0O13-7227/79/1042-0372$02.0O Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 2 Printed in U.S.A.
The Secretion Rate of Prolactin in the Rat during Suckling and Its Metabolic Clearance Rate after Increasing Intervals of Nonsuckling* C. E. GROSVENOR, F. MENA,f AND N. S. WHITWORTH^ Department of Physiology and Biophysics, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163
ABSTRACT. The amount of pituitary PRL depleted by suckling is known to increase in direct relationship to the length of the previous nonsuckling interval. Therefore, we examined the extent to which the nonsuckling interval influenced the plasma concentration of PRL attained during prolonged suckling, the metabolic clearance rate of continuously infused rat PRL, and the secretion rate of PRL into the circulation. Neither the plateau level of PRL attained in the plasma during suckling nor the clearance rate of infused PRL were altered by increasing the nonsuckling interval from 2 to 4 to 8 h. The secretion rates also were similar (554, 530, and 537 ng/min for the 2-, 4-, and 8-h nonsuckled groups, respectively). However, the number of minutes the steady secretion rate of PRL into the plasma could be maintained varied in direct relationship to the length of the nonsuckling interval. The steady secretion waned after 30 min of suckling in die 2-h nonsuckled group, after 60 min in the 4-h
T
HAT suckling stimulates the adenohypophysis to release PRL is evident both from a reduction in pituitary levels and a rise in plasma levels of the hormone (1-3). Little information, however, is available concerning the quantitative relationships between the two (3) and whether the various factors which influence the amount of PRL depleted by the pituitary (2) also affect its level in the circulation. One such factor is the length of the nonsuckling interval, which has been shown to be directly related to the amount of PRL depleted from the pituitary during suckling (2, 4-6). In the present study, the suckling-induced rise in plasma PRL concentration, the metabolic clearance rate (MCR), and the secretion rate (SR) of PRL have been analyzed after various intervals of time before suckling.
nonsuckled group, and did not subside at all during the 90-min suckling period in the 8-h group. The total amount of PRL secreted into the plasma approximated 17, 32, and 48 jug for the three groups, values which are quantitatively similar to amounts depleted from the pituitary during the first few minutes of suckling after similar nonsuckling periods. To reconcile the rapid and extensive depletion of PRL from the pituitary with the steady, potentially long term output of much smaller amounts of PRL into the plasma, we postulate that the first few minutes of suckling prepares PRL for release into the circulation (pituitary depletion stage); the amount so prepared depends upon the length of the previous nonsuckling interval. Once prepared, the PRL is secreted into the circulation in a steady minute by minute fashion, independent of the previous nonsuckling interval. This steady secretion can continue until the pool of prepared PRL is exhausted. (Endocrinology 104: 372, 1979)
Materials and Methods Primiparous lactating rats of the Holtzman strain, each with six pups, were maintained in individual cages in a room at 23-25 C with alternating 14-h light: 10-h darkness. Food and water were available at all times. Each rat (weight range, 260-320 g) was tested on days 13-14 of lactation. Plasma PRL concentration during suckling
Received June 26,1978. Address all correspondence and requests for reprints to: Dr. C. E. Grosvenor, Department of Physiology and Biophysics, University of Tennessee Center for the Health Sciences, 894 Union Avenue (NA 427), Memphis, Tennessee 38163. • This work was supported by USPHS Grant HD-04358 (C.E.G.) and PLAMIRH 96.182.1.78 (F. M.). f Present address: Institute of Biomedical Investigations, University of Mexico, Mexico City. | Present address: Department of Ob-Gyn, University of Mississippi, Jackson, Mississippi.
Pups were separated from their mothers for 2,4, or 8 h before suckling. At the end of these periods, the pups were returned to their mothers, and suckling was allowed for 90 min (five to seven rats per group). To insure uniform and sustained teat stimulation throughout the 90-min suckling period, foster litters which were 14 days of age and previously isolated from their mothers for 8 h were inserted at 30 and 60 min. Blood (0.35 ml each sample) was obtained before and at intervals after the onset of suckling from each unrestrained conscious mother through a right atrial catheter implanted 2-3 days previously (see Ref. 7 for details). The catheter was flushed with heparinized saline (0.35 ml) after each blood sample was taken. Plasma PRL concentration after iu infusion of rat PRL On day 14 postpartum, mother and pups were separated for 2, 4, and 8 h. At the end of these periods, each mother was anesthetized with urethane and implanted in the right atrium
372
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373
SECRETION AND MCR OF PRL IN RAT with a double lumen catheter. Each rat was then attached to an infusion pump (Harvard Apparatus model 600-900) and rat PRL (NIAMDD preparation, B-l; 7 IU/mg dissolved in 0.9% NaCl) was continuously infused via one lumen of the intraatrial catheter at a dose of 738 ng/min. This dose is equivalent to 472 ng RP-1 PRL (11 IU/mg)/min. The infusion volume was 5.9 jiil/min. At intervals during the infusion, the infusion pump was stopped for 15 sec. This was to ensure uniform distribution of the infused PRL throughout the circulation of the rat before the withdrawal of each blood sample (0.35 ml) through the other channel of the double lumen catheter. The blood remaining in the catheter after each blood sample had been taken was flushed back into the rat with 0.35 ml heparinized saline. The delivery volume of the infusion pump at the setting used was checked periodically by measuring the volume of saline delivered over a 2-h period from 1-ml syringes and through catheters identical to those used during the infusion experiments. The volume delivered did not measurably differ from the rate of 5.9 jul/min at setting 5 on the pump when the results of the various trials were compared. RIA of rat PRL The plasma was separated from each blood sample and stored at -20 C until assayed for PRL by RIA. The concentration of immunoreactive rat PRL was determined in duplicate 25-jul samples of each PRL solution infused and in duplicate 25jttl plasma samples using the NIAMDD rat PRL RIA system (see Ref. 8 for procedures and validation data). All PRL values are expressed as nanogram equivalents of the RP-1 standard. Samples of the B-l PRL solutions which were infused were obtained at the start of each experiment. Immunoassay of these solutions yielded values which were 113% and 90% of their expected concentrations.
