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Comparison between rat prolactin radioimmunoassay and bioassay values under different experimental and physiological conditions
131
Molecuiur und Cellular Endocrinology, 39 (1985) 131- 140 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01259
Comparison between rat prolactin radioimmunoassay and bioassay values under different experimental and physiological conditions Robert E. Owens, Felipe F. Casanueva Department o/Physiology,
Faculty of Medicine, University of Manitoba, (Received
Keywords:
lymphoma
cell lactogenic
* and Henry G. Friesen
bioassay;
16 August
estrogen;
1984; accepted
molecular
770 Bannatyne Aoenue. Winnipeg (Canada)
24 October
1984)
forms; estrous cycle; morphine;
TRH;
CR-154; ether;
haloperidot.
Summary Serum rat PRL concentrations were compared using values determined by RIA and the Nb2 lymphoma cell bioassay (Nb2BA). Rat serum samples were obtained under different physiological conditions and after the administration of pharmacological agents known to affect PRL secretion. Of the treatments examined, estrogen, morphine bromocriptine and haloperidol significantly altered the relationship between Nb2BA and RIA estimates of PRL. The estrogen-induced increase in PRL levels of ovariectomized females and the proestrus surge of PRL in intact females led to higher bioassay than RIA estimates of PRL. Treatment with haloperidol, bromocriptine and morphine altered the relationship, favoring immunoreactive more than bioactive hormone, and reversing the pretreatment Nb2BA/RIA ratio. No discrepancies between estimates of PRL by the two assays were noted in untreated males, diestrous, estrous and ovariectomized females, or following ether or TRH administration. These results confirm previous observations of discrepancies between rat serum bioassay and RIA estimates, and the data suggest that different forms of prolactin are present in the circulation at different times.
There is increasing evidence that more than one form of PRL is present in the serum and pituitaries of the rat. Previous reports have documented discrepancies in estimates of PRL by pigeon crop sac bioassay and RIA (Asawaroengchai and Nicoll, 1977; Asawaroengchai et al., 1978; Leung et al., 1978; Gala and Kuo, 1972). Recently PRL molecular heterogeneity due to charge or molecular weight differences and dissociations between RIA and bioactivity (BA) potencies have been reported (Sinha and Gilligan,
* To whom reprint requests should be addressed. Present address: Department of Internal Medicine, Endocrine Unit, Faculty of Medicine, Compostela University, Santiago de Compostela (Spain). 0303-7207/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
1981; Lawson, 1980; Wallis et al., 1980; Mittra, 1980a, b; Lawson et al., 1980; Haisenlender et al., 1984). Early attempts to study circulating forms of PRL in the rat by comparing RIA and bioassay estimates were limited primarily to pituitary extracts and pooled samples, as available bioassays lacked the sensitivity required to measure levels of hormone in small serum samples. These bioassays were cumbersome and with poor reproducibility. Recently, using the sensitive, lactogen-specific, Nb2 rat lymphoma cell bioassay (Nb2BA), Klindt et al. (1982) have reported important discrepancies in RIA/BA values in male rats. That finding, however, was not entirely confirmed by another report (Lawson et al., 1982). Ltd.
132
In an attempt to elucidate the underling mechanism of this Nb>ZBA/RIA discrepancy, and to determine if it occurs in different secretory states, we have compared the Nb2BA and RIA estimates of PRL in serum samples obtained from rats under a variety of physiological or experimental conditions and after the administration of pharmacological agents known either to stimulate or inhibit PRL secretion.
posed of rats in different physiological states and/or after pharmacological treatments known to affect serum PRL concentrations. With the exception of the ether-treated groups, freely moving unanesthetized rats were employed. The experimental protocol was cleared through the Institutional Animal Care Committee. Blood collection For the estrous cycle study, animals were bled twice in the morning and twice in the afternoon (see Table 1). The ovariectomized females in the groups designated ovariectomy and ovariectomy plus estradiol valerate were bled at the same time periods as the untreated animals on a day which was 1 month post-ovariectomy and 2 weeks following estradiol valerate (2 mg/rat). All other experiments were performed in the morning starting at 0900 h. Daily vaginal smears were taken for at least 8 days, or two complete estrous cycles,
Materials and methods Animals Sprague-Dawley rats (Canadian Breeding Labs, Montreal) weighing 200-300 g were utilized for this study. The animals were housed under environmentally controlled conditions, with a 12 h light and 12 h dark cycle. All animals were fed Purina rat chow and water ad libitum. The study included 15 experimental groups (Table 1) com-
TABLE
1
EXPERIMENTAL GROUPS CONCENTRATIONS Treatment
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. a b ’ d ’ ’ 8 b
group
Untreated males Diestrous females Proestrous females Estrous females Gvariectomy + E, a Ovariectomy Bromotxiptine b females Ether males Ether females TRH ’ males TRH females Morphine d males Morphine females Haloperidol’ males Haloperidol females
AND
PROBABILITY
LEVELS
FOR
N
Dose/route
Schedule of blood collection
6 7 5 8 6 5 6 6 6 5 5 6 8 6 7
NA NA NA NA 2 mg/s.c. ’ NA 1 mg,/kg,/i.p. 2.5 min 2.5 min 20 pg/i.p. 20 pg/i.p. 10 mg/kg/i.p. 10 mg/kg/i.p. 2 mg/kg/i.p. 2 mg/kg/i.p.
1030.1130,1400,1500 1030,1130,140@, 1500 1030,1130,1400,1500 1030,1130,1400,1500 1030,1130,1400,1500 1030.1130,1400,1500 0 B, 30,60,90 min 0, 2.5, 5.15 min 0, 2.55, 15 min 0, 10, 20, 30 min 0, 10, 20, 30 min 0,20,40,60 min 0,20,40,60 min 0, 30,60,90 min 0,30,60,90 min
OF TIMEX ASSAY WITHIN
EFFECT
Reference
h h h h h h
h
Smith et al. (1975) Lawson and Stevens (1980) Muller et al. (1981) Grosvenor et al. (1979) Mueller et al. (1973) Bruni et al. (1977)
Delestrogen, estradiol valerate (Squibb Canada Inc., Montreal, Canada). Bromocriptine mesylate, CB-154 (Sandoz Ltd., Basle, Switzerland). TRH relefact, Protirelin (Hoechst Canada Inc., Montreal, Canada). Morphine sulphate (Abbott Laboratories, Ltd., Montreal, Canada). Haloperidol, Haidol (McNeil Laboratories, Ltd., Stouffville, Ontario, Canada). Estradiof valerate administered 2 weeks before the day of blood collection which was 1 month Time 0 designates pretreatment biood samples. Other times designate minutes post-treatment. In the references cited the same times for blood collection and/or drug dosages were used.
ON PRL
Source of variation p = (time X assay)
Butcher et al. (1974)
Dickerman
GROUP
et al. (1972)
post~va~~tomy.
