surgery, Biilowsvej 13, DK-1870 Frederiksberg C, Denmark (Received
22 November
1988)
Abstract--l.
Prostacyclin production in mammary gland of two lactating goats measured as the excretion in milk of 6-ketoprostaglandin F,, (6-KPGF,,) was followed for 16 days before, during and after exogenous administration of recombinant bovine growth hormone (GH). 2. 6-KPGF,, was detected in all milk samples in concentrations ranging from 32-99pg/ml milk independently of the time of sampling. 3. GH-treatment significantly increased milk yield, the concentration and excretion of 6-KPGF,, in milk. 4. The concentration of milk 6-KPGF,, was positively correlated with milk yield in the high (R* = 0.35). but not in the low yielding goat (Rz = 0.003). 5. The possible role of prostacyclin as a local vasodilator in the mammary gland of goats is discussed.
INTRODUCMON
Local and extra mammary vasoactive factors controlling mammary blood flow have been reviewed and discussed comprehensively (Dhondt et al., 1973; Linzell, 1974; Davis and Collier, 1985). Prostaglandins and related compounds (the eicosanoids) are involved in many different physiological and pathophysiological functions. Prostaglandins may be involved in controlling mammary vascular activity. Prostaglandin F,, (PGF,,) is produced and metabolized by the goat (Maule-Walker and Peaker, 1981) and cow mammary gland (Manns, 1975; Anderson et al., 1985) and is considered a locally active inhibitor of mammary function pre-partum and in late lactation in the goat (Maule-Walker and Peaker, 1981). The involvement of the eicosanoids in inflammatory conditions have been investigated in the last 15 years, and it is now apparent that the eicosanoids are fundamental to the inflammatory process as mediators (Higgins, 1985). Thromboxane B, (TBX,) the stable metabolite of TXA,, was found in the milk of healthy cows, and both PGF,, and TXB, were found in elevated concentrations in the milk of cows with acute coliform mastitis (Anderson et al., 1985). Prostacyclin (PGI,) is the major prostanoid synthesized from arachidonic acid by vascular tissue and is known to bc a potent mediator producing vasoand bronchiedilatation and inhibition of platelet aggregation (Moncada and Vane, 1979). It is more effective in arteries than in veins (Skidgel and Printz, 1978). *Present address and address for correspondence: National Institute of Animal Science, Department of Animal Physiology and Biochemistry, Forsogsanheg Foulum, PO Box 39, DK-8833 0rum Sdrl, Denmark. Telephone: 06-65-2500. 477
It is known that bovine aortic endothelial and smooth muscle cells produce PGIr (IngermanWojenski et al., 1981), but it is not known if a production and/or metabolism in the mammary gland of ruminants is taking place or if PGI, plays a role as a local vasodilator in the mammary gland in situ. However, it has been found in cultured endothelial cells from bovine carotid aorta that the vasodilator effect of kallikrein is mediated in part by prostacyclin production (Morita et al., 1984) and kallikrein has been shown to cause vasodilation in the udder of small ruminants and cows (Dhondt et al., 1973). It has been suggested that a major vasodepressor/ vasodilator system, “the kallikrein-kinin-prostaglandin system”, antagonized the effect of the blood pressure elevation system, “the adrenergic nervousrenin-angiotensin-aldosteron complex” (McGiff et al., 1981). Another prostaglandin known to have vasodilator effects (PGEr) was only produced in small amounts compared to the production of PGI, in cultured bovine carotid aortic endothelial cells (Morita et al., 1984). The synthesis of the eicosanoids is activated through a variety of stimuli including physical, chemical and hormonal influences (Hamberg and Samuelsson, 1973). In different physiological pathways, prostaglandins seem to modulate the action of hormones rather than act as hormones themselves (Stryer, 1975). The purpose of the present experiment was to investigate whether PGI,, measured as its stable metabolite 6-keto-prostaglandin F,, (6-KPGF,,), was excreted in the milk of healthy goats, and if the excretion of milk 6-KPGF,, was correlated to milk yield. As exogenous growth hormone (GH) or somatotropin is known to stimulate mammary blood flow and milk yield in lactating goats (Mepham et al., 1984), this hormone was injected into the goats in order to detect a possible effect on the excretion of 6-KPGF,, in milk.