Results The plasma PRL concentration rose rapidly during suckling to a maximum of 242, 255, and 262 ng/ml above presuckled levels within the first 30 min of suckling after 2, 4, and 8 h, respectively, of nonsuckling (Fig. 1 and Table 1). Plasma PRL levels in the 8-h nonsuckled group were sustained throughout the entire 90 min of suckling, but declined significantly (P < 0.05) in the 2-h group at 60 min and in the 4-h group at 90 min (Fig. 1). The data were statistically evaluated by trend analysis and Duncan's multiple range test. The plasma levels in the 2-, 4-, and 8-h nonsuckled groups also rose quickly and reached stable concentrations of 206, 227, and 230 ng/ml, respectively, above preinfusion levels within 20 min of continuous iv infusion of 472 ng RP1 rat PRL/min (Fig. 2). The profile of the rise and attainment of an equilibrium concentration of PRL in the plasma of rats which were suckled was similar to that of rats infused with rat PRL (compare Figs. 1 and 2). The MCR for each group was computed from the stable concentration of plasma PRL attained during continuous infusion of rat PRL divided into the infusion rate of 472 ng/min. This yielded mean MCRs of 2.29, 2.08, and 2.05 ml/min for the 2-, 4-, and 8-h nonsuckled groups, respectively (Table 1). The SR of PRL during suckling was then calculated for each group from the mean stable
MCR
300
The MCR of immunoreactive rat PRL was calculated as follows (9):
8h N-S I5I
MCR (ml/min) = Infusion dose (ng/min) Increment in plasma concentration at equilibrium (ng/ml) We showed previously (8) that the plasma concentration of immunoreactive rat PRL in the rat reached equilibrium after approximately 20 min of continuous infusion. The plasma PRL concentration used in the computation of MCR for each rat was the average of the two or three individual values obtained during the equilibrium period minus the PRL concentration in the plasma before the start of PRL infusion.
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1
New litters Inserted
SR of PRL From the mean MCR together with the mean plateaued plasma concentration of PRL during suckling minus the PRL concentration before suckling, we could calculate the mean SR of PRL during suckling for each group of rats as follows: SR (ng/min) = MCR (ml/min) X plasma cone. (ng/ml) at steady state during suckling
60
10 20 30 Min.
of
90
Suckling
FIG. 1. Influence of increasing the length of the nonsuckling interval (N-S) from 2 to 4 to 8 h on the suckling-induced rise in plasma PRL concentration. Values are means ± SEM; each has subtracted from it the PRL concentration in the plasma at zero time. Number in parentheses refers to the number of rats.
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Endo • 1979 Vol 104 • No 2
GROSVENOR, MENA, AND WHITWORTH
TABLE 1. Estimate of SR of PRL from MCR and equilibrium concentration of PRL during suckling after 2, 4, or 8 h of nonsuckling in the rat h of Nonsuckling
Equilibrium PRL levels during PRL infu- MCR (ml/min; mean ± SE) sion (ng/ml; mean ± SE)°
2 4 8
206 ± 19.2 227 ± 23.4 230 ± 23.8
Equilibrium PRL levels during suckling = (ng/ml; mean ± SE) a
2.29 ± 0.20 2.08 ± 0.22 2.05 ± 0.22
242 ± 27.1 255 ± 20.6 262 ± 22.1
SR (ng/min)
min of Steady se- == cretion
X
554 530 537
Total secretion into plasma (fig) 17 32 48
30 60 90
Plasma concentrations of PRL during suckling and MCRs are the same data as in Figs. 1 and 2. ° Each value has subtracted from it the plasma concentration of PRL before infusion of PRL or suckling.