06609 0.8349 0.0082 0.3345 0.0025 0.3486 0.0932 0.6727 0.1969 0.4592 0.2761 0.0058 0.0796 ox!61 0.0031
133
before the animals were used. Bleeding day for males was chosen for convenience. Blood samples were collected as previously published (Casanueva et al., 1982) and left overnight at 4°C to clot. After centrifugation, serum was stored at -20°C until assayed for prolactin. Quantitation of PRL The concentrations of immunoreactive rat PRL were measured by a double antibody RIA using the protocol and materials supplied by NIAMDD. Bioactive PRL was quantitated by the previously described Nb2 lymphoma cell bioassay (Nb2BA) (Tanaka et al., 1980). The average of duplicate determinations of each sample was expressed in terms of the NIAMMD rat prolactin RP-1 standard for both assays. All samples from a given treatment were assayed together. The coefficients of variation for inter- and intra-assay variability were 14% and 7% for the RIA and 20% and 9% for the bioassay, respectively. Statistical analysis The Nb2BA and RIA estimates of PRL levels for individual rats from each group at each time point were used to calculate the mean Nb2BA and RIA PRL values. The mean from the individual Nb2BA/RIA ratios was obtained at each time and for each experimental group. Three statistical approaches were used to evaluate the data. First, a comparison of mean Nb,ZBA, RIA and Nb2/RIA ratio at each time period was carried out, followed by an estimate of the Nb2BA/RIA ratio over time for the various groups. Nb2BA and RIA comparisons at each time were made using a paired t-test. Individual Nb2BA/RIA values were determined for each blood sample collected, and the means of these values for the different treatments compared by one-way ANOVA, with differences between means tested by Tukey’s procedure. ANOVA (repeated measures design) with two repeated factors, time and assay, was applied to the data from each of the 15 groups to test whether the differences between RIA and Nb2BA were consistent averaged across the time period. The observation of a significant (p < 0.05) time X assay interaction for a particular group indicated that the difference between the Nb2BA and RIA estimates of PRL
changed significantly over amined for that group.
the
time
period
ex-
Results From each experimental group four kinds of statistical information were obtained: (a) mean RIA PRL values compared with mean BA PRL values at each time sampled (figures, upper panel); (b) mean BA/RIA ratio at each time sampled compared with the basal BA/RIA ratio (figures, lower panel); (c) RIA PRL values over time compared as a whole group against BA PRL values over time (time x assay interaction, Table 1); and (d) correlation coefficient of the regression line of BA and RIA estimates into the experimental group. Untreated males Serum PRL as measured by both RIA and Nb2BA increased over the time period examined in untreated males (Fig. 1). The Nb2BA estimates of PRL were consistently higher than those of the RIA, but only the initial blood sample at 1030 h was significantly higher (p -C0.01). Examined as a group, the Nb2BA values over time did not behave
UNTREATED
BESTRUS
PROESTRUS
ESTRUS
I
,d TIME OF DAY
Fig. 1. Serum concentrations of PRL as measured by Nb2BA (------)andRIA() at 1030, 1130, 1400 and 1500 h of one day in untreated males and in females during diestrus, proestrus and estrus. Each point represents the mean* SE. NbZBA/RIA (mean* SE) values represented by the bars are depicted in the lower panel directly beneath the time period which they represent. *p < 0.05 indicates that mean Nb2BA and RIA estimates are different at that time period.
134
differently from RIA values over time ( p = 0.6609, Table 1). The mean rfr SE Nb2BA/RIA ratio did not change significantly during the day (Fig. 1). The overall NbZBA/RIA ratio for this group was 1.48 si: 0.11, with a range for individual samples of 0.85-2.35. The calculated correlation coefficient of the regression line of Nb2BA and RIA estimates of this group was 0.99. Estrous cycle During diestrus (Fig. I), PRL levels were low compared to other periods of the estrous cycle, and the Nb2BA measurements were consistently but not significantly higher than RIA estimates. The differences between the two assays examined together over the entire bleeding period remained constant ( p = 0.8349, Table 1). The Nb2BA/RIA ratios also did not change during diestrus. The mean k SE Nb2BA/RIA value in this group was 1.93 + 0.13, with a range of 0.5-3.67. The cortelation coefficient was 0.85. During proestrus (1400, 1500 h), PRL levels were IO-fold higher by RIA and 16fold higher by Nb2BA than those measured at 1030 h. PRL estimate by Nb2BA was 2-fold higher than the RIA estimate ( p -C0.01) at 1400 h, and was not different at the remaining time periods. The divergence of the estimated PRL levels by the two assays in this group led to a significant time X assay interaction ( p = 0.0082, Table 1). The mean Nb,ZBA/RIA ratio did not change during proestrus (Fig. 1). The overall Nb2BA/RIA ratio in this group was 1.95 + 0.16, with a range of 0.88-3.86. For this group the regression line of the Nb2BA and RIA measurements of PRL yielded a correlation coefficient of 0.90. On the day of estrus (Fig. l), PRL concentrations were relatively low in the morning and afterwards elevated by 1400 h. The Nb2BA estimates of PRL were consistently higher than RIA values, being significantly different at 1030, 1130 and 1400 h (p c 0.05). Nb2BA and RIA values followed a similar pattern, and the differences between both groups of estimates remained constant ( p = 0.3345, Table 1). The Nb2BA/RIA ratio did not change with respect to the basal ratio during the day of estrus. The mean * SE ratio for Nb2BA/RIA was 2.08 + 0.14, with a range of
individual values from 0.59 to 3.57. A correlation coefficient of 0.87 was calculated for Nb2BA and RIA values. Ooariectomy and ovariectomy plus estradial valerate In the females ovariectomized 1 month previously, PRL values were low at each time examined (Fig. 2). The Nb2BA values were higher than those of RIA at each point, and significantly so at 1030 h (p < 0.01). There were no differences between Nb2BA/RIA ratios in the four time periods studied. The mean + SE NbZBA/RIA value for all the samples was 1.84 + 0.19, with a range of 0.63-3.38. The correlation coefficient for this group was 0.89. Estradiol valerate administered to ovariectomized females 2 weeks previously, significantly elevated PRL values compared with the untreated
1800
1800
---
Nb2BA RIA l
ovx
*A
2ook
TIME OF DAY 3.0 r-
Fig. 2. Serum concentrations of PRL as measured by Nb2BA -) at 1030,1130,1400 and 1500 h for (------)andRIA( females 1 months post-ovariectomy without treatment (0) (OVX) and 1 month post-ovariectomy with 2 mg of estradiol valerate per animal after 2 weeks (A) (OVX + I!.,). Each point represents the mean&SE. In the lower panel mean_+SE Nb,ZBA/RIA values are depicted for OVX (solid bars) and OVX + EZ (open bars) directly beneath the time period which they represent. * p c 0.05 indicates that mean Nb2BA and RIA estimates are different at that time period.
NbZ/RIA
80+47 6k 1
148+ ilk
75 5
115+25 213+76
2.66kO.43 1.61k0.36
155+50 9Ok26 *
RIA
NbZ/RIA
1.58kO.08 0.76*0.55 20
Nb2
19 17
RIA
91k 51+
0
58k12 92k29
* p < 0.05 RIA vs. N62BA values at different time periods. ** p -c 0.05 Nb2BA/RIA ratio vs. same ratio at time 0.