K. CHRISTENSENet
478 MATERIALS AND METHODS Animals
Two Norwegian lactating goats from the herd of the Institute were used in the present studies. The goats were in 25th-27th week of their second lactation period and yielded 1 (Goat 1) or 2 1(Goat 2) milk daily, in the following called low and high yielding goats, respectively. They were milked twice a day at 8a.m. and 3p.m. and the milk yield was recorded. They were housed in individual stalls and fed concentrates according to milk yield jn two equal portions at the time of milking and received hay ad Ii&turn. They had free access to water. The udder was healthy in both goats. Experimental
procedures
The experiment was divided into three periods: a pre-treatment control period (period I) lasting three days, a treatment period (period 2) lasting 6 days and a post-treatment control period (period 3) lasting 7 days. In the treatment period the experimental animals were injected daily with 10 IU recombinant bovine growth hormone (0.88 IU/mg determined by radioimmunoassay. Lot. No. 58920, Lilly Industries Ltd., Windlesham, UK). The hormone was solubilized in 4ml 0.9% NaCl containing 0.025 M NaHCO, and 0.025 M Na,CG, just prior to the injection given subcutanously in the shoulder region at approximately 9 a.m. Analyricat
procedures
Milk samples of IOml were collected from the morning and afternoon milking, respectively, centrifuged immediately at 17OOg for 15 min at 4°C to remove cells and separate the fat from milk plasma. The fat free milk plasma (2 x 3 ml) was frozen at -20°C until analysis was performed within 3 weeks. Prostacyclin metabolite formation was followed by spontaneous breakdown to the stable 6-keto-PGF,, (PaceAsciak, 1976). The latter was determined in milk and used to reflect milk concentrations of PGI,. Milk 6keto-PGF,, (&KPGF,,) con~ntrations were measured by means of a commercial RIA kit (Amersham TRK code 790). Prior to RIA, milk plasma extraction was performed at SepPak Cl8 columns (Waters). Two ml milk plasma were acidified to pH 3 with glacial acetic acid and eluted on the activated columns with a mixture of IOml water, torn1 ethanol (15%). lOm1 petroleum ether and 4ml methyl formate as previously described (Powell, 1980). After evaporation the concentrate was diluted in 1ml assay buffer. Mean recovery was 73.4%. All analyses were performed in duplicate. Statistical
analysis
Analysis of variance (ANOVA) and analysis of regression (GLM) were performed using SAS as described by Freund and Littell (1981). The two-way ANOVA comprised the independent variables treatment, goat and treatment x goat interactions. One-way ANOVA was performed for each goat. Student’s f-test was used for statistical comparisons. RESULTS
6-KPGF,, was detected in all milk samples in concentrations ranging from 32-99 pg/ml milk. The concentration of 6-KPGF,, in the milk from the morning and the afternoon milkings (N = 30) were 58.0 f 2.32 and 62.6 + 2.61 pg/ml milk, respectively, the dtfference being non-significant (P > 0.05). The relative variations in the concentration of 6-KPGF,, expressed as the coefficient of variation (CV,% = SD/mean x 100) was similar in the morning and afternoon milk: 22.7 and 23.6% respectively. Therefore, the mean concentration of 6-KPGF,, in the morning and afternoon milk was used for further calculations.
al.