concentration of PRL which was reached within the first 30 min of suckling x the average MCR. This yielded SRs of PRL during suckling of 554, 530, and 537 ng/min for rats previously not suckled for 2, 4, and 8 h (Table 1). We did not attempt to quantitate the SR of PRL in the 2- and 4-h previously nonsuckled groups during the periods of suckling where the plasma concentration declined from the previously attained equilibrium level. From the number of minutes the SR of PRL was sustained, it was possible to estimate the total output of PRL from the pituitary into the plasma. This yield outputs of 17, 32, and 48 /xg PRL during continuous suckling in rats previously not suckled for 2, 4, and 8 h, respectively (Table 1). These estimates probably are slightly lower than actual levels because the SR during the period of decline in plasma PRL concentration was not estimated in the 2- and 4-h groups, and in the 8-h group it is not known how much longer than 90 min the steady secretion of PRL would have been maintained. Discussion Yanai and Nagasawa (10) reported that plasma PRL concentrations rose at similar rates during suckling in rats previously not suckled for 8, 16, or 24 h. Similar results were obtained by Mena etal. (11) using nonsuckling periods of 8 and 16 h. The suckling-induced pattern of PRL concentration in plasma obtained by several other investigators after nonsuckling periods ranging from 6.5-12.5 h reveals a striking similarity when plotted on the same scale (8). In the present study, the patterns of plasma PRL concentrations during 30 min of suckling were virtually identical regardless of whether the previous nonsuckling period was 2, 4, or 8 h in length. Thus, it would appear that the suckling-induced pattern of change in plasma PRL concentrations in the rat is quite similar, though the preceding nonsuckling period may vary widely in length. In each of these studies, including the present, the concentration of PRL increased rapidly in the plasma to a maximum within 20-30 min of suckling. The concentration thereafter tended to plateau. The results in the present study also indicate that the clearance rate of PRL in the lactating rat is not altered by
300
I
I I
-
1[ i
200
100
'
4h N-S I7I 8h N-S |5| 2h N-SI9I
472ngRatPrl/min
15
'
30
45
60
Minutes FIG. 2. Influence of increasing the length of the nonsuckling interval (N-S) from 2 to 4 to 8 h on the plasma concentration of PRL resulting from continuous infusion of rat PRL into the circulation. Values are means ± SEM; each has subtracted from it the PRL concentration in the plasma at zero time. Number in parentheses refer to the number of rats.
lengthening nonsuckling intervals (up to 8 h in length). In each of the nonsuckled groups tested, the rate of increase and the equilibrium concentration of PRL attained in the plasma after continuous iv infusion of rat PRL clearly resembled those which occurred during suckling. This similarity corroborates our earlier observations (8, 12) and indicates that PRL is released into the circulation during suckling in small (500-600 ng/min) amounts in a steady minute by minute fashion rather than in a large amount over a short span of time. The maintenance of this steady secretion is dependent upon sustained suckling; thus, plasma levels fall once suckling is interrupted (8).
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SECRETION AND MCR OF PRL IN RAT In calculating the SRs we used clearance data obtained from urethane-anesthetized rats and plasma PRL levels obtained during suckling in conscious rats. The use of urethane anesthesia to immobilize the rats after the various nonsuckling periods probably exerted a slight depressing influence on the clearance rate and hence, the SRs of PRL in. the conscious lactating rat may actually be slightly higher than reported. This supposition stems from recent experiments on which we use the same method as in the present study to determine the MCR of PRL in rats during mid (days 12-15) and late (days 20-23) lactation. The clearance initially was determined while they were conscious then again after they had been anesthetized with urethane. The MCR was depressed an average of about 20% in rats on both days as a result of the anesthesia, but the difference was not statistically significant (Grosvenor, C. E., and N. S. Whitworth, manuscript in preparation). From the data available thus far it is becoming clear that the secretion of PRL into the plasma during suckling differs in two important respects from the depletion of PRL from the pituitary during suckling. Firstly, the SR into plasma during suckling, computed from the equilibrium plasma PRL concentration during suckling multiplied by the M!CR, is unaffected by the length of the previous nonsuckling period, whereas the amount of PRL depleted from the pituitary ranges upward from a nondetectable change after 2 h to 80-95% after nonsuckling periods of 10-12 h (2, 4-6). Secondly, the secretion into plasma occurs steadily and in small amounts (Refs. 8,12, and as shown in the present study), whereas depletion of PRL from the pituitary occurs rapidly and in large amounts. As little as 2 min of suckling is nearly as effective as 30-90 min of suckling in provoking maximal depletion (13, 14). Electronmicroscopic examination of the rat pituitary reveals marked extrusion of PRL granules after 5 min of suckling (15), and after only 1 min of suckling, the rough endoplasmic reticulum of the PRLsecreting cell in the pituitary has collapsed, suggesting that its contents have been discharged (16). We observed in the present paper that it is the number of minutes the steady secretion of PRL into plasma can be maintained by suckling rather than the SR itself which is directly related to the previous nonsuckling interval. Thus, the steady secretion waned after 30 min in the 2-h nonsuckled group and after 60 min in the 4-h group. The gradual nature of the reduction in plasma concentration in the 2- and 4-h groups suggests that the secretion of PRL into the plasma was not abruptly terminated but rather only subsided. The disappearance rate of PRL in these groups was approximately that of a ti/2 = 75 min, in contrast to a ti/2 of 6-8 min after infusion of PRL (12). The SR of PRL in the 8-h nonsuckled group was sustained throughout the 90 min that suckling was