Morphine males Morphine females
Time (min):
TRH males TRH females
10 Nb,Z/RIA
RIA
Nb2
RIA
32+11
0
1.46kO.12
Time (min):
194+110
RIA
Nb2
RIA
114+60
30
0
CB-154 females
Time (min):
178+64 65+21
Nb2
168k38 193zk84
Nb2
31 f 14
Nb2
SERUM PRL VALUES (ng/mI) AFTER CB-154, TRH AND MORPHINE
TABLE 2
**
1.06*0.10 ** 0.71 jzO.10 **
Nb2/RIA
1.51k0.14 0.81kO.38
NbZ/RIA
0.74*0.14
NbZ/RIA
124+22 101+61
RIA
40
4
2
109+21 91+56
Nb2
155 f 30 176+48
Nb2
11+
Nb2
(MEAN f SE)
94+25 213k48
RIA
20
17*
RIA
60
ADMINISTRATION
Nb,Z/RIA **
0.86+0.06 ** 0.65kO.18 **
NbZ/RIA
2.05 kO.46 0.81 kO.20
Nb,Z/RIA
0.53*0.07
5
106+49 22+ 7
RIA
60
124k12 48k17
RIA
30
1s+
RIA
90
6
89+41 9* 9
Nb2
186+21 43k21
Nb2
14+
Nb2
NbZ/RIA
0.88kO.04 ** 0.35+0.11 **
NbZ/RIA
1.52kO.15 1.07kO.32
NbZ/RIA
0.55&0.10 **
E
136
ovariectomized animals (Fig. 2). Nb2BA PRL estimates were significantly higher (p c 0.05) than those of the RIA at 1130, 1400 and 1500 h. The divergence of the two assays during the day resulted in a significant time x assay interaction (p = 0.0025, Table l), suggesting that the differences between estimates were not constant. The Nb2BA/RIA ratios were not different with respect to the basal ratio at the time periods evaluated. The mean + SE Nb,[LBA/RIA value for this subgroup was 1.59 f 0.11, with a range of 0.74-2.74. The correlation coefficient of Nb2BA and RIA estimates of PRL for the group was 0.96.
MALES
180 t
-
TRH The Nb2BA estimates of serum PRL after TRH treatment of male rats were not significantly higher than values determined by RIA (Table 2). The differences between the two assays were found to be consistent when examined over time (p = 0.4592, Table 1). The Nb2BA/RIA values for TRH-treated males ranged from 1.04 to 3.89, with an overall mean + SE of 1.67 f 0.13. The correlation coefficient for this group was 0.93. After treatment of diestrous females with TRH there was an increase in the mean level of PRL as measured by both assays (Table 2). However, the large degree of variation between the animals prohibited the observation of a clear response to this treatment. The differences between the estimates of PRL by Nb2BA and RIA remained unchanged over time (p = 0.2761, Table 1). The mean Nb2BA/RIA values in diestrous females were not
Nb2BA RIA T
0
t
/I',
TIME (mid
d
Bromocriptine (CB-154) Administration of bromocriptine depressed circulating Nb2BA and RIA PRL levels within 30 min (Table 2). Examined over time, the relationship between the estimates of PRL by RIA and Nb2BA were not different ( p = 0.0932, Table 1). The treatment induced an inversion in the BA/RIA ratio; in fact Nb2BA/RIA values were altered ( p < 0.05) at 30, 60 and 90 min as compared to pretreatment values (time 0). The range of Nb2BA/RIA values observed was 0.31-1.84, with a mean + SE of 0.86 + 0.10. The correlation between Nb2BA and RIA estimates of PRL for the bromocriptine group was very good, with a coefficient of 0.99.
---.
220
FEMALES
Y-
r
Fig. 3. Effect of ether on Nb2BA (- - - - - -) and RIA () estimates of PRL in males and diestrous females. Immediately following the collection of a control sample (time 0), animals were exposed to ether for 2 min. Each point represents the mean f SE. In the lower panel, directly beneath the time they represent, mean + SE Nb,ZBA/RIA values are depicted. * p c 0.05 indicates that mean Nb2BA and RIA estimates are different at that time period.
altered by TRH ad~nistration. Overall the Nb,ZBA/RIA ratio for this group was 0.97 + 0.11, with a range of values of 0.41-1.88. The Nb2BA and RIA estimates analyzed by linear regression had a correlation coefficient of 0.97. Ether In males (Fig. 3), levels of PRL by RIA following a 2 min ether exposure rose steadily over the four times examined. The estimates of Nb2BA followed a very similar pattern and were significantly higher than RIA values at each time ( p < 0.05 at 0 and 2.5 min; p < 0.01 at 5 and 15 min). Nevertheless, as groups both assays behave similarly and the differences between both estimates evaluated over time were constant (p = 0.6727, Table 1). No significant changes in the Nb2BA/RIA ratio were noted. The Nb,ZBA/RIA values for this group ranged from 1.10 to 2.34, with a mean i SE of 1.52 + 0.06. The correlation coefficient for ether-treated males was 0.97.
137
In diestrous females, RIA and Nb2BA PRL levels rose rapidly following ether exposure to reach a peak at 5 min. Nb2BA estimates were higher, but significantly so (p < 0.01) only in the pretreatment sample. There were no changes in the Nb2BA/RIA ratio, although this value appeared to be moving toward a 1 : 1 ratio with each subsequent sample. The range of Nb,ZBA/RIA values for ether-treated females was 0.86-2.18, with an overall mean + SE of 1.30 f 0.08. The correlation coefficient of the estimates of the two assays was 0.97. Morphine In males (Table 2) there was no effect of morphine on mean levels of PRL estimated by RIA, but the differences between estimates by Nb2 and RIA over time were altered significantly ( p = 0.0058, Table 1). At 20 and 40 min posttreatment a reversal situation with higher estimates by RIA than Nb2BA occurred. In fact the pretreatment Nb,ZBA/RIA of 2.26 + 0.43 dropped rapidly to 1.05 f 0.10 by 20 min and to 0.86 f 0.06 by 40 min post-treatment. The Nb2BA/RIA values were all significantly ( p < 0.05) depressed following morphine administration compared to control time. The mean value of the BA/RIA ratio was 1.26 * 0.16, and the values ranged from 0.61 to 4.13. The correlation of Nb2BA and RIA PRL estimates yielded a correlation coefficient of 0.92. In diestrous females treated with morphine a similar shift was observed. The Nb2BA estimates were higher than the RIA estimates before morphine treatment (time 0), but the relationship was reversed after treatment, with higher RIA estimates that were significant at 20 min ( p -z 0.05). Over time, the differences between the estimates of PRL by both assays were not changed by morphine, although the probability approached the level of significance (p = 0.0796, Table 1). As expected, with an inversion of the Nb2BA/RIA ratio, the Nb2BA/RIA values were significantly depressed ( p -c 0.05) compared to controls following morphine treatment. The range of Nb,ZBA/RIA values for morphine-treated females was 0.25-3.64, with a mean + SE of 1.12 f 0.26. The correlation of PRL estimates by the two assays was high for this group, with a coefficient of 0.98.
Haloperidol As can be seen in Fig. 4, haloperidol administration caused an increase in circulating PRL levels. In males RIA estimates were higher (p -c 0.05) than Nb2BA values at the three time points examined after haloperidol treatment. The differences were not consistent over time, the Nb2BA and the RIA estimates behaving as different populations under haloperidol (p = 0.0061, Table 1). The NbZBA/RIA ratio reflected that pattern, as a significant ( p < 0.05) decrease from a pretreatment ratio of 1.44 f 0.24 to 0.65 f 0.04,0.73 f 0.04 and 0.63 f 0.06 at 30, 60 and 90 min respectively. The range of Nb2BA/RIA values for haloperidol males was 0.36-2.13, with a mean f SE of 0.86 & 0.04. Assay estimates for this group correlated well, with a coefficient of 0.95.
MALES
24oor
I-
I
FEMALES
2200 2Ow t
moo
r -mA
‘i
1400
> 4 c"
1200 1000 000
Nb2sA
--
t 800
k t
ax 200
l
,,+--'rf-__**
0300000 3.0
TIME
j
0 l
mm
1.0L 0.6
Fig. 4. Effect of haloperidol on Nb2BA (- - - - - -) and RIA ) estimates of PRL in males and diestrous females. (Immediately following the collection of a control sample (time 0), haloperidol was administered (2 mg/kg i.p.). Each point represents the meanf SE. In the lower panel, Nb,ZBA/RIA (mean* SE) values represented by the bars are depicted directly beneath the time period which they represent. * p -z 0.05 indicates that mean Nb2BA and RIA estimates are different at that time point. ** p -z 0.05 vs. the control (time 0) Nb,ZBA/RIA value.
