PROLACTIN INHIBITION TEST WITH l-DOPA: DECREASE AND RESTORATION OF PLASMA PROLACTIN LEVELS IN THE RAT BY A PERIPHERAL PROCESS
A. A.
P. C. SAHULEKA, G. H. VAN GALEN H. G. KWA Netherlands The Cancer Institute, Antoni van Leeuwenhoekhuis, Biology, Sarphatistraat 108, Amsterdam, The Netherlands
VAN DER
GUGTEN,
AND
Department of
(Received 27 June 1975) SUMMARY
l-DOPA, within 30 min after administration, induced a highly significant decrease of plasma prolactin levels (phase 1) in a number of groups of rats, differing in age and/or endocrine status, apparently by direct inhibition of prolactin release from the pituitary. Three hours after administration of l-DOPA these low plasma prolactin concentrations in treated animals had increased (phase 2) and did not differ significantly from levels in control animals, indicating that the effect of l-DOPA on plasma prolactin levels is only of short duration. During this process some interesting phenomena were observed, especially in the animals treated with oestrone. The elimination rate of prolactin from plasma was very high (t\m=1/2\ 2\m=.\8min), as indicated by decreasing concentrations of the hormone during phase 1. Pituitary prolactin content did not change during phase 1, suggesting that prolactin synthesis was also stopped. Notwithstanding the high elimination rate, plasma prolactin regained initial concentrations in phase 2, suggesting release of a substantial part of the pituitary prolactin content. The latter, however, remained constant during the whole experiment (i.e. before l-DOPA administration and during phase 1 as well as phase 2). The results suggested another working mechanism of l-DOPA in decreasing plasma prolactin levels, namely by stimulating the uptake of this hormone in the periphery. After the effect of l-DOPA had ceased, most of the prolactin from the periphery returned into the bloodstream, causing a rapid restoration of plasma prolactin levels without substantial release from the pituitary. The nature of the processes responsible for the peripheral uptake of prolactin is discussed. =
INTRODUCTION
A number of experiments in the rat shows that prolactin secretion is inhibited by the increase of brain dopamine produced by l-DOPA administration, van Maanen & Smelik (1968) demonstrated that dopamine was the predominant catecholamine in the ventral hypo¬ thalamus and that reserpine implants deplete dopamine in this region. Donoso, Bishop, Fawcett, Krulich & McCann (1971) demonstrated that any induced alteration of dopamine synthesis (e.g. l-DOPA treatment) was associated with a converse change in plasma prolactin concentration. The routes by which the pituitary gland is inhibited from secreting prolactin are still under discussion. Some authors suggest that dopamine does not affect prolactin release by direct action on the pituitary gland, but increases the activity of prolactin-release inhibiting factor (PIF) in the hypothalamic-hypophysial portal blood (Kamberi, Mical & Porter, 1971a, b,
c). Starting from this concept, Lu & Meites (1971, 1972) explained that the observed effect of l-DOPA (the precursor of dopamine) in decreasing plasma prolactin was caused by increased brain catecholamine synthesis, associated with an increase in hypothalamic PIF synthesis and release. The latter was increased to such an extent that it became measur¬ able, by bioassay, in the general circulation of intact female rats. Other authors, however, suggest (van Maanen & Smelik, 1968) or have demonstrated (MacLeod, 1974; MacLeod & Lehmeyer, 1974; Takahara, Arimura & Schally, 1974a, b) that dopamine acts directly on the hypophysis, blocking prolactin release. Hill-Samli & MacLeod (1974) even propose that dopamine itself represents the PIF. Most studies dealing with the effect on prolactin secretion of l-DOPA, dopamine and substances that influence their conversion, report only the ultimate effect at either a hypo¬ thalamic or hypophysial level. Plasma prolactin levels in these cases are used as indicators of the prolactin-release inhibiting effect of any of these drugs in vivo. Little attention has been paid to the processes involved in the acute elimination of prolactin from the blood circulation (after l-DOPA administration) as well as to those further involved in restoring plasma prolactin levels to initial (pretreatment) values. It has recently been demonstrated that initial plasma prolactin levels are poor indicators of the capacity of the pituitary to synthesize and release prolactin. Increase of plasma prolactin, induced by perphenazine, reflected differences in the regulation of prolactin release between groups of rats, varying in endocrine status, which had similar prolactin levels before perphenazine treatment, possibly reflecting differences between the groups in ability to mobilize PIF synthesis and release (van der Gugten, Sahuleka, van Galen & Kwa, 1976). In the present experiments, employing comparable groups of rats, the reaction pattern of plasma prolactin to a single intraperitoneal injection of l-DOPA was investigated. In view of rather unexpected results in the first series of experiments, the influence of L-DOPA treatment on both pituitary and plasma prolactin content was analysed at various intervals. MATERIALS AND METHODS
