SECRETION RATE AND METABOLIC CLEARANCE RATE OF PROLACTIN IN THE RAT DURING MID- AND LATE LACTATION C. E. GROSVENOR AND N. S. WHITWORTH Department of Physiology and Biophysics, University of Tennessee Center Sciences, Memphis, Tennessee 38163, U.S.A.
(Received 1
December
for
the Health
1978)
SUMMARY
prolactin concentration in the plasma of lactating rats rose less rapidly and attained a significantly lower plateau level in response to suckling on day 20\p=n-\21of lactation than it did on day 13\p=n-\14of lactation. Neither differences in suckling stimulation of the older pups nor a higher metabolic clearance rate (MCR) of prolactin were implicated in the reduced prolactin concentration seen in the late-lactating rats. The MCR was, in fact, slightly reduced in both conscious and late-lactating rats anaesthetized with urethane when compared with those in mid-lactation. The MCR of prolactin was not significantly altered by urethane anaesthesia in rats on either day of lactation. However, the secretion rate of prolactin, computed from the MCR multiplied by the equilibrum concentration of prolactin during suckling, was considerably reduced (665 to 392 ng/min) from mid- to late lactation. We conclude from these data that the reduced plasma concentration of prolactin in response to suckling in late lactation is the result of an impairment within the prolactin secretory mechanism. The
INTRODUCTION
The prolactin concentration in plasma typically rises seven- to tenfold within 15-30 min of the commencement of suckling in the rat during mid-lactation ; continued suckling effected by substituting hungry foster litters maintains the high plasma levels for a varying number of minutes depending upon the length of the previous period of non-suckling (Grosvenor & Whitworth, 1974; Grosvenor, Mena & Whitworth, 1979). There is some evidence that prolactin secretion into the circulation may be reduced in the rat during late lactation when circulating levels found at this time are considered (Amenomori, Chen & Meites, 1970; Ford & Melampy, 1973; Lu, Chen, Huang, Grandison, Marshall & Meites, 1976). Subramanian & Reece (1975) noted that the concentration of prolactin in plasma rose less rapidly during suckling and attained a much lower peak concentration during late lactation than it did in rats during early lactation. The fall in pituitary prolactin concentration after suckling, however, was similar in both groups. To account for the lower prolactin levels in the circulation during late lactation, it was suggested (Subramanian & Reece, 1975) that prolactin released by suckling was removed more rapidly from the circulation. In the present study it has been confirmed that the plasma prolactin concentration reaches a level during suckling in late lactation which is considerably lower than that obtained in the same rats during mid-lactation. We then examined the extent to which alterations in metabolic clearance rate, in secretion rate and in suckling-stimulation of older pups, in contrast with that of younger pups, might be implicated in the reduced plasma levels which occur during suckling in the late-lactating rat. In addition, the metabolic clearance rates of prolactin in conscious and in anaesthetized rats during the two periods of lactation have been compared. *
of
Present address : Departments of Obstetrics and Gynecology and Physiology and Biophysics, University Medical Centre, Jackson, Mississippi 39216, U.S.A.
Mississippi
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 14 h light : 10 h darkness. Food and water were available at all times.