Table I. Analysts of variance of independent vanablea in rclatwn to growth hormone treatment of two lactating goats (F-values and Pr > F. l = P < 0.05; ** = P < 0.01; *** = P < 0.001) Source of variation Treatment Goat Treatment
rq ‘-. 2
I x goat
2
Milk yield daily 46.66”* 2257*21.05***
Concentration of &KPGF,, 3.92* 12.73** 2.40
Excretion of O-KPGF,, 15.10*** 94.05*** i.ss*
The daily milk yield, concentration of milk 6-KPGF,, and total daily excretion of 6-KPGF,, in milk before, during and after GH-treatment of the two goats are shown graphically in Fig. 1 for individual days. The greatest response of GH-treatment on the measured parameters was obtained on days 5 and 6 of the GH-treatment period and the first day after cessation of treatment. Even 7 days after GH-treatment some effect on milk yield and production of 6KPGF,, was observed. Analysis of variance was performed on the values obtained before (days l--3). during (days S-10) and after (days 14-16) GH-treatment. The results of the statistical analysis are shown in Table 1. Because of statistically significant interactions between treatment and goats, mean values and SDS are shown in Table 2. GH-treatment significantly increased milk yield in both goats as shown in Table 2, but more pronounced in the goat yielding 2 I milk daily (high yielding) than in the goat yielding 1 I milk daily (low yielding), ciz. 29.0 and 13.6%, respectively. The basal concentration of milk &KPGF,, was significantly lower in the high than in the low yielding goat (P < 0.05). GH-treatment increased the concentration of milk 6KPGF,, by 53.5% in the high yielding goat to a level comparable to that of the low yielding goat in which GH-treatment did not affect the concentration of milk 6-KPGF,, (cf. Table 2). It is interesting to note that the coefficient of variation for the concentration of 6-KPGF,, in milk was similar in the two goats and decreased, as a consequence of GH-treatment, from 15.9-I 1.4.2.7% in goat 1 and from 16.3-12.1-3.3% in goat 2 for the periods before, during and after GH-treatment, respectively. The total excretion of 6-KPGF,, was significantly (P < 0.001) greater in the high than in the low yielding goat. GH-treatment significantly increased the total production of 6-KPGF,, (Table 1) but, as shown in Table 2, GH-treatment caused an increase in 6-KPGF,, excretion in milk of 94.4% in the high yielding goat and 23.6% in the low yielding goat. The correlation coefficient (R’) of the concentration of milk 6-KPGF,, to milk yield was 0.35 in the high yielding goat and 0.003 in the low yielding goat.
DISCUSSION The blood flow of any organ may be regulated both by changes in local vasodilatation and in systemic blood pressure, the latter being a function of cardiac output and vascular resistance. Very little information is available about regulatory factors of the m~mary blood flow. This made us investigate prostacyclin (PGI,) as a possible local vasodilator in the mammary gland of lactating goats.
479
Prostacyclin excretion in milk
Goat 2
100
a0
3 ii? 60 s E A
40
20
0 12.345671)s
10
11
12
13
14
IS
16
10
I,
12
13
14
IS
16
~150
E & 4 4
100
30
0 123456788
TS
TE
days
Fig. 1. Daily milk yield (ml), concentration of milk 6-KPGF,, (pg/ml) and daily excretion of 6-KPGF,, (ng) in milk from a low yielding (goat I) and a high yielding (goat 2) goat in relation to growth hormone treatment (‘I’S= treatment start; TE = treatment end).
K. CHRISTENSEN et al.
480
Table 2. Dailv milk vield. concentration and dailv excretmn of 6-KPGF,. in the milk before (days l--3 in period I). durmg (days X- IO m ‘periods 2-3) and after (days 14- 16 in period 3) growth hormone treatmeni of goats in + SD) _ Goat 2 (high yielding)
Goat 1 (low yielding) GH-treatment Milk (ml) &KPGF,, (p&ml) 6-KPGF,, (ng)
Before
During
After
Pt
Before
During
After
Pt
865 i 61” 69 i 11 55 + 7
9x3 & 44b 70 2 X 68 + I3
94X*43ab 74 If-2 70 * 7
* NS NS
2070 i 2s* 43 i 7” 89 + 18’
2671 i 64b 66k8” 173*30b
2417 k 72’ 6122’ 152+7’
*** * **
tone-wav ANOVA. significant levels as in Table I. IX&rent superscripts within the same row and goat are signifi~ntly different iP S 0.05). NS z ~o~si8nifi~~~t (P > 0.05).