138
In females there were a 75-fold increase in both RIA and Nb2BA PRL values after haloperidol administration. After the peak, Nb2BA decreases faster than RIA estimates, with significantly higher RIA readings at 60 and 90 min post-treatment ( p < 0.05 and p < 0.005, respectively). This was not reflected in the ratio, as the Nb2BA/RIA values remained very close to 1.0 throughout the study. The range of Nb2BA/RIA values for haloperidol-treated females was 0.53-1.41, with an overall mean k SE of 0.84 k 0.09. Examined over time, haloperidol also altered ( p = 0.0031, Table 1) the relationship between estimates of PRL by Nb2BA and RIA in the females. The correlation coefficient for this group was 0.93. The overall correlation coefficient of the Nb2BA and RIA estimates of PRL for the whole study, disregarding sex, time and treatment, was 0.89. The cumulative mean & SE Nb2BA/RIA value was 1.5 * 0.1. Discussion The data presented in this study support previous reports (Leung et al., 1978; Leung, 1980; Lawson, 1980; Lawson et al., 1980; Tanaka et al., 1980; Klindt et al., 1982), suggesting that BA and RIA estimates of rat PRL are often discrepant. Of the treatments examined, estrogen, morphine, bromocriptine and haloperidol altered the relationship between Nb2BA and RIA estimates of PRL. The estrogen-induced increase in PRL levels of ovariectomized females and the similarly induced proestrus surge in intact females favored a form or forms of PRL with more bioactivity than immunoreactivity (Smith et al., 1975). That estrogen administration leads to an alteration in the circulating forms of PRL is supported by previous work, in which a shift in the percentages of the various molecular sizes of PRL was noted in the pituitaries of ovariectomized estrogen-treated females, with estrogen causing an increase in the larger forms, primarily ‘big’ PRL (Lawson and Stevens, 1980). Estrogen increased PRL secretion by a complex action with pituitary lactotroph proliferation, a dual antidopaminergic action (Cramer et al., 1979) and an action at genomic level (Vician et al., 1979). Of the two suggested pituitary PRL pools
(Swearingen, 1971; Morin et al., 1975), a newly synthesized, more readily releasable and an older, more stable pool, dopamine has been shown to act predominantly to suppress the former, although it suppresses both pools somewhat (Jaques and Gala, 1979). At present we do not know if the greater Nb2BA estimates of PRL compared to RIA values when estrogen is administered are due to dopamine antagonism, lactotroph proliferation or both. The dopamine agonist bromocriptine administered to diestrous females significantly altered the Nb2BA/RIA relationship in serum, favoring a smaller decrease in immunoreactive PRL as compared to bioactive PRL. It seems surprising that the dopamine antagonist haloperidol and the dopamine agonist bromocriptine had similar effects on the relationship of the Nb2BA and RIA estimates of PRL in serum while causing opposite effects on the circulating concentration of the hormone. Possibly the effects reflect the common site of action of these two agents. Morphine administration favored immunoreactive more than bioactive PRL forms, inverting the NbZBA/RIA ratio. Morphine appears to act through reduction of the hypothalamic tuberoinfundibular dopaminergic system (Deyo et al., 1979), and if the data from the present study suggest a different mechanism of action compared with estrogen, the confirmation of this point remains to be elucidated. Of all the different treatments examined in this study, TRH and ether administration did not change the profile of BA/RIA values. TRH elicits PRL secretion directly at pituitary level and apparently through a non-dopaminergic antagonist mechanism (de1 Pozo and Brownell, 1979; Morin et al., 1975), and Dannies et al. (1976) suggested that TRH acts by stimulating primarily the release of the old storage PRL pool. On the other hand, ether stimulates PRL secretion via hypothalamic activation and probably mobilizes mainly the releasable pool (Shin, 1979). In accordance with TRH, ether seems to activate PRL release through a non-dopaminergic mechanism of action (Grosvenor et al., 1979; Shin, 1979). In summary, differences between estimates of PRL by the two assays were altered, favoring bioactive more than immunoreactive hormone, in ovariectomized females after 2 weeks of estradiol
139
valerate and in intact females during proestrus. Significant changes were also noted between the estimates of PRL by RIA and Nb2BA following the administration of morphine and haloperidol, but the RIA estimates in these two cases surpassed those of Nb2BA. These two treatments therefore induced a complete reversal of the Nb2BA and RIA relationship. Differences between the estimates of PRL by the two assays were not altered in females in estrus, ovariectomized females or bromocriptine-treated females. Similarly, no changes were noted in either males or females following ether administration. It is interesting to note that all the treatments which significantly and consistently altered the circulating forms of PRL have been shown to act at least partially through a dopaminergic system. However, the possibility that the BA-RIA discrepancies found in this work were not due to secretory processes but to peripheral (blood or tissue) modifications of the PRL molecule has not been ruled out. It is noteworthy that the pattern of PRL secretion in males in several of the experimental groups obviously differed from that of the females receiving the same treatment. Similarly, the levels of PRL obtained for males in the various treatment groups were abnormally elevated. It is possible that this observation was due, at least in part, to nonspecific stress and the degree of previous adaptation of the rats to handling. This was apparent in the present experiment, as the basal PRL concentrations obtained from females of the different treatment groups, which were handled daily to obtain vaginal smears, were at acceptable levels. Basal PRL concentrations from males, on the other hand, which were scarcely handled before experimentation, were elevated. Although several authors have previously reported alterations in the relationship of PRL estimates by bioassay and RIA, the cumbersome bioassays used precluded a systematic approach with large numbers of animals. The general conclusion of such studies was that, although bioassay values were greater than the estimates by RIA, the acceptable correlation between the two methods validated the RIA as an evaluating tool. In the present study we have taken advantage of the availability of a new PRL bioassay (Nb2BA) which
shares with RIA the reproducibility, precision and ability to run a larger number of samples per assay. The screening thus performed allowed us to detect not only Nb,ZBA-RIA discrepancies, but, more importantly, that those discrepancies are stimulus-dependent. In fact, depending on the stimulus employed, the Nb2BA/RIA ratio can be greater than, equal to or less than 1, and was completely reversed in some experiments. The high precision and reproducibility of the Nb2BA allows us to rule out analytical imprecision as the cause of the divergences. However, the exact meaning and intimate mechanisms subserving them are at present far from clearly understood. The fact that some divergences have been identified must not obscure the finding that on the whole the differences were modest and that a high correlation was generally found between BA and RIA estimates of PRL. Thus, the continued use of RIAs for measuring serum PRL in physiological and experimental studies appears justified. Acknowledgements This research was supported by grants from the Medical Research Council of Canada and USPHS HD-07843-11. F.F.C. is the recipient of a Canadian Medical Research Council Fellowship. We thank Mrs. J. Mueller for typing the manuscript. References Asawaroengchai, 301-308.
H.
and Nicoll, C.S. (1977) J. Endocrinol.