Prolactin concentration in plasma during suckling in mid- and late lactation separated from their mothers for 5-6 h before suckling on day 13-14 (mid-lacta¬ Pups and tion) again on day 20-21 (late lactation). They were returned to their mothers and suck¬ ling was allowed for 60 min. To ensure uniform and sustained teat stimulation throughout the 60 min suckling period, the original litters were removed and foster litters which were of similar age and which had been isolated from their mothers for 5-6 h were exchanged after 30 min. Blood (0-35 ml each sample) was obtained before and at intervals during suckling from each unrestrained conscious mother through a right atrial catheter which had been implanted on day 12 post partum (see Grosvenor, Whitworth & Mena, 1975 for details). The catheter was flushed with heparinized saline (0-35 ml) after each blood sample had been taken. The following experiment was performed to test for differences in suckling intensity and/ or frequency since this may contribute to any alteration in the prolactin concentration in the plasma that occurs in late lactation. Two groups of rats were implanted with right atrial catheters on day 18-19 of lactation. On day 20-21 their pups were removed for 5-6 h. One group of mothers then received 20- to 21-day-old pups and the other 13- to 14-day-old pups which had been removed 5-6 h earlier from their mothers. Suckling was then permitted for 45 min. Plasma samples (0-35 ml each) were obtained before and at intervals during this period. were
Metabolic clearance rate ofprolactin in conscious and unconscious mid- and late-lactating rats Each mother in this experiment was implanted with a double lumen catheter in the right atrium on either day 12 or 18 of lactation. On day 14 or 20 post partum, mother and pups were separated for 5-6 h. Each rat was attached to an infusion pump (Harvard Apparatus Model 600-900) and rat prolactin (NIAMDD preparation, B-l, 7 i.u./mg dissolved in 0-9% NaCl) was continuously infused through one lumen of the intra-atrial catheter at a dose of 738 ng/min. This dose is equivalent to 472 ng RP-l/min (11 i.u./mg). The infusion volume was 5-9 µ /min. After 25 and 35 min of infusion, the pump was stopped for 15 s. This was to ensure uniform distribution of the infused prolactin 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. Shortly after the second blood sample was obtained, each rat was anaesthetized within 2-3 min with urethane (ethyl carbamate, Sigma Chemicals ; 0-4-0-5 ml 25% solution) adminis¬ tered through one lumen of the catheter while the prolactin infusion continued through the other lumen. Additional plasma samples then were obtained 20 and 30 min after the ure¬ thane had been injected, i.e. after 55 and 65 min of infusion. The basal plasma concentration of prolactin was monitored in a similarly prepared group of conscious rats at day 14 and 20 post partum for 35 min and then for an additional 35 min following urethane anaesthesia.
Radioimmunoassay of rat prolactin The plasma was separated from each blood sample and stored at —20 °C until assayed for prolactin by radioimmunoassay. The concentration of immunoreactive rat prolactin was determined in duplicate 25 µ samples of each prolactin solution infused and in duplicate 25 µ samples of plasma using the NIAMDD rat prolactin radioimmunoassay system (see Grosvenor & Whitworth, 1974, for procedures and validation data). All prolactin values are expressed as ng equivalents of the RP-1 standard. Samples of the NIAMDD B-l prolactin
solutions which were infused were obtained at the start of each experiment. Immunoassay of these solutions yielded values which were 85-114% of the expected potency of 7 i.u./mg. Metabolic clearance rate (MCR) The MCR of immunoreactive rat prolactin was calculated as follows
(Tait, 1963):
infusion dose (ng/min) plasma concentration at equilibrium (ng/ml) We showed previously (Grosvenor & Whitworth, 1974) that the plasma concentration of immunoreactive rat prolactin in the rat reached equilibrium after approximately 20 min of continuous infusion. The plasma prolactin concentration used in the computation of MCR for MCR
(ml/min)
=
-
increment in
each rat while conscious and while unconscious was the average of the two individual values obtained during each state minus the prolactin concentration in the plasma before the start of prolactin infusion.
Secretion rate ofprolactin during suckling From the mean MCR data obtained in conscious rats, together with the mean plasma con¬ centration of prolactin during suckling, it was possible to calculate the mean secretion rate (SR) of prolactin during suckling for the group of rats on day 14 and 20 post partum as follows : SR (ng/min) MCR (ml/min) multiplied by plasma concentration at steady state (ng/ml) during suckling. Statistical comparisons were made using Student's r-test. =
RESULTS
plasma prolactin concentration in rats on day 13-14 post partum rose steadily during suckling from a basal level of 27 ng/ml and reached a plateau level of 266 ng/ml after 30 min. The prolactin concentration in the same rats when retested 1 week later rose more slowly in response to suckling from a basal level of 24 ng/ml and attained a plateau level of 180 ng/ml which was significantly lower than that observed on day 14 (Fig. 1, Table 1). The use of 14-day-old pups to provide the suckling stimulation in lactating rats on day 21 did not alter the plasma prolactin profile from that seen when 21-day-old pups provided the stimulus (Fig. 2). Thus a difference in the suckling stimulation from the older pups did not appear to be a factor in the reduced prolactin concentration observed on day 21.