Animals and treatments Rats of the highly inbred strain R-Amsterdam were used. They were fed standard laboratory diet (Hope Farms, Woerden, Holland) and received tap water or oestrogenized drinking water (Oestrone, Organon, 2 mg/1) ad libitum. They were housed, three or four to a Macrolon cage (38x25x15 cm) with wood-shavings and Sterolite as bedding, in a light- (06.0018.00 h) and temperature- (23+1-5 °C) controlled room. Three different experiments were carried out on 31 groups of rats as summarized in Table 1. In Experiment 1, 128 animals were divided into 12 experimental and 12 control groups. Gonadectomy of both male and female rats was carried out 18-25 days before treatment with l-DOPA. Oestrone in drinking water was supplied to the appropriate groups starting on the day of operation. In Experiment 2, two groups of oestrone-treated castrated rats bearing prolactin-producing pituitary tumours, were used. In the first group, in-situ pituitary tumours had been induced by fortnightly s.c. implantation of an oestrone-cholesterol pellet containing 200 µ% oestrone, supplemented with oestrone in drinking water (2 mg/1), both given for approximately 1 year. In the second group, growth of primary transplants of these tumours was sustained by the same pellet treatment, reduced to once a month, combined with oestrone in drinking water, both during approximately 1 year. The latter régime is not sufficient to induce tumour formation of the hypophysis in situ. In Experiment 3, 40 8-weekold castrated rats were divided into four experimental groups killed at various times after l-DOPA treatment, and one control group. Castration was carried out and oestrone treatment (2 mg/1 drinking water) was initiated 24 days before L-DOPA administration.
On the
day of experiment each rat was injected i.p. with either 0-5 ml solvent/100 g body weight (0-005 M-Na2S205 adjusted with 1 M-HC1 to pH 3-5; controls) or 6 mg l-DOPA/100 g body weight (suspension of 6 mg 3,4-dihydroxy-L-phenylalanine, Merck/0-5 ml solvent). For each rat the time of administration was defined as 0 h. Blood samples were taken from the retro-bulbar plexus of the eye under light ether anaesthesia, the first sample (time 0) 10 s before injection of vehicle or L-DOPA, the other samples at the intervals indicated on the Figs. At 180 min (Experiment 1) and at 0, 15, 30, 45 and 120 min (for the various groups in Experiment 3) the animals were killed, their pituitary glands dissected out, weighed indi¬ vidually on a Mettler balance and homogenized with saline (1 ml/10 mg wet tissue) in a small glass tube using 10 strokes of a Teflon pestle. Hypophyses and pituitary tumours of the animals in Experiment 2 were not studied for prolactin content. Heparinized blood samples and homogenized pituitaries were centrifuged (10 min, 500 g, 4 °C). All individual plasma samples and pituitary extracts were stored at —20 °C, until assayed for prolactin by radioimmunoassay. Table 1. Details
of the experimental groups No. of animals treated with l-DOPA
Young male rats 1. Intact 2. Castrated 3. Castrated + oestrone b. Adult male rats 1. Intact 2. Castrated 3. Castrated + oestrone
No. of (6 mg/100 g body Age (weeks) controls wt, i.p.) Experiment 1
Oestrone treatment
(days)
(g;
Body
wt
means ±
s.d.)
a.
c.
Young female rats
1. Intact 2. Spayed 3. Spayed + oestrone d. Adult female rats 1. Intact 2. Spayed 3. Spayed + oestrone e.
Castrated adult male rats 1. Bearing pituitary tumours in situ 2. Bearing pituitary tumour
7-8 8
3 4 3
42 23 23
3 3 3
7 8
8
4 3 3
25 18 18
4 2 3
7
24
19
330 ±39 329 ±24 298 ±22
127±10 7 7
157+ 7
25
12 6 7
106±15 203 ± 8
202±16 18
181+ 7
320-350 270-330
234 ± 25 254 ±23
24
145±14
Experiment 2 60-64 52-56
transplants
f. Young male rats 1. Castrated + oestrone
99±11 153 + 14 135 + 16
—
—
Experiment 3 8
8
Oestrone treatment: 2 mg/1 drinking water; in addition, for every 2 weeks (group 1) or every month (group 2).