73,
Asawaroengchai, H., Russell, SM. and Nicoll. C.S. (1978) Endocrinology 102,407-414. Bruni. J.F., Van Vugt, D., Marshall, S. and Meites. J. (1977) Life Sci. 21, 46-466. Butcher, R.L., Collins, W.E. and Fugo, N.W. (1974) Endocrinology 94, 1704-1708. Casanueva, F., Cocchi, D., Locatelli. V., Flauto, C., Zambotti, F., Bestetti. G.. Rossi, G.L. and Muller. E.E. (1982) Endocrinology 110, 590-599. Cramer, G.M., Parker, C.R. and Porter, J.C. (1979) Endocrinology 104. 419-422. Dannies, S.P.. Gautvik, K.M. and Tashjian, A.H. (1976) Endocrinology 98, 1147-1159. de1 Pozo, E. and Brownell, J. (1979) Hormone Res. 10. 143-172. Deyo. S.N.. Swift, R.M. and Miller, R.J. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76. 3006-3011. Dickerman, S., Clark. J.. Dickerman. E. and Meites, J. (1972) Neuroendocrinoloay 9. 332-340.
140
Gala, R.R. and Kuo, E.Y.H. (1972) Proc. Sot. Exp. Biol. Med 139, 1349-1352. Grosvenor, C.E., Mena, F. and Whitworth, N.S. (1979) Endocrinology 105, 884-887. Haisenlender, D.H., Lawson, D.M. and Gala, R.R. (1984) Life Sci. 24, 31-36. Jaques, S. and Gala, R.R. (1979) Acta Endocrinol. (Kbh.) 92, 437-440. Klindt, J., Robertson, MS. and Friesen, H.G. (1982) Endocrinology 111, 350-352. Lawson, D.M. (1980) Proc. Sot. Exp. Biol. Med. 165, 364-367. Lawson, D.M. and Stevens, R.W. (1980) Life Sci. 27, 1489-1494. Lawson, D.M., Gala, R.R., Chin, M.L. and Haisenlender, D.H. (1980) Life Sci. 37, 1147-1151. Lawson, D.M., Sensui, N., Haisenlender, D.H. and Gala, R.R. (1982) Life Sci. 331, 3063-3080. Leung, F.C. (1980) Endocrinology 106.61-67. Leung, F.C., Russell, SM. and Nicoll, C.S. (1978) Endocrinology 103, 1619-1628. Mittra, I. (1980a) B&hem. Biophys. Res. Commun. 95, 1750-1759. Mittra, I. (1980b) B&hem. Biophys. Res. Commun. 95, 1760-1767.
Morin, A., Tixier-Vidal, A., Gourdji, D., Kerdelhue, B. and Grouselle, D. (1975) Mol. Cell. Endocrinol. 3, 351-373. Mueller, G.P., Chen, M.J. and Meites, J. (1973) Proc. Sot. Exp. Biol. Med. 144, 613-618. Muller, E.E., Cammani, F., Genaxzani, A.R., Casanueva, F., Cocchi, D., Lccatelli, V., Massara, F. and Mantegaua, P. (1981) Life Sci. 29, 867-883. Shin, S.H. (1979) Life Sci. 25, 1829-1835. Shin, S.H. (1980) Neuroendocrinology 31, 375-379. Sinha, Y.N. and Gilligan, T.A. (1981) Endocrinology 108, 1091-1094. Smith, M.S., Freeman, M.E. and Neill, J.D. (1975) Endocrinology 96, 219-226. Swearingen, K.C. (1981) Endocrinology 89, 1380-1388. Tanaka, T., Shiu, R.P.C., Gout, P.W., Beer, C.T., Noble, R.L. and Friesen, H.G. (1980) J. Clin. Endocrinol. Metab. 51, 1058-1063. Vician, L., Shupnik, M.A. and Gorski, J. (1979) Endocrinology 104,736-743. Wallis, M., Danniels, M. and Ellis, S.A. (1980) Biochem. J. 189, 605-614.
Molecuiur und Cellular Endocrinology, 39 (1985) 131- 140 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01259
Comparison between rat prolactin radioimmunoassay and bioassay values under different experimental and physiological conditions Robert E. Owens, Felipe F. Casanueva Department o/Physiology,
Faculty of Medicine, University of Manitoba, (Received
Keywords:
lymphoma
cell lactogenic
* and Henry G. Friesen
bioassay;
16 August
estrogen;
1984; accepted
molecular
770 Bannatyne Aoenue. Winnipeg (Canada)
24 October
1984)
forms; estrous cycle; morphine;
TRH;
CR-154; ether;
haloperidot.
Summary Serum rat PRL concentrations were compared using values determined by RIA and the Nb2 lymphoma cell bioassay (Nb2BA). Rat serum samples were obtained under different physiological conditions and after the administration of pharmacological agents known to affect PRL secretion. Of the treatments examined, estrogen, morphine bromocriptine and haloperidol significantly altered the relationship between Nb2BA and RIA estimates of PRL. The estrogen-induced increase in PRL levels of ovariectomized females and the proestrus surge of PRL in intact females led to higher bioassay than RIA estimates of PRL. Treatment with haloperidol, bromocriptine and morphine altered the relationship, favoring immunoreactive more than bioactive hormone, and reversing the pretreatment Nb2BA/RIA ratio. No discrepancies between estimates of PRL by the two assays were noted in untreated males, diestrous, estrous and ovariectomized females, or following ether or TRH administration. These results confirm previous observations of discrepancies between rat serum bioassay and RIA estimates, and the data suggest that different forms of prolactin are present in the circulation at different times.
There is increasing evidence that more than one form of PRL is present in the serum and pituitaries of the rat. Previous reports have documented discrepancies in estimates of PRL by pigeon crop sac bioassay and RIA (Asawaroengchai and Nicoll, 1977; Asawaroengchai et al., 1978; Leung et al., 1978; Gala and Kuo, 1972). Recently PRL molecular heterogeneity due to charge or molecular weight differences and dissociations between RIA and bioactivity (BA) potencies have been reported (Sinha and Gilligan,
* To whom reprint requests should be addressed. Present address: Department of Internal Medicine, Endocrine Unit, Faculty of Medicine, Compostela University, Santiago de Compostela (Spain). 0303-7207/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
1981; Lawson, 1980; Wallis et al., 1980; Mittra, 1980a, b; Lawson et al., 1980; Haisenlender et al., 1984). Early attempts to study circulating forms of PRL in the rat by comparing RIA and bioassay estimates were limited primarily to pituitary extracts and pooled samples, as available bioassays lacked the sensitivity required to measure levels of hormone in small serum samples. These bioassays were cumbersome and with poor reproducibility. Recently, using the sensitive, lactogen-specific, Nb2 rat lymphoma cell bioassay (Nb2BA), Klindt et al. (1982) have reported important discrepancies in RIA/BA values in male rats. That finding, however, was not entirely confirmed by another report (Lawson et al., 1982). Ltd.