The average
Metabolic clearance rate The prolactin concentration rose to an average of 168 ng/ml above pre-infusion levels follow¬ ing 35 min of infusion of rat prolactin (equivalent to 472 ng RP-1 rat prolactin (5-19 mu.)/ min) to conscious rats on day 14 post partum (Table 1). Infusing the same amount of prolactin in a group of conscious rats which had been lactating for 21 days gave average prolactin values of 187 ng/ml above basal levels (Table 1). The concentration was essentially the same after the 25 and 35 min infusion in each group. Infusion of prolactin into 14- and 21-daylactating rats under urethane anaesthesia gave average prolactin values at equilibrium of 204 and 232 ng/ml respectively. None of these equilibrium prolactin values differed significantly. The basal levels of prolactin in rats sampled similarly but not receiving prolactin infusion were quite stable and were not influenced by urethane anaesthesia (Fig. 3). The MCR was then calculated for each rat from the average prolactin concentration attained above the pre-infusion level, before and after urethane anaesthesia, divided into the infusion rate of rat prolactin (equivalent to 472 ng RP-1 rat prolactin (5-19 mu.)/min). This yielded rates before and after urethane anaesthesia of 2-8 and 2-3 ml/min for day 14 postpartum rats and 2-5 and 2-0 ml/min for day 21 post-partum rats (Table 1). The decrease in MCR as a result of anaesthesia was consistent within both groups of rats but the 0-5 ml/min difference »vas not statistically significant for rats on either day of lactation.
300
I
200
ce
"*
. cd Vi
S
100
CL.
% 60 45 15 30 Time of suckling (min) Fig. 1. Effect of suckling six pups on the plasma concentration of prolactin on day 14 (·) and on day 21 (o) of lactation. The same group of eight rats was tested on both days. The arrow indicates the exchange of the original litters with hungry foster litters. Values are means ± s.e.m.
200
|¿2 O 1-
100
-
I
.3 CL
15
30
45
Time of suckling
(min) (·) and four 21-day-old
Fig. 2. Effect of suckling four 14-day-old pups pups (O) concentration of prolactin in rats 21 days post partum. Values are means ± s.e.m. Secretion rate
ofprolactin
on
the
plasma
The average MCR obtained from conscious lactating rats multiplied by the average prolactin concentration at equilibrium achieved during suckling, above basal levels before suckling, yielded estimates of rates of secretion of prolactin into the circulation of 665 and 392 ng/min
for rats on day 14 and day 21 of lactation respectively (Table 1). Individual values for secre¬ tion rates could not be determined, however, since MCR and prolactin profiles during suck¬ ling were not obtained from the same rats. Table 1. Metabolic clearance rate (MCR)* ofprolactin in conscious and unconscious rats and secretion rates ofprolactin in conscious lactating rats on days 14 and 21 of lactation. Values are means
± s.e.m.