We have demonstrated a significant output of the prostacyclin metabolite &KPGF,, in the milk of healthy goats. The concentration of (I-KPGF,, was not necessarily positively correlated with milk yield (cf. Table 2). This gives rise to the following speculations on the possible function of PGI, as a vasodilator in the mammary gland during lactation. It is well established that the mammary blood flow increases with increasing milk yield during the normal lactation period (Linzell, 1974), and this appears to be related to increased activity of the mammary gland (active hyperaemia). Peaker (1980) proposed that changes in metabolic activity lead to changes in the production of vasodilators. The most potent mammary vasodilators so far investigated in u&o seem to be plasmakinins and bradykinin (Dhondt et al., 1973). Morita et al. (1984) proposed, on the basis of their in uitro experiments with bovine carotid aortic endothelial cells, that the vasodilator effect of kallikrein was mediated in part by prostacyclin production. The latter authors further concluded that bradykinin and kallidin also stimulated the release of prostacyclin, although the effects were far less than that of kallikrein. It is thus possible that PGI, is a potent vasodilator in the mammary gland also under normal physiological conditions, and perhaps involved in the regulation of mammary blood flow induced by changes in mammary metabolic activity during lactation. We actually found a greater total daily excretion of &KPGF,, in the milk of the high yielding goat in which mammary blood flow is also expected to be higher compared to that of the low yielding goat. Furthermore, GH-treatment increased the milk concentration of 6-KPGF,, in the high yielding goat, which also responded with a greater increase in milk yield than the low yielding goat (Table 2). It is well known that GH stimulates milk yield by increasing mammary metabolic activity and mammary blood flow. However, basal concentrations of $KPGF,, in the milk was signifi~ntly lower (P < 0.05) in the high than in the low yielding goat throughout the experiment, as shown in Table 2. It is therefore unlikely that local PGI, synthesis in mammary endothelial tissue, and hence 6-KPGF,, excretion in milk, is related to mammary metabolic activity alone. In that case, one would expect concentrations of 6-KPGF1, in the milk to be at least as high in the high as in the low yielding goat. Changes in PGI, production might also be related to changes in systemic blood pressure, especially to changes in arteriolar resistance. As discussed by Mepham et al. (1984) there is a possibility that GH-treatment increases cardiac output and thereby the blood supply of the mammary gland. The resulting increase in systemic pressure
would then depend on the resistance of the arteriolar smooth muscle cells which may be regulated by extrinsic or local control mechanisms. Apparently, the basal stress on the mammary blood vessels is greater in the low than in the high yielding goat (Table 2). which may be attributed to inherent differences in muscular elasticity or regulation of smooth muscle tone. This would explain why a greater mammary blood flow can be maintained in the high yielding goat despite the lower basal production of PGI,. Apparently, GH does not act directly on mammary tissue (McDowell and Hart, 1984). Johnsson and Hart (1986) proposed that circulating concentrations of somatomedins and other local factors under GH influence might function in local vascular regulation and membrane transport in the mammary gland of ruminants. Could PGI, be a mediator of GH in regulating vascular tone and hence blood flow in the mammary gland? We suggest that prostacyclin might be a potent vasodilator in the mammary gland of goats, not only as a consequence of increased mammary metabolic activity, but also as a consequence of increased vascular stress. We encourage further investigations on the possible role of prostacyclin in regulating mammary blood fiow and hence nutrient supply of the udder.
REFERENCES
Anderson K. L., Kindahl H.. Petroni A., Smith A. R. and Gustafsson B. K. (1985) Arachidonic acid metabolites in milk of cows during acute coliform mastitis. Am. J. Vet. Res. 46, 1573-.1577. Davis S. R. and Collier R. J. (1985) Mammary blood flow and regulation of substrate supply for milk synthesis. J. Dairy Sci. 68, 1041-1058. Dhondt G., Houvenaghel A., Peeters G. and Verschooten F. (1973) Influence of vasoactive hormones on blood flow through the mam~dry artery in lactating cows. Arch. int. P~armaco~~~. 204, 89-104. Freund R. J. and Littell R, C. (1981) SAS for Linear models. A guide to the ANOVA and GLM procedures. SAS Institute Inc., North Carolina, USA. Hamberg M. and Samuelsson B. (1973) Detection and isolation of an endoperoxide intermediate in prostaglandin synthesis. Proc. Narn. Arad. Sri. 70, 899-903. Higgins A. J. (1985) The biology. pathophysiology and control of eicosanoids in inflammation. Review paper. J. Vef. Pharmacol. Therap. 8, I-15. Ingerman-Wojenski C., Silver M. J., Smith J. B. and Macarak E. (1981) Bovine endothelial cells in culture produce thromboxane as well as prostacyclin. J. riin. Invest. 67, 1292-1296. Johnsson 1. D. and Hart I. C. (1986) Manipulation of milk yield with growth hormone. Recent Admnces in Animal Nutrition, pp. 105-123. Butterworths, London.