32
Experiment 2, a 200/ig pellet
was
implanted
s.c.
Radioimmunoassay procedures Radioimmunoassay was carried out as described earlier (Kwa, van der Gugten & Verhof¬ stad, 1969; Kwa, van der Gugten, Sala & Verhofstad, 1972; van der Gugten et al. 1976), using 0-3 ng 125I-labelled rat prolactin per tube. Slopes of inhibition curves of all samples (5 points each) were checked for parallelism with the slope of the reference curves, in order
detect any decrease in immunoreactivity of the endogenous prolactin that might occur after L-DOPA treatment. This would show up in aberrant slopes of the inhibition curves that are no longer characteristic of intact prolactin (Bryant & Greenwood, 1968; van der Gugten & Kwa, 1970). Statistical analysis Student's /-test was used to assess the statistical significance of the results, which are ex¬ pressed as means ± s.d. to
Table 2. Plasma prolactin levels 10 s before and 30 min
after l-DOPA administration Experiment 1 (means ± s.d.)
a.
Group Young male rats
1. Intact 2. Castrated 3. Castrated + oestrone b. Adult male rats 1. Intact 2. Castrated 3. Castrated + oestrone c. Young female rats 1. Intact 2. Spayed 3. Spayed + oestrone d. Adult female rats 1. Intact 2. Spayed 3. Spayed + oestrone Number of animals in
10 s before
30 min after
(ng/ml)
(ng/ml)
13-6 + 7-6 7-9 + 5-1
(10) (10) 184-5±65-8 (12) 300±7-6
(10)
001 001 0001
(7) (7) (7)
0001 0001 0001
(8)
(7) (7)
0001 001 0001
4-7±2-8 (12) 1-2±0-7 (6) l-4±0-7 (7)
0001 0-01 001
4·5±1·7
9-3±6-2
37-3±22-4 (12) 19-6 + 7-9 (10) 270-4±69-7 (10)
1-5±1-5 6-7±4-l 19-6±9-9
48-2±28-6 (16) 15-7 ±8-6 (8) 52-2±35-5 (10)
parentheses. Oestrone
(P
A. A.
P. C. SAHULEKA, G. H. VAN GALEN H. G. KWA Netherlands The Cancer Institute, Antoni van Leeuwenhoekhuis, Biology, Sarphatistraat 108, Amsterdam, The Netherlands
VAN DER
GUGTEN,
AND
Department of
(Received 27 June 1975) SUMMARY
l-DOPA, within 30 min after administration, induced a highly significant decrease of plasma prolactin levels (phase 1) in a number of groups of rats, differing in age and/or endocrine status, apparently by direct inhibition of prolactin release from the pituitary. Three hours after administration of l-DOPA these low plasma prolactin concentrations in treated animals had increased (phase 2) and did not differ significantly from levels in control animals, indicating that the effect of l-DOPA on plasma prolactin levels is only of short duration. During this process some interesting phenomena were observed, especially in the animals treated with oestrone. The elimination rate of prolactin from plasma was very high (t\m=1/2\ 2\m=.\8min), as indicated by decreasing concentrations of the hormone during phase 1. Pituitary prolactin content did not change during phase 1, suggesting that prolactin synthesis was also stopped. Notwithstanding the high elimination rate, plasma prolactin regained initial concentrations in phase 2, suggesting release of a substantial part of the pituitary prolactin content. The latter, however, remained constant during the whole experiment (i.e. before l-DOPA administration and during phase 1 as well as phase 2). The results suggested another working mechanism of l-DOPA in decreasing plasma prolactin levels, namely by stimulating the uptake of this hormone in the periphery. After the effect of l-DOPA had ceased, most of the prolactin from the periphery returned into the bloodstream, causing a rapid restoration of plasma prolactin levels without substantial release from the pituitary. The nature of the processes responsible for the peripheral uptake of prolactin is discussed. =
INTRODUCTION
A number of experiments in the rat shows that prolactin secretion is inhibited by the increase of brain dopamine produced by l-DOPA administration, van Maanen & Smelik (1968) demonstrated that dopamine was the predominant catecholamine in the ventral hypo¬ thalamus and that reserpine implants deplete dopamine in this region. Donoso, Bishop, Fawcett, Krulich & McCann (1971) demonstrated that any induced alteration of dopamine synthesis (e.g. l-DOPA treatment) was associated with a converse change in plasma prolactin concentration. The routes by which the pituitary gland is inhibited from secreting prolactin are still under discussion. Some authors suggest that dopamine does not affect prolactin release by direct action on the pituitary gland, but increases the activity of prolactin-release inhibiting factor (PIF) in the hypothalamic-hypophysial portal blood (Kamberi, Mical & Porter, 1971a, b,