Infusion of prolactin (472 ng (5-19 mu.)/min) Conscious
Suckling
Unconscious
Equilibrium plasma Equilibrium plasma Equilibrium plasma Secretion concn during concn during concn during rate* MCR MCR lactation infusionf (ng/ml) (ml/min) infusionf (ng/ml) (ml/min) sucklingt (ng/ml) (ng/min) Day of 14 21 *
168 ±24
2-81 ±0-49 2-52 ±0-29
187±24
204±15 232 ±50
2-31 ±0-14 2-03 ±0-37
237±18 156±17J
664 392
See text for method of calculation.
t Represents concentration attained minus the concentration in plasma before the infusion of prolactin or commencement of suckling. Î Significantly less than on day 14 (P
(Received 1
December
for
the Health
1978)
SUMMARY
prolactin concentration in the plasma of lactating rats rose less rapidly and attained a significantly lower plateau level in response to suckling on day 20\p=n-\21of lactation than it did on day 13\p=n-\14of lactation. Neither differences in suckling stimulation of the older pups nor a higher metabolic clearance rate (MCR) of prolactin were implicated in the reduced prolactin concentration seen in the late-lactating rats. The MCR was, in fact, slightly reduced in both conscious and late-lactating rats anaesthetized with urethane when compared with those in mid-lactation. The MCR of prolactin was not significantly altered by urethane anaesthesia in rats on either day of lactation. However, the secretion rate of prolactin, computed from the MCR multiplied by the equilibrum concentration of prolactin during suckling, was considerably reduced (665 to 392 ng/min) from mid- to late lactation. We conclude from these data that the reduced plasma concentration of prolactin in response to suckling in late lactation is the result of an impairment within the prolactin secretory mechanism. The
INTRODUCTION
The prolactin concentration in plasma typically rises seven- to tenfold within 15-30 min of the commencement of suckling in the rat during mid-lactation ; continued suckling effected by substituting hungry foster litters maintains the high plasma levels for a varying number of minutes depending upon the length of the previous period of non-suckling (Grosvenor & Whitworth, 1974; Grosvenor, Mena & Whitworth, 1979). There is some evidence that prolactin secretion into the circulation may be reduced in the rat during late lactation when circulating levels found at this time are considered (Amenomori, Chen & Meites, 1970; Ford & Melampy, 1973; Lu, Chen, Huang, Grandison, Marshall & Meites, 1976). Subramanian & Reece (1975) noted that the concentration of prolactin in plasma rose less rapidly during suckling and attained a much lower peak concentration during late lactation than it did in rats during early lactation. The fall in pituitary prolactin concentration after suckling, however, was similar in both groups. To account for the lower prolactin levels in the circulation during late lactation, it was suggested (Subramanian & Reece, 1975) that prolactin released by suckling was removed more rapidly from the circulation. In the present study it has been confirmed that the plasma prolactin concentration reaches a level during suckling in late lactation which is considerably lower than that obtained in the same rats during mid-lactation. We then examined the extent to which alterations in metabolic clearance rate, in secretion rate and in suckling-stimulation of older pups, in contrast with that of younger pups, might be implicated in the reduced plasma levels which occur during suckling in the late-lactating rat. In addition, the metabolic clearance rates of prolactin in conscious and in anaesthetized rats during the two periods of lactation have been compared. *
of
Present address : Departments of Obstetrics and Gynecology and Physiology and Biophysics, University Medical Centre, Jackson, Mississippi 39216, U.S.A.
Mississippi
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 14 h light : 10 h darkness. Food and water were available at all times.