Prostacyclin Linzell J. L. (1974) Mammary blood flow and methods of identifying and measuring precursors of milk. In Lacration Vol. 1, (Edited by Larson B. L. and Smith V. R.), pp. 143-225. Academic Press, New York. Manns J. G. (1975) The excretion of prostaglandin F,, in milk of cows. Prostaglandins 9, 463474. Maule-Walker F. M. and Peaker M. (1981) Prostaglandins and lactation. Acfa wf. Scund. Suppl. 77, 2999310. McDowell G. H. and Hart I. C. (1984) Responses to infusion of growth hormone into the mammary-arteries of lactatine sheeu. Can. J. Anim. Sci. 64, 306-307. McGiff J.-C., Spokas E. G. and Wong P. Y.-K. (1981) Prostaglandin mechanisms in blood pressure regulation and hypertension. The PG System. NATO Sci. A$ Die. A36, 2355246. Mepham T. B., Lawrence S. E., Peters A. R. and Hart I. C. (1984) Effects of exogeneous growth hormone on mammary function in lactating goats. Horm. metaboi. Res. 16, 248-253.
excretion
in milk
481
Moncada S. and Vane J. R. (1979) The role of prostacyclin in vascular tissue. Fed. Proc. 38, 6671. Morita I., Kanayasu T. and Murota S.-I. (1984) Kallikrein stimulates prostacyclin production in bovine vascular endothelial cells. Biochem. biophys. Acta 792, 304309. Pace-Asciak C. (1976) Isolation, structure and biosynthesis of 6-keto-prostaglandin F,, in rat stomach. J. Am. Chem. Sci. 98, 2348-2349. Peaker M. (1980) The effect of raised intramammary pressure on mammary function in the goat in relation to the cessation of lactation. J. Physio/. 301, 415428. Powell W. S. (1980) Rapid extraction of oxygenated metabolites of arachidonic-acid from biological samples using octadecvlsilvl silica. Prostazlundins 20. 947-957. Skidgel R: A.-and Printz M. k. (1978) PGIz production by rat blood vessels: diminished prostacyclin formation in veins compared to arteries. Prostaglundins 16, 1 -16. Stryer L. (1975) Biochemistry, pp. 821-822. W. H. Freeman & Co.. San Francisco.
1989
0
0300-9629/89 S3.00 + 0.00 1989 Pergamon Press plc
THE EXCRETION OF PROSTACYCLIN (PGI,) IN MILK AND ITS POSSIBLE ROLE AS A VASODILATOR IN THE MAMMARY GLAND OF GOATS K.
CHRISTENSEN,*
M.
0.
NIELSEN
and N. JmLwt
The Royal Veterinary and Agricultural University, Department of Animal Physiology and Biochemistry; and tDepartment of Surgery, Biilowsvej 13, DK-1870 Frederiksberg C, Denmark (Received
22 November
1988)
Abstract--l.
Prostacyclin production in mammary gland of two lactating goats measured as the excretion in milk of 6-ketoprostaglandin F,, (6-KPGF,,) was followed for 16 days before, during and after exogenous administration of recombinant bovine growth hormone (GH). 2. 6-KPGF,, was detected in all milk samples in concentrations ranging from 32-99pg/ml milk independently of the time of sampling. 3. GH-treatment significantly increased milk yield, the concentration and excretion of 6-KPGF,, in milk. 4. The concentration of milk 6-KPGF,, was positively correlated with milk yield in the high (R* = 0.35). but not in the low yielding goat (Rz = 0.003). 5. The possible role of prostacyclin as a local vasodilator in the mammary gland of goats is discussed.
INTRODUCMON
Local and extra mammary vasoactive factors controlling mammary blood flow have been reviewed and discussed comprehensively (Dhondt et al., 1973; Linzell, 1974; Davis and Collier, 1985). Prostaglandins and related compounds (the eicosanoids) are involved in many different physiological and pathophysiological functions. Prostaglandins may be involved in controlling mammary vascular activity. Prostaglandin F,, (PGF,,) is produced and metabolized by the goat (Maule-Walker and Peaker, 1981) and cow mammary gland (Manns, 1975; Anderson et al., 1985) and is considered a locally active inhibitor of mammary function pre-partum and in late lactation in the goat (Maule-Walker and Peaker, 1981). The involvement of the eicosanoids in inflammatory conditions have been investigated in the last 15 years, and it is now apparent that the eicosanoids are fundamental to the inflammatory process as mediators (Higgins, 1985). Thromboxane B, (TBX,) the stable metabolite of TXA,, was found in the milk of healthy cows, and both PGF,, and TXB, were found in elevated concentrations in the milk of cows with acute coliform mastitis (Anderson et al., 1985). Prostacyclin (PGI,) is the major prostanoid synthesized from arachidonic acid by vascular tissue and is known to bc a potent mediator producing vasoand bronchiedilatation and inhibition of platelet aggregation (Moncada and Vane, 1979). It is more effective in arteries than in veins (Skidgel and Printz, 1978). *Present address and address for correspondence: National Institute of Animal Science, Department of Animal Physiology and Biochemistry, Forsogsanheg Foulum, PO Box 39, DK-8833 0rum Sdrl, Denmark. Telephone: 06-65-2500. 477