132
In an attempt to elucidate the underling mechanism of this Nb>ZBA/RIA discrepancy, and to determine if it occurs in different secretory states, we have compared the Nb2BA and RIA estimates of PRL in serum samples obtained from rats under a variety of physiological or experimental conditions and after the administration of pharmacological agents known either to stimulate or inhibit PRL secretion.
posed of rats in different physiological states and/or after pharmacological treatments known to affect serum PRL concentrations. With the exception of the ether-treated groups, freely moving unanesthetized rats were employed. The experimental protocol was cleared through the Institutional Animal Care Committee. Blood collection For the estrous cycle study, animals were bled twice in the morning and twice in the afternoon (see Table 1). The ovariectomized females in the groups designated ovariectomy and ovariectomy plus estradiol valerate were bled at the same time periods as the untreated animals on a day which was 1 month post-ovariectomy and 2 weeks following estradiol valerate (2 mg/rat). All other experiments were performed in the morning starting at 0900 h. Daily vaginal smears were taken for at least 8 days, or two complete estrous cycles,
Materials and methods Animals Sprague-Dawley rats (Canadian Breeding Labs, Montreal) weighing 200-300 g were utilized for this study. The animals were housed under environmentally controlled conditions, with a 12 h light and 12 h dark cycle. All animals were fed Purina rat chow and water ad libitum. The study included 15 experimental groups (Table 1) com-
TABLE
1
EXPERIMENTAL GROUPS CONCENTRATIONS Treatment
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. a b ’ d ’ ’ 8 b
group
Untreated males Diestrous females Proestrous females Estrous females Gvariectomy + E, a Ovariectomy Bromotxiptine b females Ether males Ether females TRH ’ males TRH females Morphine d males Morphine females Haloperidol’ males Haloperidol females
AND
PROBABILITY
LEVELS
FOR
N
Dose/route
Schedule of blood collection
6 7 5 8 6 5 6 6 6 5 5 6 8 6 7
NA NA NA NA 2 mg/s.c. ’ NA 1 mg,/kg,/i.p. 2.5 min 2.5 min 20 pg/i.p. 20 pg/i.p. 10 mg/kg/i.p. 10 mg/kg/i.p. 2 mg/kg/i.p. 2 mg/kg/i.p.
1030.1130,1400,1500 1030,1130,140@, 1500 1030,1130,1400,1500 1030,1130,1400,1500 1030,1130,1400,1500 1030.1130,1400,1500 0 B, 30,60,90 min 0, 2.5, 5.15 min 0, 2.55, 15 min 0, 10, 20, 30 min 0, 10, 20, 30 min 0,20,40,60 min 0,20,40,60 min 0, 30,60,90 min 0,30,60,90 min
OF TIMEX ASSAY WITHIN
EFFECT
Reference
h h h h h h
h
Smith et al. (1975) Lawson and Stevens (1980) Muller et al. (1981) Grosvenor et al. (1979) Mueller et al. (1973) Bruni et al. (1977)
Delestrogen, estradiol valerate (Squibb Canada Inc., Montreal, Canada). Bromocriptine mesylate, CB-154 (Sandoz Ltd., Basle, Switzerland). TRH relefact, Protirelin (Hoechst Canada Inc., Montreal, Canada). Morphine sulphate (Abbott Laboratories, Ltd., Montreal, Canada). Haloperidol, Haidol (McNeil Laboratories, Ltd., Stouffville, Ontario, Canada). Estradiof valerate administered 2 weeks before the day of blood collection which was 1 month Time 0 designates pretreatment biood samples. Other times designate minutes post-treatment. In the references cited the same times for blood collection and/or drug dosages were used.
ON PRL
Source of variation p = (time X assay)
Butcher et al. (1974)
Dickerman
GROUP
et al. (1972)
post~va~~tomy.
06609 0.8349 0.0082 0.3345 0.0025 0.3486 0.0932 0.6727 0.1969 0.4592 0.2761 0.0058 0.0796 ox!61 0.0031
133
before the animals were used. Bleeding day for males was chosen for convenience. Blood samples were collected as previously published (Casanueva et al., 1982) and left overnight at 4°C to clot. After centrifugation, serum was stored at -20°C until assayed for prolactin. Quantitation of PRL The concentrations of immunoreactive rat PRL were measured by a double antibody RIA using the protocol and materials supplied by NIAMDD. Bioactive PRL was quantitated by the previously described Nb2 lymphoma cell bioassay (Nb2BA) (Tanaka et al., 1980). The average of duplicate determinations of each sample was expressed in terms of the NIAMMD rat prolactin RP-1 standard for both assays. All samples from a given treatment were assayed together. The coefficients of variation for inter- and intra-assay variability were 14% and 7% for the RIA and 20% and 9% for the bioassay, respectively. Statistical analysis The Nb2BA and RIA estimates of PRL levels for individual rats from each group at each time point were used to calculate the mean Nb2BA and RIA PRL values. The mean from the individual Nb2BA/RIA ratios was obtained at each time and for each experimental group. Three statistical approaches were used to evaluate the data. First, a comparison of mean Nb,ZBA, RIA and Nb2/RIA ratio at each time period was carried out, followed by an estimate of the Nb2BA/RIA ratio over time for the various groups. Nb2BA and RIA comparisons at each time were made using a paired t-test. Individual Nb2BA/RIA values were determined for each blood sample collected, and the means of these values for the different treatments compared by one-way ANOVA, with differences between means tested by Tukey’s procedure. ANOVA (repeated measures design) with two repeated factors, time and assay, was applied to the data from each of the 15 groups to test whether the differences between RIA and Nb2BA were consistent averaged across the time period. The observation of a significant (p < 0.05) time X assay interaction for a particular group indicated that the difference between the Nb2BA and RIA estimates of PRL
changed significantly over amined for that group.
the
time
period
ex-
Results From each experimental group four kinds of statistical information were obtained: (a) mean RIA PRL values compared with mean BA PRL values at each time sampled (figures, upper panel); (b) mean BA/RIA ratio at each time sampled compared with the basal BA/RIA ratio (figures, lower panel); (c) RIA PRL values over time compared as a whole group against BA PRL values over time (time x assay interaction, Table 1); and (d) correlation coefficient of the regression line of BA and RIA estimates into the experimental group. Untreated males Serum PRL as measured by both RIA and Nb2BA increased over the time period examined in untreated males (Fig. 1). The Nb2BA estimates of PRL were consistently higher than those of the RIA, but only the initial blood sample at 1030 h was significantly higher (p -C0.01). Examined as a group, the Nb2BA values over time did not behave
UNTREATED
BESTRUS
PROESTRUS
ESTRUS
I
,d TIME OF DAY
Fig. 1. Serum concentrations of PRL as measured by Nb2BA (------)andRIA() at 1030, 1130, 1400 and 1500 h of one day in untreated males and in females during diestrus, proestrus and estrus. Each point represents the mean* SE. NbZBA/RIA (mean* SE) values represented by the bars are depicted in the lower panel directly beneath the time period which they represent. *p < 0.05 indicates that mean Nb2BA and RIA estimates are different at that time period.