c). Starting from this concept, Lu & Meites (1971, 1972) explained that the observed effect of l-DOPA (the precursor of dopamine) in decreasing plasma prolactin was caused by increased brain catecholamine synthesis, associated with an increase in hypothalamic PIF synthesis and release. The latter was increased to such an extent that it became measur¬ able, by bioassay, in the general circulation of intact female rats. Other authors, however, suggest (van Maanen & Smelik, 1968) or have demonstrated (MacLeod, 1974; MacLeod & Lehmeyer, 1974; Takahara, Arimura & Schally, 1974a, b) that dopamine acts directly on the hypophysis, blocking prolactin release. Hill-Samli & MacLeod (1974) even propose that dopamine itself represents the PIF. Most studies dealing with the effect on prolactin secretion of l-DOPA, dopamine and substances that influence their conversion, report only the ultimate effect at either a hypo¬ thalamic or hypophysial level. Plasma prolactin levels in these cases are used as indicators of the prolactin-release inhibiting effect of any of these drugs in vivo. Little attention has been paid to the processes involved in the acute elimination of prolactin from the blood circulation (after l-DOPA administration) as well as to those further involved in restoring plasma prolactin levels to initial (pretreatment) values. It has recently been demonstrated that initial plasma prolactin levels are poor indicators of the capacity of the pituitary to synthesize and release prolactin. Increase of plasma prolactin, induced by perphenazine, reflected differences in the regulation of prolactin release between groups of rats, varying in endocrine status, which had similar prolactin levels before perphenazine treatment, possibly reflecting differences between the groups in ability to mobilize PIF synthesis and release (van der Gugten, Sahuleka, van Galen & Kwa, 1976). In the present experiments, employing comparable groups of rats, the reaction pattern of plasma prolactin to a single intraperitoneal injection of l-DOPA was investigated. In view of rather unexpected results in the first series of experiments, the influence of L-DOPA treatment on both pituitary and plasma prolactin content was analysed at various intervals. MATERIALS AND METHODS
Animals and treatments Rats of the highly inbred strain R-Amsterdam were used. They were fed standard laboratory diet (Hope Farms, Woerden, Holland) and received tap water or oestrogenized drinking water (Oestrone, Organon, 2 mg/1) ad libitum. They were housed, three or four to a Macrolon cage (38x25x15 cm) with wood-shavings and Sterolite as bedding, in a light- (06.0018.00 h) and temperature- (23+1-5 °C) controlled room. Three different experiments were carried out on 31 groups of rats as summarized in Table 1. In Experiment 1, 128 animals were divided into 12 experimental and 12 control groups. Gonadectomy of both male and female rats was carried out 18-25 days before treatment with l-DOPA. Oestrone in drinking water was supplied to the appropriate groups starting on the day of operation. In Experiment 2, two groups of oestrone-treated castrated rats bearing prolactin-producing pituitary tumours, were used. In the first group, in-situ pituitary tumours had been induced by fortnightly s.c. implantation of an oestrone-cholesterol pellet containing 200 µ% oestrone, supplemented with oestrone in drinking water (2 mg/1), both given for approximately 1 year. In the second group, growth of primary transplants of these tumours was sustained by the same pellet treatment, reduced to once a month, combined with oestrone in drinking water, both during approximately 1 year. The latter régime is not sufficient to induce tumour formation of the hypophysis in situ. In Experiment 3, 40 8-weekold castrated rats were divided into four experimental groups killed at various times after l-DOPA treatment, and one control group. Castration was carried out and oestrone treatment (2 mg/1 drinking water) was initiated 24 days before L-DOPA administration.