It is known that bovine aortic endothelial and smooth muscle cells produce PGIr (IngermanWojenski et al., 1981), but it is not known if a production and/or metabolism in the mammary gland of ruminants is taking place or if PGI, plays a role as a local vasodilator in the mammary gland in situ. However, it has been found in cultured endothelial cells from bovine carotid aorta that the vasodilator effect of kallikrein is mediated in part by prostacyclin production (Morita et al., 1984) and kallikrein has been shown to cause vasodilation in the udder of small ruminants and cows (Dhondt et al., 1973). It has been suggested that a major vasodepressor/ vasodilator system, “the kallikrein-kinin-prostaglandin system”, antagonized the effect of the blood pressure elevation system, “the adrenergic nervousrenin-angiotensin-aldosteron complex” (McGiff et al., 1981). Another prostaglandin known to have vasodilator effects (PGEr) was only produced in small amounts compared to the production of PGI, in cultured bovine carotid aortic endothelial cells (Morita et al., 1984). The synthesis of the eicosanoids is activated through a variety of stimuli including physical, chemical and hormonal influences (Hamberg and Samuelsson, 1973). In different physiological pathways, prostaglandins seem to modulate the action of hormones rather than act as hormones themselves (Stryer, 1975). The purpose of the present experiment was to investigate whether PGI,, measured as its stable metabolite 6-keto-prostaglandin F,, (6-KPGF,,), was excreted in the milk of healthy goats, and if the excretion of milk 6-KPGF,, was correlated to milk yield. As exogenous growth hormone (GH) or somatotropin is known to stimulate mammary blood flow and milk yield in lactating goats (Mepham et al., 1984), this hormone was injected into the goats in order to detect a possible effect on the excretion of 6-KPGF,, in milk.
K. CHRISTENSENet
478 MATERIALS AND METHODS Animals
Two Norwegian lactating goats from the herd of the Institute were used in the present studies. The goats were in 25th-27th week of their second lactation period and yielded 1 (Goat 1) or 2 1(Goat 2) milk daily, in the following called low and high yielding goats, respectively. They were milked twice a day at 8a.m. and 3p.m. and the milk yield was recorded. They were housed in individual stalls and fed concentrates according to milk yield jn two equal portions at the time of milking and received hay ad Ii&turn. They had free access to water. The udder was healthy in both goats. Experimental
procedures
The experiment was divided into three periods: a pre-treatment control period (period I) lasting three days, a treatment period (period 2) lasting 6 days and a post-treatment control period (period 3) lasting 7 days. In the treatment period the experimental animals were injected daily with 10 IU recombinant bovine growth hormone (0.88 IU/mg determined by radioimmunoassay. Lot. No. 58920, Lilly Industries Ltd., Windlesham, UK). The hormone was solubilized in 4ml 0.9% NaCl containing 0.025 M NaHCO, and 0.025 M Na,CG, just prior to the injection given subcutanously in the shoulder region at approximately 9 a.m. Analyricat
procedures
Milk samples of IOml were collected from the morning and afternoon milking, respectively, centrifuged immediately at 17OOg for 15 min at 4°C to remove cells and separate the fat from milk plasma. The fat free milk plasma (2 x 3 ml) was frozen at -20°C until analysis was performed within 3 weeks. Prostacyclin metabolite formation was followed by spontaneous breakdown to the stable 6-keto-PGF,, (PaceAsciak, 1976). The latter was determined in milk and used to reflect milk concentrations of PGI,. Milk 6keto-PGF,, (&KPGF,,) con~ntrations were measured by means of a commercial RIA kit (Amersham TRK code 790). Prior to RIA, milk plasma extraction was performed at SepPak Cl8 columns (Waters). Two ml milk plasma were acidified to pH 3 with glacial acetic acid and eluted on the activated columns with a mixture of IOml water, torn1 ethanol (15%). lOm1 petroleum ether and 4ml methyl formate as previously described (Powell, 1980). After evaporation the concentrate was diluted in 1ml assay buffer. Mean recovery was 73.4%. All analyses were performed in duplicate. Statistical
analysis
Analysis of variance (ANOVA) and analysis of regression (GLM) were performed using SAS as described by Freund and Littell (1981). The two-way ANOVA comprised the independent variables treatment, goat and treatment x goat interactions. One-way ANOVA was performed for each goat. Student’s f-test was used for statistical comparisons. RESULTS
6-KPGF,, was detected in all milk samples in concentrations ranging from 32-99 pg/ml milk. The concentration of 6-KPGF,, in the milk from the morning and the afternoon milkings (N = 30) were 58.0 f 2.32 and 62.6 + 2.61 pg/ml milk, respectively, the dtfference being non-significant (P > 0.05). The relative variations in the concentration of 6-KPGF,, expressed as the coefficient of variation (CV,% = SD/mean x 100) was similar in the morning and afternoon milk: 22.7 and 23.6% respectively. Therefore, the mean concentration of 6-KPGF,, in the morning and afternoon milk was used for further calculations.