Prolactin concentration in plasma during suckling in mid- and late lactation separated from their mothers for 5-6 h before suckling on day 13-14 (mid-lacta¬ Pups and tion) again on day 20-21 (late lactation). They were returned to their mothers and suck¬ ling was allowed for 60 min. To ensure uniform and sustained teat stimulation throughout the 60 min suckling period, the original litters were removed and foster litters which were of similar age and which had been isolated from their mothers for 5-6 h were exchanged after 30 min. Blood (0-35 ml each sample) was obtained before and at intervals during suckling from each unrestrained conscious mother through a right atrial catheter which had been implanted on day 12 post partum (see Grosvenor, Whitworth & Mena, 1975 for details). The catheter was flushed with heparinized saline (0-35 ml) after each blood sample had been taken. The following experiment was performed to test for differences in suckling intensity and/ or frequency since this may contribute to any alteration in the prolactin concentration in the plasma that occurs in late lactation. Two groups of rats were implanted with right atrial catheters on day 18-19 of lactation. On day 20-21 their pups were removed for 5-6 h. One group of mothers then received 20- to 21-day-old pups and the other 13- to 14-day-old pups which had been removed 5-6 h earlier from their mothers. Suckling was then permitted for 45 min. Plasma samples (0-35 ml each) were obtained before and at intervals during this period. were
Metabolic clearance rate ofprolactin in conscious and unconscious mid- and late-lactating rats Each mother in this experiment was implanted with a double lumen catheter in the right atrium on either day 12 or 18 of lactation. On day 14 or 20 post partum, mother and pups were separated for 5-6 h. Each rat was attached to an infusion pump (Harvard Apparatus Model 600-900) and rat prolactin (NIAMDD preparation, B-l, 7 i.u./mg dissolved in 0-9% NaCl) was continuously infused through one lumen of the intra-atrial catheter at a dose of 738 ng/min. This dose is equivalent to 472 ng RP-l/min (11 i.u./mg). The infusion volume was 5-9 µ /min. After 25 and 35 min of infusion, the pump was stopped for 15 s. This was to ensure uniform distribution of the infused prolactin 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. Shortly after the second blood sample was obtained, each rat was anaesthetized within 2-3 min with urethane (ethyl carbamate, Sigma Chemicals ; 0-4-0-5 ml 25% solution) adminis¬ tered through one lumen of the catheter while the prolactin infusion continued through the other lumen. Additional plasma samples then were obtained 20 and 30 min after the ure¬ thane had been injected, i.e. after 55 and 65 min of infusion. The basal plasma concentration of prolactin was monitored in a similarly prepared group of conscious rats at day 14 and 20 post partum for 35 min and then for an additional 35 min following urethane anaesthesia.
Radioimmunoassay of rat prolactin The plasma was separated from each blood sample and stored at —20 °C until assayed for prolactin by radioimmunoassay. The concentration of immunoreactive rat prolactin was determined in duplicate 25 µ samples of each prolactin solution infused and in duplicate 25 µ samples of plasma using the NIAMDD rat prolactin radioimmunoassay system (see Grosvenor & Whitworth, 1974, for procedures and validation data). All prolactin values are expressed as ng equivalents of the RP-1 standard. Samples of the NIAMDD B-l prolactin
solutions which were infused were obtained at the start of each experiment. Immunoassay of these solutions yielded values which were 85-114% of the expected potency of 7 i.u./mg. Metabolic clearance rate (MCR) The MCR of immunoreactive rat prolactin was calculated as follows
(Tait, 1963):
infusion dose (ng/min) plasma concentration at equilibrium (ng/ml) We showed previously (Grosvenor & Whitworth, 1974) that the plasma concentration of immunoreactive rat prolactin in the rat reached equilibrium after approximately 20 min of continuous infusion. The plasma prolactin concentration used in the computation of MCR for MCR
(ml/min)
=
-
increment in
each rat while conscious and while unconscious was the average of the two individual values obtained during each state minus the prolactin concentration in the plasma before the start of prolactin infusion.
Secretion rate ofprolactin during suckling From the mean MCR data obtained in conscious rats, together with the mean plasma con¬ centration of prolactin during suckling, it was possible to calculate the mean secretion rate (SR) of prolactin during suckling for the group of rats on day 14 and 20 post partum as follows : SR (ng/min) MCR (ml/min) multiplied by plasma concentration at steady state (ng/ml) during suckling. Statistical comparisons were made using Student's r-test. =
RESULTS
plasma prolactin concentration in rats on day 13-14 post partum rose steadily during suckling from a basal level of 27 ng/ml and reached a plateau level of 266 ng/ml after 30 min. The prolactin concentration in the same rats when retested 1 week later rose more slowly in response to suckling from a basal level of 24 ng/ml and attained a plateau level of 180 ng/ml which was significantly lower than that observed on day 14 (Fig. 1, Table 1). The use of 14-day-old pups to provide the suckling stimulation in lactating rats on day 21 did not alter the plasma prolactin profile from that seen when 21-day-old pups provided the stimulus (Fig. 2). Thus a difference in the suckling stimulation from the older pups did not appear to be a factor in the reduced prolactin concentration observed on day 21.