375
applied, though it too would be expected to decline with even longer suckling episodes. The total amount of PRL secreted into the plasma, therefore, approximated 17, 32, and 48 jug (187, 354, and 528 mU) after 2, 4, and 8 h of nonsuckling, respectively. These amounts correspond favorably with the 308 mU (28 jug in terms of RP-1 standard) depleted after 6 h of nonsuckling (14) and the 530 mU depleted after 10 h of nonsuckling (3). They also correspond well with those of Amenomori et al. (17) who reported that about 60 /xg were depleted after 12 h of nonsuckling, though the potency of the reference standard was not stated in their study. Thus, with the use of prolonged continuous suckling, effected in our laboratory by inserting hungry foster pups at 30-min intervals, a fairly good quantitative relationship seems to exist between the greater depletion of immunoreactive PRL as the nonsuckling period increases (3, 14, 17) and the total amount that is secreted into the circulation. Our data also indicate that of the large amount of immunoreactive PRL depleted during the first few minutes of suckling only a small fraction appears in the circulation as RIAdetectable PRL during the same time, but that with longer periods of suckling a better quantitative relationship is achieved. Though quantitatively related, the rapid depletion of PRL within the pituitary during suckling bears little resemblance to the steady, potentially long term secretion of PRL into the circulation provoked by the same stimulus. To reconcile these differences, we postulate that the first few minutes of suckling prepares PRL for release into the circulation (pituitary depletion stage); the amount so prepared depends upon the length of the previous nonsuckling interval. Once prepared, the PRL is secreted into the circulation in a steady minute by minute fashion, independent of the previous nonsuckling interval. This steady secretion can continue until the pool of prepared PRL is exhausted. This postulation is similar to the model of pituitary cell function originally proposed by Nicoll (18). In his model, the depletion of PRL precedes release but not all of the depleted PRL necessarily is released into the circulation. The nature of the depleted or prepared PRL is unknown at the present time. One possibility is that it may be rapidly transformed after its depletion into a polymer or it may become bound to a carrier protein within the pituitary so it cannot be readily detected by RIA. The PRL secreted into the circulation then may represent uncoupled or depolymerized PRL which, once again, is detectable by RIA. Forms of PRL with different bioassay:RIA ratios have been shown to exist within and to be secreted by the rat pituitary (19). Acknowledgments We are grateful to the NIAMDD Rat Pituitary Hormone Distribu-
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GROSVENOR, MENA, AND WHITWORTH
376
tion Program for the gift of rat PRL, and to Tanya McGee and Lee Brinkley for expert technical assistance.
References 1. Neill, J. D., Prolactin: its secretion and control, In Handbook of Physiology, vol. 4, pt. 2, 1974, p. 469. 2. Grosvenor, C. E., and F. Mena, Effect of suckling upon the secretion and release of prolactin from the pituitary of the lactating rat, Bien Symp Anim Reprod 32: 115, 1971. 3. Nicoll, C. S., F. Mena, C. W. Nichols, Jr., S. H. Green, M. Tai, and S. M. Russell, Analysis of suckling-induced changes in adenohypophyseal prolactin concentration in the lactating rat by three assay methods, Ada Endocrinol (Kbh) 83: 512, 1976. 4. Sar, M., and J. Meites, Effects of suckling on pituitary release of prolactin, GH, and TSH in postpartum lacting rats, NeuroendocrinologyA: 25, 1969. 5. Convey, E. M., and R. P. Reece, Restoration of pituitary lactogen released in response to suckling, Proc Soc Exp Biol Med 131: 543, 1969. 6. Grosvenor, C. E., and C. W. Turner, Pituitary lactogenic hormone concentration and milk secretion in lactating rats, Endocrinology 63: 535, 1958. 7. Grosvenor, C. E., N. S. Whitworth, and F. Mena, Milk secretory response of the conscious lactating rat following intravenous injections of rat prolactin, J Dairy Sci 58: 1803, 1975. 8. Grosvenor, C. E., and N. S. Whitworth, Evidence for a steady rate of secretion of prolactin following suckling in the rat, J Dairy Sci 57: 900, 1974. 9. Tait, J. F., Review: the use of isotopic steroids for the measurement
10. 11. 12. 13.
14. 15. 16. 17. 18. 19.
Endo • 1979 Vol 104 • No 2
of production rates in vivo, J Clin Endocrinol Metab 25: 1285, 1963. Yanai, R., and H. Nagasawa, Effect of non-suckling period on pituitary prolactin secretory activity in rats, Horm Res 4:169,1973. Mena, F., A. Enjalbert, L. Carbonell, M: Priam, and C. Kordon, Effect of suckling on plasma prolactin and hypothalamic monoamine levels in the rat, Endocrinology 99: 445, 1976. Grosvenor, C. E., and N. S. Whitworth, Incorporation of rat prolactin into rat milk in vivo and in vitro, J Endocrinol 70: 1, 1976. Grosvenor, C. E., V. Mena, and D. A. Schaefgen, Effect of nonsuckling interval and duration of suckling in the suckling-induced fall in pituitary prolactin concentration in the rat, Endocrinology 81: 449, 1967. Subramanian, M. G., and R. P. Reece, Anterior pituitary and plasma prolactin in rats after 2 to 90 minutes of suckling, Proc Soc Exp Biol Med 149: 754, 1975. Shiino, M., G. Williams, and E. G. Rennels, Ultrastructural observation of pituitary release of prolactin in the rat by suckling stimulus, Endocrinology 90: 176, 1972. Chang, N. G., and M. B. Nikitovitch-Winer, Correlation between suckling-induced changes in the ultrastructure of mammotrophs and prolactin release, Cell Tissue Res 166: 399, 1976. Amenomori, Y., C. L. Chen, and J. Meites, Serum prolactin levels in rats during different reproductive states, Endocrinology 86: 506, 1970. Nicoll, C. S., Some observations and speculation on the mechanism of "depletion," "repletion" and release of adenohypophysial hormones, Gen Comp Endocrinol (Suppl) 3: 86, 1972. Asawaroengchai, H., S. M. Russell, and C. S. Nicoll, Electrophoretically separable forms of rat prolactin with different bioassay and radioimmunoassay activities, Endocrinology 102: 407, 1978.