al.
Table I. Analysts of variance of independent vanablea in rclatwn to growth hormone treatment of two lactating goats (F-values and Pr > F. l = P < 0.05; ** = P < 0.01; *** = P < 0.001) Source of variation Treatment Goat Treatment
rq ‘-. 2
I x goat
2
Milk yield daily 46.66”* 2257*21.05***
Concentration of &KPGF,, 3.92* 12.73** 2.40
Excretion of O-KPGF,, 15.10*** 94.05*** i.ss*
The daily milk yield, concentration of milk 6-KPGF,, and total daily excretion of 6-KPGF,, in milk before, during and after GH-treatment of the two goats are shown graphically in Fig. 1 for individual days. The greatest response of GH-treatment on the measured parameters was obtained on days 5 and 6 of the GH-treatment period and the first day after cessation of treatment. Even 7 days after GH-treatment some effect on milk yield and production of 6KPGF,, was observed. Analysis of variance was performed on the values obtained before (days l--3). during (days S-10) and after (days 14-16) GH-treatment. The results of the statistical analysis are shown in Table 1. Because of statistically significant interactions between treatment and goats, mean values and SDS are shown in Table 2. GH-treatment significantly increased milk yield in both goats as shown in Table 2, but more pronounced in the goat yielding 2 I milk daily (high yielding) than in the goat yielding 1 I milk daily (low yielding), ciz. 29.0 and 13.6%, respectively. The basal concentration of milk &KPGF,, was significantly lower in the high than in the low yielding goat (P < 0.05). GH-treatment increased the concentration of milk 6KPGF,, by 53.5% in the high yielding goat to a level comparable to that of the low yielding goat in which GH-treatment did not affect the concentration of milk 6-KPGF,, (cf. Table 2). It is interesting to note that the coefficient of variation for the concentration of 6-KPGF,, in milk was similar in the two goats and decreased, as a consequence of GH-treatment, from 15.9-I 1.4.2.7% in goat 1 and from 16.3-12.1-3.3% in goat 2 for the periods before, during and after GH-treatment, respectively. The total excretion of 6-KPGF,, was significantly (P < 0.001) greater in the high than in the low yielding goat. GH-treatment significantly increased the total production of 6-KPGF,, (Table 1) but, as shown in Table 2, GH-treatment caused an increase in 6-KPGF,, excretion in milk of 94.4% in the high yielding goat and 23.6% in the low yielding goat. The correlation coefficient (R’) of the concentration of milk 6-KPGF,, to milk yield was 0.35 in the high yielding goat and 0.003 in the low yielding goat.
DISCUSSION The blood flow of any organ may be regulated both by changes in local vasodilatation and in systemic blood pressure, the latter being a function of cardiac output and vascular resistance. Very little information is available about regulatory factors of the m~mary blood flow. This made us investigate prostacyclin (PGI,) as a possible local vasodilator in the mammary gland of lactating goats.
479
Prostacyclin excretion in milk
Goat 2
100
a0
3 ii? 60 s E A
40
20
0 12.345671)s
10
11
12
13
14
IS
16
10
I,
12
13
14
IS
16
~150
E & 4 4
100
30
0 123456788
TS
TE
days
Fig. 1. Daily milk yield (ml), concentration of milk 6-KPGF,, (pg/ml) and daily excretion of 6-KPGF,, (ng) in milk from a low yielding (goat I) and a high yielding (goat 2) goat in relation to growth hormone treatment (‘I’S= treatment start; TE = treatment end).