On the
day of experiment each rat was injected i.p. with either 0-5 ml solvent/100 g body weight (0-005 M-Na2S205 adjusted with 1 M-HC1 to pH 3-5; controls) or 6 mg l-DOPA/100 g body weight (suspension of 6 mg 3,4-dihydroxy-L-phenylalanine, Merck/0-5 ml solvent). For each rat the time of administration was defined as 0 h. Blood samples were taken from the retro-bulbar plexus of the eye under light ether anaesthesia, the first sample (time 0) 10 s before injection of vehicle or L-DOPA, the other samples at the intervals indicated on the Figs. At 180 min (Experiment 1) and at 0, 15, 30, 45 and 120 min (for the various groups in Experiment 3) the animals were killed, their pituitary glands dissected out, weighed indi¬ vidually on a Mettler balance and homogenized with saline (1 ml/10 mg wet tissue) in a small glass tube using 10 strokes of a Teflon pestle. Hypophyses and pituitary tumours of the animals in Experiment 2 were not studied for prolactin content. Heparinized blood samples and homogenized pituitaries were centrifuged (10 min, 500 g, 4 °C). All individual plasma samples and pituitary extracts were stored at —20 °C, until assayed for prolactin by radioimmunoassay. Table 1. Details
of the experimental groups No. of animals treated with l-DOPA
Young male rats 1. Intact 2. Castrated 3. Castrated + oestrone b. Adult male rats 1. Intact 2. Castrated 3. Castrated + oestrone
No. of (6 mg/100 g body Age (weeks) controls wt, i.p.) Experiment 1
Oestrone treatment
(days)
(g;
Body
wt
means ±
s.d.)
a.
c.
Young female rats
1. Intact 2. Spayed 3. Spayed + oestrone d. Adult female rats 1. Intact 2. Spayed 3. Spayed + oestrone e.
Castrated adult male rats 1. Bearing pituitary tumours in situ 2. Bearing pituitary tumour
7-8 8
3 4 3
42 23 23
3 3 3
7 8
8
4 3 3
25 18 18
4 2 3
7
24
19
330 ±39 329 ±24 298 ±22
127±10 7 7
157+ 7
25
12 6 7
106±15 203 ± 8
202±16 18
181+ 7
320-350 270-330
234 ± 25 254 ±23
24
145±14
Experiment 2 60-64 52-56
transplants
f. Young male rats 1. Castrated + oestrone
99±11 153 + 14 135 + 16
—
—
Experiment 3 8
8
Oestrone treatment: 2 mg/1 drinking water; in addition, for every 2 weeks (group 1) or every month (group 2).
32
Experiment 2, a 200/ig pellet
was
implanted
s.c.
Radioimmunoassay procedures Radioimmunoassay was carried out as described earlier (Kwa, van der Gugten & Verhof¬ stad, 1969; Kwa, van der Gugten, Sala & Verhofstad, 1972; van der Gugten et al. 1976), using 0-3 ng 125I-labelled rat prolactin per tube. Slopes of inhibition curves of all samples (5 points each) were checked for parallelism with the slope of the reference curves, in order
detect any decrease in immunoreactivity of the endogenous prolactin that might occur after L-DOPA treatment. This would show up in aberrant slopes of the inhibition curves that are no longer characteristic of intact prolactin (Bryant & Greenwood, 1968; van der Gugten & Kwa, 1970). Statistical analysis Student's /-test was used to assess the statistical significance of the results, which are ex¬ pressed as means ± s.d. to
Table 2. Plasma prolactin levels 10 s before and 30 min
after l-DOPA administration Experiment 1 (means ± s.d.)
a.
Group Young male rats
1. Intact 2. Castrated 3. Castrated + oestrone b. Adult male rats 1. Intact 2. Castrated 3. Castrated + oestrone c. Young female rats 1. Intact 2. Spayed 3. Spayed + oestrone d. Adult female rats 1. Intact 2. Spayed 3. Spayed + oestrone Number of animals in
10 s before
30 min after
(ng/ml)
(ng/ml)
13-6 + 7-6 7-9 + 5-1
(10) (10) 184-5±65-8 (12) 300±7-6
(10)
001 001 0001
(7) (7) (7)
0001 0001 0001
(8)
(7) (7)
0001 001 0001
4-7±2-8 (12) 1-2±0-7 (6) l-4±0-7 (7)
0001 0-01 001
4·5±1·7
9-3±6-2
37-3±22-4 (12) 19-6 + 7-9 (10) 270-4±69-7 (10)
1-5±1-5 6-7±4-l 19-6±9-9
48-2±28-6 (16) 15-7 ±8-6 (8) 52-2±35-5 (10)
parentheses. Oestrone
(P