134
differently from RIA values over time ( p = 0.6609, Table 1). The mean rfr SE Nb2BA/RIA ratio did not change significantly during the day (Fig. 1). The overall NbZBA/RIA ratio for this group was 1.48 si: 0.11, with a range for individual samples of 0.85-2.35. The calculated correlation coefficient of the regression line of Nb2BA and RIA estimates of this group was 0.99. Estrous cycle During diestrus (Fig. I), PRL levels were low compared to other periods of the estrous cycle, and the Nb2BA measurements were consistently but not significantly higher than RIA estimates. The differences between the two assays examined together over the entire bleeding period remained constant ( p = 0.8349, Table 1). The Nb2BA/RIA ratios also did not change during diestrus. The mean k SE Nb2BA/RIA value in this group was 1.93 + 0.13, with a range of 0.5-3.67. The cortelation coefficient was 0.85. During proestrus (1400, 1500 h), PRL levels were IO-fold higher by RIA and 16fold higher by Nb2BA than those measured at 1030 h. PRL estimate by Nb2BA was 2-fold higher than the RIA estimate ( p -C0.01) at 1400 h, and was not different at the remaining time periods. The divergence of the estimated PRL levels by the two assays in this group led to a significant time X assay interaction ( p = 0.0082, Table 1). The mean Nb,ZBA/RIA ratio did not change during proestrus (Fig. 1). The overall Nb2BA/RIA ratio in this group was 1.95 + 0.16, with a range of 0.88-3.86. For this group the regression line of the Nb2BA and RIA measurements of PRL yielded a correlation coefficient of 0.90. On the day of estrus (Fig. l), PRL concentrations were relatively low in the morning and afterwards elevated by 1400 h. The Nb2BA estimates of PRL were consistently higher than RIA values, being significantly different at 1030, 1130 and 1400 h (p c 0.05). Nb2BA and RIA values followed a similar pattern, and the differences between both groups of estimates remained constant ( p = 0.3345, Table 1). The Nb2BA/RIA ratio did not change with respect to the basal ratio during the day of estrus. The mean * SE ratio for Nb2BA/RIA was 2.08 + 0.14, with a range of
individual values from 0.59 to 3.57. A correlation coefficient of 0.87 was calculated for Nb2BA and RIA values. Ooariectomy and ovariectomy plus estradial valerate In the females ovariectomized 1 month previously, PRL values were low at each time examined (Fig. 2). The Nb2BA values were higher than those of RIA at each point, and significantly so at 1030 h (p < 0.01). There were no differences between Nb2BA/RIA ratios in the four time periods studied. The mean + SE NbZBA/RIA value for all the samples was 1.84 + 0.19, with a range of 0.63-3.38. The correlation coefficient for this group was 0.89. Estradiol valerate administered to ovariectomized females 2 weeks previously, significantly elevated PRL values compared with the untreated
1800
1800
---
Nb2BA RIA l
ovx
*A
2ook
TIME OF DAY 3.0 r-
Fig. 2. Serum concentrations of PRL as measured by Nb2BA -) at 1030,1130,1400 and 1500 h for (------)andRIA( females 1 months post-ovariectomy without treatment (0) (OVX) and 1 month post-ovariectomy with 2 mg of estradiol valerate per animal after 2 weeks (A) (OVX + I!.,). Each point represents the mean&SE. In the lower panel mean_+SE Nb,ZBA/RIA values are depicted for OVX (solid bars) and OVX + EZ (open bars) directly beneath the time period which they represent. * p c 0.05 indicates that mean Nb2BA and RIA estimates are different at that time period.
NbZ/RIA
80+47 6k 1
148+ ilk
75 5
115+25 213+76
2.66kO.43 1.61k0.36
155+50 9Ok26 *
RIA
NbZ/RIA
1.58kO.08 0.76*0.55 20
Nb2
19 17
RIA
91k 51+
0
58k12 92k29
* p < 0.05 RIA vs. N62BA values at different time periods. ** p -c 0.05 Nb2BA/RIA ratio vs. same ratio at time 0.
Morphine males Morphine females
Time (min):
TRH males TRH females
10 Nb,Z/RIA
RIA
Nb2
RIA
32+11
0
1.46kO.12
Time (min):
194+110
RIA
Nb2
RIA
114+60
30
0
CB-154 females
Time (min):
178+64 65+21
Nb2
168k38 193zk84
Nb2
31 f 14
Nb2
SERUM PRL VALUES (ng/mI) AFTER CB-154, TRH AND MORPHINE
TABLE 2
**
1.06*0.10 ** 0.71 jzO.10 **
Nb2/RIA
1.51k0.14 0.81kO.38
NbZ/RIA
0.74*0.14
NbZ/RIA
124+22 101+61
RIA
40
4
2
109+21 91+56
Nb2
155 f 30 176+48
Nb2
11+
Nb2
(MEAN f SE)
94+25 213k48
RIA
20
17*
RIA
60
ADMINISTRATION
Nb,Z/RIA **
0.86+0.06 ** 0.65kO.18 **
NbZ/RIA
2.05 kO.46 0.81 kO.20
Nb,Z/RIA
0.53*0.07
5
106+49 22+ 7
RIA
60
124k12 48k17
RIA
30
1s+
RIA
90
6
89+41 9* 9
Nb2
186+21 43k21
Nb2
14+
Nb2
NbZ/RIA
0.88kO.04 ** 0.35+0.11 **
NbZ/RIA
1.52kO.15 1.07kO.32
NbZ/RIA
0.55&0.10 **
E
136
ovariectomized animals (Fig. 2). Nb2BA PRL estimates were significantly higher (p c 0.05) than those of the RIA at 1130, 1400 and 1500 h. The divergence of the two assays during the day resulted in a significant time x assay interaction (p = 0.0025, Table l), suggesting that the differences between estimates were not constant. The Nb2BA/RIA ratios were not different with respect to the basal ratio at the time periods evaluated. The mean + SE Nb,[LBA/RIA value for this subgroup was 1.59 f 0.11, with a range of 0.74-2.74. The correlation coefficient of Nb2BA and RIA estimates of PRL for the group was 0.96.
MALES
180 t
-
TRH The Nb2BA estimates of serum PRL after TRH treatment of male rats were not significantly higher than values determined by RIA (Table 2). The differences between the two assays were found to be consistent when examined over time (p = 0.4592, Table 1). The Nb2BA/RIA values for TRH-treated males ranged from 1.04 to 3.89, with an overall mean + SE of 1.67 f 0.13. The correlation coefficient for this group was 0.93. After treatment of diestrous females with TRH there was an increase in the mean level of PRL as measured by both assays (Table 2). However, the large degree of variation between the animals prohibited the observation of a clear response to this treatment. The differences between the estimates of PRL by Nb2BA and RIA remained unchanged over time (p = 0.2761, Table 1). The mean Nb2BA/RIA values in diestrous females were not
Nb2BA RIA T
0
t
/I',
TIME (mid
d
Bromocriptine (CB-154) Administration of bromocriptine depressed circulating Nb2BA and RIA PRL levels within 30 min (Table 2). Examined over time, the relationship between the estimates of PRL by RIA and Nb2BA were not different ( p = 0.0932, Table 1). The treatment induced an inversion in the BA/RIA ratio; in fact Nb2BA/RIA values were altered ( p < 0.05) at 30, 60 and 90 min as compared to pretreatment values (time 0). The range of Nb2BA/RIA values observed was 0.31-1.84, with a mean + SE of 0.86 + 0.10. The correlation between Nb2BA and RIA estimates of PRL for the bromocriptine group was very good, with a coefficient of 0.99.
---.
220
FEMALES
Y-
r
Fig. 3. Effect of ether on Nb2BA (- - - - - -) and RIA () estimates of PRL in males and diestrous females. Immediately following the collection of a control sample (time 0), animals were exposed to ether for 2 min. Each point represents the mean f SE. In the lower panel, directly beneath the time they represent, mean + SE Nb,ZBA/RIA values are depicted. * p c 0.05 indicates that mean Nb2BA and RIA estimates are different at that time period.
altered by TRH ad~nistration. Overall the Nb,ZBA/RIA ratio for this group was 0.97 + 0.11, with a range of values of 0.41-1.88. The Nb2BA and RIA estimates analyzed by linear regression had a correlation coefficient of 0.97. Ether In males (Fig. 3), levels of PRL by RIA following a 2 min ether exposure rose steadily over the four times examined. The estimates of Nb2BA followed a very similar pattern and were significantly higher than RIA values at each time ( p < 0.05 at 0 and 2.5 min; p < 0.01 at 5 and 15 min). Nevertheless, as groups both assays behave similarly and the differences between both estimates evaluated over time were constant (p = 0.6727, Table 1). No significant changes in the Nb2BA/RIA ratio were noted. The Nb,ZBA/RIA values for this group ranged from 1.10 to 2.34, with a mean i SE of 1.52 + 0.06. The correlation coefficient for ether-treated males was 0.97.