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Vol. 104, No. 2 Printed in U.S.A.
The Secretion Rate of Prolactin in the Rat during Suckling and Its Metabolic Clearance Rate after Increasing Intervals of Nonsuckling* C. E. GROSVENOR, F. MENA,f AND N. S. WHITWORTH^ Department of Physiology and Biophysics, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163
ABSTRACT. The amount of pituitary PRL depleted by suckling is known to increase in direct relationship to the length of the previous nonsuckling interval. Therefore, we examined the extent to which the nonsuckling interval influenced the plasma concentration of PRL attained during prolonged suckling, the metabolic clearance rate of continuously infused rat PRL, and the secretion rate of PRL into the circulation. Neither the plateau level of PRL attained in the plasma during suckling nor the clearance rate of infused PRL were altered by increasing the nonsuckling interval from 2 to 4 to 8 h. The secretion rates also were similar (554, 530, and 537 ng/min for the 2-, 4-, and 8-h nonsuckled groups, respectively). However, the number of minutes the steady secretion rate of PRL into the plasma could be maintained varied in direct relationship to the length of the nonsuckling interval. The steady secretion waned after 30 min of suckling in die 2-h nonsuckled group, after 60 min in the 4-h
T
HAT suckling stimulates the adenohypophysis to release PRL is evident both from a reduction in pituitary levels and a rise in plasma levels of the hormone (1-3). Little information, however, is available concerning the quantitative relationships between the two (3) and whether the various factors which influence the amount of PRL depleted by the pituitary (2) also affect its level in the circulation. One such factor is the length of the nonsuckling interval, which has been shown to be directly related to the amount of PRL depleted from the pituitary during suckling (2, 4-6). In the present study, the suckling-induced rise in plasma PRL concentration, the metabolic clearance rate (MCR), and the secretion rate (SR) of PRL have been analyzed after various intervals of time before suckling.
nonsuckled group, and did not subside at all during the 90-min suckling period in the 8-h group. The total amount of PRL secreted into the plasma approximated 17, 32, and 48 jug for the three groups, values which are quantitatively similar to amounts depleted from the pituitary during the first few minutes of suckling after similar nonsuckling periods. To reconcile the rapid and extensive depletion of PRL from the pituitary with the steady, potentially long term output of much smaller amounts of PRL into the plasma, we postulate that the first few minutes of suckling prepares PRL for release into the circulation (pituitary depletion stage); the amount so prepared depends upon the length of the previous nonsuckling interval. Once prepared, the PRL is secreted into the circulation in a steady minute by minute fashion, independent of the previous nonsuckling interval. This steady secretion can continue until the pool of prepared PRL is exhausted. (Endocrinology 104: 372, 1979)
Materials and Methods Primiparous lactating rats of the Holtzman strain, each with six pups, were maintained in individual cages in a room at 23-25 C with alternating 14-h light: 10-h darkness. Food and water were available at all times. Each rat (weight range, 260-320 g) was tested on days 13-14 of lactation. Plasma PRL concentration during suckling
Received June 26,1978. Address all correspondence and requests for reprints to: Dr. C. E. Grosvenor, Department of Physiology and Biophysics, University of Tennessee Center for the Health Sciences, 894 Union Avenue (NA 427), Memphis, Tennessee 38163. • This work was supported by USPHS Grant HD-04358 (C.E.G.) and PLAMIRH 96.182.1.78 (F. M.). f Present address: Institute of Biomedical Investigations, University of Mexico, Mexico City. | Present address: Department of Ob-Gyn, University of Mississippi, Jackson, Mississippi.
Pups were separated from their mothers for 2,4, or 8 h before suckling. At the end of these periods, the pups were returned to their mothers, and suckling was allowed for 90 min (five to seven rats per group). To insure uniform and sustained teat stimulation throughout the 90-min suckling period, foster litters which were 14 days of age and previously isolated from their mothers for 8 h were inserted at 30 and 60 min. Blood (0.35 ml each sample) was obtained before and at intervals after the onset of suckling from each unrestrained conscious mother through a right atrial catheter implanted 2-3 days previously (see Ref. 7 for details). The catheter was flushed with heparinized saline (0.35 ml) after each blood sample was taken. Plasma PRL concentration after iu infusion of rat PRL On day 14 postpartum, mother and pups were separated for 2, 4, and 8 h. At the end of these periods, each mother was anesthetized with urethane and implanted in the right atrium
372
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373
SECRETION AND MCR OF PRL IN RAT with a double lumen catheter. Each rat was then attached to an infusion pump (Harvard Apparatus model 600-900) and rat PRL (NIAMDD preparation, B-l; 7 IU/mg dissolved in 0.9% NaCl) was continuously infused via one lumen of the intraatrial catheter at a dose of 738 ng/min. This dose is equivalent to 472 ng RP-1 PRL (11 IU/mg)/min. The infusion volume was 5.9 jiil/min. At intervals during the infusion, the infusion pump was stopped for 15 sec. This was to ensure uniform distribution of the infused PRL throughout the circulation of the rat before the withdrawal of each blood sample (0.35 ml) through the other channel of the double lumen catheter. The blood remaining in the catheter after each blood sample had been taken was flushed back into the rat with 0.35 ml heparinized saline. The delivery volume of the infusion pump at the setting used was checked periodically by measuring the volume of saline delivered over a 2-h period from 1-ml syringes and through catheters identical to those used during the infusion experiments. The volume delivered did not measurably differ from the rate of 5.9 jul/min at setting 5 on the pump when the results of the various trials were compared. RIA of rat PRL The plasma was separated from each blood sample and stored at -20 C until assayed for PRL by RIA. The concentration of immunoreactive rat PRL was determined in duplicate 25-jul samples of each PRL solution infused and in duplicate 25jttl plasma samples using the NIAMDD rat PRL RIA system (see Ref. 8 for procedures and validation data). All PRL values are expressed as nanogram equivalents of the RP-1 standard. Samples of the B-l PRL solutions which were infused were obtained at the start of each experiment. Immunoassay of these solutions yielded values which were 113% and 90% of their expected concentrations.