The average
Metabolic clearance rate The prolactin concentration rose to an average of 168 ng/ml above pre-infusion levels follow¬ ing 35 min of infusion of rat prolactin (equivalent to 472 ng RP-1 rat prolactin (5-19 mu.)/ min) to conscious rats on day 14 post partum (Table 1). Infusing the same amount of prolactin in a group of conscious rats which had been lactating for 21 days gave average prolactin values of 187 ng/ml above basal levels (Table 1). The concentration was essentially the same after the 25 and 35 min infusion in each group. Infusion of prolactin into 14- and 21-daylactating rats under urethane anaesthesia gave average prolactin values at equilibrium of 204 and 232 ng/ml respectively. None of these equilibrium prolactin values differed significantly. The basal levels of prolactin in rats sampled similarly but not receiving prolactin infusion were quite stable and were not influenced by urethane anaesthesia (Fig. 3). The MCR was then calculated for each rat from the average prolactin concentration attained above the pre-infusion level, before and after urethane anaesthesia, divided into the infusion rate of rat prolactin (equivalent to 472 ng RP-1 rat prolactin (5-19 mu.)/min). This yielded rates before and after urethane anaesthesia of 2-8 and 2-3 ml/min for day 14 postpartum rats and 2-5 and 2-0 ml/min for day 21 post-partum rats (Table 1). The decrease in MCR as a result of anaesthesia was consistent within both groups of rats but the 0-5 ml/min difference »vas not statistically significant for rats on either day of lactation.
300
I
200
ce
"*
. cd Vi
S
100
CL.
% 60 45 15 30 Time of suckling (min) Fig. 1. Effect of suckling six pups on the plasma concentration of prolactin on day 14 (·) and on day 21 (o) of lactation. The same group of eight rats was tested on both days. The arrow indicates the exchange of the original litters with hungry foster litters. Values are means ± s.e.m.
200
|¿2 O 1-
100
-
I
.3 CL
15
30
45
Time of suckling
(min) (·) and four 21-day-old
Fig. 2. Effect of suckling four 14-day-old pups pups (O) concentration of prolactin in rats 21 days post partum. Values are means ± s.e.m. Secretion rate
ofprolactin
on
the
plasma
The average MCR obtained from conscious lactating rats multiplied by the average prolactin concentration at equilibrium achieved during suckling, above basal levels before suckling, yielded estimates of rates of secretion of prolactin into the circulation of 665 and 392 ng/min
for rats on day 14 and day 21 of lactation respectively (Table 1). Individual values for secre¬ tion rates could not be determined, however, since MCR and prolactin profiles during suck¬ ling were not obtained from the same rats. Table 1. Metabolic clearance rate (MCR)* ofprolactin in conscious and unconscious rats and secretion rates ofprolactin in conscious lactating rats on days 14 and 21 of lactation. Values are means
± s.e.m.
Infusion of prolactin (472 ng (5-19 mu.)/min) Conscious
Suckling
Unconscious
Equilibrium plasma Equilibrium plasma Equilibrium plasma Secretion concn during concn during concn during rate* MCR MCR lactation infusionf (ng/ml) (ml/min) infusionf (ng/ml) (ml/min) sucklingt (ng/ml) (ng/min) Day of 14 21 *
168 ±24
2-81 ±0-49 2-52 ±0-29
187±24
204±15 232 ±50
2-31 ±0-14 2-03 ±0-37
237±18 156±17J
664 392
See text for method of calculation.
t Represents concentration attained minus the concentration in plasma before the infusion of prolactin or commencement of suckling. Î Significantly less than on day 14 (P