K. CHRISTENSEN et al.
480
Table 2. Dailv milk vield. concentration and dailv excretmn of 6-KPGF,. in the milk before (days l--3 in period I). durmg (days X- IO m ‘periods 2-3) and after (days 14- 16 in period 3) growth hormone treatmeni of goats in + SD) _ Goat 2 (high yielding)
Goat 1 (low yielding) GH-treatment Milk (ml) &KPGF,, (p&ml) 6-KPGF,, (ng)
Before
During
After
Pt
Before
During
After
Pt
865 i 61” 69 i 11 55 + 7
9x3 & 44b 70 2 X 68 + I3
94X*43ab 74 If-2 70 * 7
* NS NS
2070 i 2s* 43 i 7” 89 + 18’
2671 i 64b 66k8” 173*30b
2417 k 72’ 6122’ 152+7’
*** * **
tone-wav ANOVA. significant levels as in Table I. IX&rent superscripts within the same row and goat are signifi~ntly different iP S 0.05). NS z ~o~si8nifi~~~t (P > 0.05).
We have demonstrated a significant output of the prostacyclin metabolite &KPGF,, in the milk of healthy goats. The concentration of (I-KPGF,, was not necessarily positively correlated with milk yield (cf. Table 2). This gives rise to the following speculations on the possible function of PGI, as a vasodilator in the mammary gland during lactation. It is well established that the mammary blood flow increases with increasing milk yield during the normal lactation period (Linzell, 1974), and this appears to be related to increased activity of the mammary gland (active hyperaemia). Peaker (1980) proposed that changes in metabolic activity lead to changes in the production of vasodilators. The most potent mammary vasodilators so far investigated in u&o seem to be plasmakinins and bradykinin (Dhondt et al., 1973). Morita et al. (1984) proposed, on the basis of their in uitro experiments with bovine carotid aortic endothelial cells, that the vasodilator effect of kallikrein was mediated in part by prostacyclin production. The latter authors further concluded that bradykinin and kallidin also stimulated the release of prostacyclin, although the effects were far less than that of kallikrein. It is thus possible that PGI, is a potent vasodilator in the mammary gland also under normal physiological conditions, and perhaps involved in the regulation of mammary blood flow induced by changes in mammary metabolic activity during lactation. We actually found a greater total daily excretion of &KPGF,, in the milk of the high yielding goat in which mammary blood flow is also expected to be higher compared to that of the low yielding goat. Furthermore, GH-treatment increased the milk concentration of 6-KPGF,, in the high yielding goat, which also responded with a greater increase in milk yield than the low yielding goat (Table 2). It is well known that GH stimulates milk yield by increasing mammary metabolic activity and mammary blood flow. However, basal concentrations of $KPGF,, in the milk was signifi~ntly lower (P < 0.05) in the high than in the low yielding goat throughout the experiment, as shown in Table 2. It is therefore unlikely that local PGI, synthesis in mammary endothelial tissue, and hence 6-KPGF,, excretion in milk, is related to mammary metabolic activity alone. In that case, one would expect concentrations of 6-KPGF1, in the milk to be at least as high in the high as in the low yielding goat. Changes in PGI, production might also be related to changes in systemic blood pressure, especially to changes in arteriolar resistance. As discussed by Mepham et al. (1984) there is a possibility that GH-treatment increases cardiac output and thereby the blood supply of the mammary gland. The resulting increase in systemic pressure
would then depend on the resistance of the arteriolar smooth muscle cells which may be regulated by extrinsic or local control mechanisms. Apparently, the basal stress on the mammary blood vessels is greater in the low than in the high yielding goat (Table 2). which may be attributed to inherent differences in muscular elasticity or regulation of smooth muscle tone. This would explain why a greater mammary blood flow can be maintained in the high yielding goat despite the lower basal production of PGI,. Apparently, GH does not act directly on mammary tissue (McDowell and Hart, 1984). Johnsson and Hart (1986) proposed that circulating concentrations of somatomedins and other local factors under GH influence might function in local vascular regulation and membrane transport in the mammary gland of ruminants. Could PGI, be a mediator of GH in regulating vascular tone and hence blood flow in the mammary gland? We suggest that prostacyclin might be a potent vasodilator in the mammary gland of goats, not only as a consequence of increased mammary metabolic activity, but also as a consequence of increased vascular stress. We encourage further investigations on the possible role of prostacyclin in regulating mammary blood fiow and hence nutrient supply of the udder.
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