Results The plasma PRL concentration rose rapidly during suckling to a maximum of 242, 255, and 262 ng/ml above presuckled levels within the first 30 min of suckling after 2, 4, and 8 h, respectively, of nonsuckling (Fig. 1 and Table 1). Plasma PRL levels in the 8-h nonsuckled group were sustained throughout the entire 90 min of suckling, but declined significantly (P < 0.05) in the 2-h group at 60 min and in the 4-h group at 90 min (Fig. 1). The data were statistically evaluated by trend analysis and Duncan's multiple range test. The plasma levels in the 2-, 4-, and 8-h nonsuckled groups also rose quickly and reached stable concentrations of 206, 227, and 230 ng/ml, respectively, above preinfusion levels within 20 min of continuous iv infusion of 472 ng RP1 rat PRL/min (Fig. 2). The profile of the rise and attainment of an equilibrium concentration of PRL in the plasma of rats which were suckled was similar to that of rats infused with rat PRL (compare Figs. 1 and 2). The MCR for each group was computed from the stable concentration of plasma PRL attained during continuous infusion of rat PRL divided into the infusion rate of 472 ng/min. This yielded mean MCRs of 2.29, 2.08, and 2.05 ml/min for the 2-, 4-, and 8-h nonsuckled groups, respectively (Table 1). The SR of PRL during suckling was then calculated for each group from the mean stable
MCR
300
The MCR of immunoreactive rat PRL was calculated as follows (9):
8h N-S I5I
MCR (ml/min) = Infusion dose (ng/min) Increment in plasma concentration at equilibrium (ng/ml) We showed previously (8) that the plasma concentration of immunoreactive rat PRL in the rat reached equilibrium after approximately 20 min of continuous infusion. The plasma PRL concentration used in the computation of MCR for each rat was the average of the two or three individual values obtained during the equilibrium period minus the PRL concentration in the plasma before the start of PRL infusion.
n 200 E -». O)
c o CO
o £ 100
T
1
New litters Inserted
SR of PRL From the mean MCR together with the mean plateaued plasma concentration of PRL during suckling minus the PRL concentration before suckling, we could calculate the mean SR of PRL during suckling for each group of rats as follows: SR (ng/min) = MCR (ml/min) X plasma cone. (ng/ml) at steady state during suckling
60
10 20 30 Min.
of
90
Suckling
FIG. 1. Influence of increasing the length of the nonsuckling interval (N-S) from 2 to 4 to 8 h on the suckling-induced rise in plasma PRL concentration. Values are means ± SEM; each has subtracted from it the PRL concentration in the plasma at zero time. Number in parentheses refers to the number of rats.
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Endo • 1979 Vol 104 • No 2
GROSVENOR, MENA, AND WHITWORTH
TABLE 1. Estimate of SR of PRL from MCR and equilibrium concentration of PRL during suckling after 2, 4, or 8 h of nonsuckling in the rat h of Nonsuckling
Equilibrium PRL levels during PRL infu- MCR (ml/min; mean ± SE) sion (ng/ml; mean ± SE)°
2 4 8
206 ± 19.2 227 ± 23.4 230 ± 23.8
Equilibrium PRL levels during suckling = (ng/ml; mean ± SE) a
2.29 ± 0.20 2.08 ± 0.22 2.05 ± 0.22
242 ± 27.1 255 ± 20.6 262 ± 22.1
SR (ng/min)
min of Steady se- == cretion
X
554 530 537
Total secretion into plasma (fig) 17 32 48
30 60 90
Plasma concentrations of PRL during suckling and MCRs are the same data as in Figs. 1 and 2. ° Each value has subtracted from it the plasma concentration of PRL before infusion of PRL or suckling.
concentration of PRL which was reached within the first 30 min of suckling x the average MCR. This yielded SRs of PRL during suckling of 554, 530, and 537 ng/min for rats previously not suckled for 2, 4, and 8 h (Table 1). We did not attempt to quantitate the SR of PRL in the 2- and 4-h previously nonsuckled groups during the periods of suckling where the plasma concentration declined from the previously attained equilibrium level. From the number of minutes the SR of PRL was sustained, it was possible to estimate the total output of PRL from the pituitary into the plasma. This yield outputs of 17, 32, and 48 /xg PRL during continuous suckling in rats previously not suckled for 2, 4, and 8 h, respectively (Table 1). These estimates probably are slightly lower than actual levels because the SR during the period of decline in plasma PRL concentration was not estimated in the 2- and 4-h groups, and in the 8-h group it is not known how much longer than 90 min the steady secretion of PRL would have been maintained. Discussion Yanai and Nagasawa (10) reported that plasma PRL concentrations rose at similar rates during suckling in rats previously not suckled for 8, 16, or 24 h. Similar results were obtained by Mena etal. (11) using nonsuckling periods of 8 and 16 h. The suckling-induced pattern of PRL concentration in plasma obtained by several other investigators after nonsuckling periods ranging from 6.5-12.5 h reveals a striking similarity when plotted on the same scale (8). In the present study, the patterns of plasma PRL concentrations during 30 min of suckling were virtually identical regardless of whether the previous nonsuckling period was 2, 4, or 8 h in length. Thus, it would appear that the suckling-induced pattern of change in plasma PRL concentrations in the rat is quite similar, though the preceding nonsuckling period may vary widely in length. In each of these studies, including the present, the concentration of PRL increased rapidly in the plasma to a maximum within 20-30 min of suckling. The concentration thereafter tended to plateau. The results in the present study also indicate that the clearance rate of PRL in the lactating rat is not altered by
300
I
I I
-
1[ i
200
100
'
4h N-S I7I 8h N-S |5| 2h N-SI9I
472ngRatPrl/min
15
'
30
45
60
Minutes FIG. 2. Influence of increasing the length of the nonsuckling interval (N-S) from 2 to 4 to 8 h on the plasma concentration of PRL resulting from continuous infusion of rat PRL into the circulation. Values are means ± SEM; each has subtracted from it the PRL concentration in the plasma at zero time. Number in parentheses refer to the number of rats.
lengthening nonsuckling intervals (up to 8 h in length). In each of the nonsuckled groups tested, the rate of increase and the equilibrium concentration of PRL attained in the plasma after continuous iv infusion of rat PRL clearly resembled those which occurred during suckling. This similarity corroborates our earlier observations (8, 12) and indicates that PRL is released into the circulation during suckling in small (500-600 ng/min) amounts in a steady minute by minute fashion rather than in a large amount over a short span of time. The maintenance of this steady secretion is dependent upon sustained suckling; thus, plasma levels fall once suckling is interrupted (8).
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SECRETION AND MCR OF PRL IN RAT In calculating the SRs we used clearance data obtained from urethane-anesthetized rats and plasma PRL levels obtained during suckling in conscious rats. The use of urethane anesthesia to immobilize the rats after the various nonsuckling periods probably exerted a slight depressing influence on the clearance rate and hence, the SRs of PRL in. the conscious lactating rat may actually be slightly higher than reported. This supposition stems from recent experiments on which we use the same method as in the present study to determine the MCR of PRL in rats during mid (days 12-15) and late (days 20-23) lactation. The clearance initially was determined while they were conscious then again after they had been anesthetized with urethane. The MCR was depressed an average of about 20% in rats on both days as a result of the anesthesia, but the difference was not statistically significant (Grosvenor, C. E., and N. S. Whitworth, manuscript in preparation). From the data available thus far it is becoming clear that the secretion of PRL into the plasma during suckling differs in two important respects from the depletion of PRL from the pituitary during suckling. Firstly, the SR into plasma during suckling, computed from the equilibrium plasma PRL concentration during suckling multiplied by the M!CR, is unaffected by the length of the previous nonsuckling period, whereas the amount of PRL depleted from the pituitary ranges upward from a nondetectable change after 2 h to 80-95% after nonsuckling periods of 10-12 h (2, 4-6). Secondly, the secretion into plasma occurs steadily and in small amounts (Refs. 8,12, and as shown in the present study), whereas depletion of PRL from the pituitary occurs rapidly and in large amounts. As little as 2 min of suckling is nearly as effective as 30-90 min of suckling in provoking maximal depletion (13, 14). Electronmicroscopic examination of the rat pituitary reveals marked extrusion of PRL granules after 5 min of suckling (15), and after only 1 min of suckling, the rough endoplasmic reticulum of the PRLsecreting cell in the pituitary has collapsed, suggesting that its contents have been discharged (16). We observed in the present paper that it is the number of minutes the steady secretion of PRL into plasma can be maintained by suckling rather than the SR itself which is directly related to the previous nonsuckling interval. Thus, the steady secretion waned after 30 min in the 2-h nonsuckled group and after 60 min in the 4-h group. The gradual nature of the reduction in plasma concentration in the 2- and 4-h groups suggests that the secretion of PRL into the plasma was not abruptly terminated but rather only subsided. The disappearance rate of PRL in these groups was approximately that of a ti/2 = 75 min, in contrast to a ti/2 of 6-8 min after infusion of PRL (12). The SR of PRL in the 8-h nonsuckled group was sustained throughout the 90 min that suckling was
375
applied, though it too would be expected to decline with even longer suckling episodes. The total amount of PRL secreted into the plasma, therefore, approximated 17, 32, and 48 jug (187, 354, and 528 mU) after 2, 4, and 8 h of nonsuckling, respectively. These amounts correspond favorably with the 308 mU (28 jug in terms of RP-1 standard) depleted after 6 h of nonsuckling (14) and the 530 mU depleted after 10 h of nonsuckling (3). They also correspond well with those of Amenomori et al. (17) who reported that about 60 /xg were depleted after 12 h of nonsuckling, though the potency of the reference standard was not stated in their study. Thus, with the use of prolonged continuous suckling, effected in our laboratory by inserting hungry foster pups at 30-min intervals, a fairly good quantitative relationship seems to exist between the greater depletion of immunoreactive PRL as the nonsuckling period increases (3, 14, 17) and the total amount that is secreted into the circulation. Our data also indicate that of the large amount of immunoreactive PRL depleted during the first few minutes of suckling only a small fraction appears in the circulation as RIAdetectable PRL during the same time, but that with longer periods of suckling a better quantitative relationship is achieved. Though quantitatively related, the rapid depletion of PRL within the pituitary during suckling bears little resemblance to the steady, potentially long term secretion of PRL into the circulation provoked by the same stimulus. To reconcile these differences, we postulate that the first few minutes of suckling prepares PRL for release into the circulation (pituitary depletion stage); the amount so prepared depends upon the length of the previous nonsuckling interval. Once prepared, the PRL is secreted into the circulation in a steady minute by minute fashion, independent of the previous nonsuckling interval. This steady secretion can continue until the pool of prepared PRL is exhausted. This postulation is similar to the model of pituitary cell function originally proposed by Nicoll (18). In his model, the depletion of PRL precedes release but not all of the depleted PRL necessarily is released into the circulation. The nature of the depleted or prepared PRL is unknown at the present time. One possibility is that it may be rapidly transformed after its depletion into a polymer or it may become bound to a carrier protein within the pituitary so it cannot be readily detected by RIA. The PRL secreted into the circulation then may represent uncoupled or depolymerized PRL which, once again, is detectable by RIA. Forms of PRL with different bioassay:RIA ratios have been shown to exist within and to be secreted by the rat pituitary (19). Acknowledgments We are grateful to the NIAMDD Rat Pituitary Hormone Distribu-
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GROSVENOR, MENA, AND WHITWORTH
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tion Program for the gift of rat PRL, and to Tanya McGee and Lee Brinkley for expert technical assistance.
References 1. Neill, J. D., Prolactin: its secretion and control, In Handbook of Physiology, vol. 4, pt. 2, 1974, p. 469. 2. Grosvenor, C. E., and F. Mena, Effect of suckling upon the secretion and release of prolactin from the pituitary of the lactating rat, Bien Symp Anim Reprod 32: 115, 1971. 3. Nicoll, C. S., F. Mena, C. W. Nichols, Jr., S. H. Green, M. Tai, and S. M. Russell, Analysis of suckling-induced changes in adenohypophyseal prolactin concentration in the lactating rat by three assay methods, Ada Endocrinol (Kbh) 83: 512, 1976. 4. Sar, M., and J. Meites, Effects of suckling on pituitary release of prolactin, GH, and TSH in postpartum lacting rats, NeuroendocrinologyA: 25, 1969. 5. Convey, E. M., and R. P. Reece, Restoration of pituitary lactogen released in response to suckling, Proc Soc Exp Biol Med 131: 543, 1969. 6. Grosvenor, C. E., and C. W. Turner, Pituitary lactogenic hormone concentration and milk secretion in lactating rats, Endocrinology 63: 535, 1958. 7. Grosvenor, C. E., N. S. Whitworth, and F. Mena, Milk secretory response of the conscious lactating rat following intravenous injections of rat prolactin, J Dairy Sci 58: 1803, 1975. 8. Grosvenor, C. E., and N. S. Whitworth, Evidence for a steady rate of secretion of prolactin following suckling in the rat, J Dairy Sci 57: 900, 1974. 9. Tait, J. F., Review: the use of isotopic steroids for the measurement
10. 11. 12. 13.
14. 15. 16. 17. 18. 19.
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of production rates in vivo, J Clin Endocrinol Metab 25: 1285, 1963. Yanai, R., and H. Nagasawa, Effect of non-suckling period on pituitary prolactin secretory activity in rats, Horm Res 4:169,1973. Mena, F., A. Enjalbert, L. Carbonell, M: Priam, and C. Kordon, Effect of suckling on plasma prolactin and hypothalamic monoamine levels in the rat, Endocrinology 99: 445, 1976. Grosvenor, C. E., and N. S. Whitworth, Incorporation of rat prolactin into rat milk in vivo and in vitro, J Endocrinol 70: 1, 1976. Grosvenor, C. E., V. Mena, and D. A. Schaefgen, Effect of nonsuckling interval and duration of suckling in the suckling-induced fall in pituitary prolactin concentration in the rat, Endocrinology 81: 449, 1967. Subramanian, M. G., and R. P. Reece, Anterior pituitary and plasma prolactin in rats after 2 to 90 minutes of suckling, Proc Soc Exp Biol Med 149: 754, 1975. Shiino, M., G. Williams, and E. G. Rennels, Ultrastructural observation of pituitary release of prolactin in the rat by suckling stimulus, Endocrinology 90: 176, 1972. Chang, N. G., and M. B. Nikitovitch-Winer, Correlation between suckling-induced changes in the ultrastructure of mammotrophs and prolactin release, Cell Tissue Res 166: 399, 1976. Amenomori, Y., C. L. Chen, and J. Meites, Serum prolactin levels in rats during different reproductive states, Endocrinology 86: 506, 1970. Nicoll, C. S., Some observations and speculation on the mechanism of "depletion," "repletion" and release of adenohypophysial hormones, Gen Comp Endocrinol (Suppl) 3: 86, 1972. Asawaroengchai, H., S. M. Russell, and C. S. Nicoll, Electrophoretically separable forms of rat prolactin with different bioassay and radioimmunoassay activities, Endocrinology 102: 407, 1978.
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