137
In diestrous females, RIA and Nb2BA PRL levels rose rapidly following ether exposure to reach a peak at 5 min. Nb2BA estimates were higher, but significantly so (p < 0.01) only in the pretreatment sample. There were no changes in the Nb2BA/RIA ratio, although this value appeared to be moving toward a 1 : 1 ratio with each subsequent sample. The range of Nb,ZBA/RIA values for ether-treated females was 0.86-2.18, with an overall mean + SE of 1.30 f 0.08. The correlation coefficient of the estimates of the two assays was 0.97. Morphine In males (Table 2) there was no effect of morphine on mean levels of PRL estimated by RIA, but the differences between estimates by Nb2 and RIA over time were altered significantly ( p = 0.0058, Table 1). At 20 and 40 min posttreatment a reversal situation with higher estimates by RIA than Nb2BA occurred. In fact the pretreatment Nb,ZBA/RIA of 2.26 + 0.43 dropped rapidly to 1.05 f 0.10 by 20 min and to 0.86 f 0.06 by 40 min post-treatment. The Nb2BA/RIA values were all significantly ( p < 0.05) depressed following morphine administration compared to control time. The mean value of the BA/RIA ratio was 1.26 * 0.16, and the values ranged from 0.61 to 4.13. The correlation of Nb2BA and RIA PRL estimates yielded a correlation coefficient of 0.92. In diestrous females treated with morphine a similar shift was observed. The Nb2BA estimates were higher than the RIA estimates before morphine treatment (time 0), but the relationship was reversed after treatment, with higher RIA estimates that were significant at 20 min ( p -z 0.05). Over time, the differences between the estimates of PRL by both assays were not changed by morphine, although the probability approached the level of significance (p = 0.0796, Table 1). As expected, with an inversion of the Nb2BA/RIA ratio, the Nb2BA/RIA values were significantly depressed ( p -c 0.05) compared to controls following morphine treatment. The range of Nb,ZBA/RIA values for morphine-treated females was 0.25-3.64, with a mean + SE of 1.12 f 0.26. The correlation of PRL estimates by the two assays was high for this group, with a coefficient of 0.98.
Haloperidol As can be seen in Fig. 4, haloperidol administration caused an increase in circulating PRL levels. In males RIA estimates were higher (p -c 0.05) than Nb2BA values at the three time points examined after haloperidol treatment. The differences were not consistent over time, the Nb2BA and the RIA estimates behaving as different populations under haloperidol (p = 0.0061, Table 1). The NbZBA/RIA ratio reflected that pattern, as a significant ( p < 0.05) decrease from a pretreatment ratio of 1.44 f 0.24 to 0.65 f 0.04,0.73 f 0.04 and 0.63 f 0.06 at 30, 60 and 90 min respectively. The range of Nb2BA/RIA values for haloperidol males was 0.36-2.13, with a mean f SE of 0.86 & 0.04. Assay estimates for this group correlated well, with a coefficient of 0.95.
MALES
24oor
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Fig. 4. Effect of haloperidol on Nb2BA (- - - - - -) and RIA ) estimates of PRL in males and diestrous females. (Immediately following the collection of a control sample (time 0), haloperidol was administered (2 mg/kg i.p.). Each point represents the meanf SE. In the lower panel, Nb,ZBA/RIA (mean* SE) values represented by the bars are depicted directly beneath the time period which they represent. * p -z 0.05 indicates that mean Nb2BA and RIA estimates are different at that time point. ** p -z 0.05 vs. the control (time 0) Nb,ZBA/RIA value.
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In females there were a 75-fold increase in both RIA and Nb2BA PRL values after haloperidol administration. After the peak, Nb2BA decreases faster than RIA estimates, with significantly higher RIA readings at 60 and 90 min post-treatment ( p < 0.05 and p < 0.005, respectively). This was not reflected in the ratio, as the Nb2BA/RIA values remained very close to 1.0 throughout the study. The range of Nb2BA/RIA values for haloperidol-treated females was 0.53-1.41, with an overall mean k SE of 0.84 k 0.09. Examined over time, haloperidol also altered ( p = 0.0031, Table 1) the relationship between estimates of PRL by Nb2BA and RIA in the females. The correlation coefficient for this group was 0.93. The overall correlation coefficient of the Nb2BA and RIA estimates of PRL for the whole study, disregarding sex, time and treatment, was 0.89. The cumulative mean & SE Nb2BA/RIA value was 1.5 * 0.1. Discussion The data presented in this study support previous reports (Leung et al., 1978; Leung, 1980; Lawson, 1980; Lawson et al., 1980; Tanaka et al., 1980; Klindt et al., 1982), suggesting that BA and RIA estimates of rat PRL are often discrepant. Of the treatments examined, estrogen, morphine, bromocriptine and haloperidol altered the relationship between Nb2BA and RIA estimates of PRL. The estrogen-induced increase in PRL levels of ovariectomized females and the similarly induced proestrus surge in intact females favored a form or forms of PRL with more bioactivity than immunoreactivity (Smith et al., 1975). That estrogen administration leads to an alteration in the circulating forms of PRL is supported by previous work, in which a shift in the percentages of the various molecular sizes of PRL was noted in the pituitaries of ovariectomized estrogen-treated females, with estrogen causing an increase in the larger forms, primarily ‘big’ PRL (Lawson and Stevens, 1980). Estrogen increased PRL secretion by a complex action with pituitary lactotroph proliferation, a dual antidopaminergic action (Cramer et al., 1979) and an action at genomic level (Vician et al., 1979). Of the two suggested pituitary PRL pools
(Swearingen, 1971; Morin et al., 1975), a newly synthesized, more readily releasable and an older, more stable pool, dopamine has been shown to act predominantly to suppress the former, although it suppresses both pools somewhat (Jaques and Gala, 1979). At present we do not know if the greater Nb2BA estimates of PRL compared to RIA values when estrogen is administered are due to dopamine antagonism, lactotroph proliferation or both. The dopamine agonist bromocriptine administered to diestrous females significantly altered the Nb2BA/RIA relationship in serum, favoring a smaller decrease in immunoreactive PRL as compared to bioactive PRL. It seems surprising that the dopamine antagonist haloperidol and the dopamine agonist bromocriptine had similar effects on the relationship of the Nb2BA and RIA estimates of PRL in serum while causing opposite effects on the circulating concentration of the hormone. Possibly the effects reflect the common site of action of these two agents. Morphine administration favored immunoreactive more than bioactive PRL forms, inverting the NbZBA/RIA ratio. Morphine appears to act through reduction of the hypothalamic tuberoinfundibular dopaminergic system (Deyo et al., 1979), and if the data from the present study suggest a different mechanism of action compared with estrogen, the confirmation of this point remains to be elucidated. Of all the different treatments examined in this study, TRH and ether administration did not change the profile of BA/RIA values. TRH elicits PRL secretion directly at pituitary level and apparently through a non-dopaminergic antagonist mechanism (de1 Pozo and Brownell, 1979; Morin et al., 1975), and Dannies et al. (1976) suggested that TRH acts by stimulating primarily the release of the old storage PRL pool. On the other hand, ether stimulates PRL secretion via hypothalamic activation and probably mobilizes mainly the releasable pool (Shin, 1979). In accordance with TRH, ether seems to activate PRL release through a non-dopaminergic mechanism of action (Grosvenor et al., 1979; Shin, 1979). In summary, differences between estimates of PRL by the two assays were altered, favoring bioactive more than immunoreactive hormone, in ovariectomized females after 2 weeks of estradiol
139
valerate and in intact females during proestrus. Significant changes were also noted between the estimates of PRL by RIA and Nb2BA following the administration of morphine and haloperidol, but the RIA estimates in these two cases surpassed those of Nb2BA. These two treatments therefore induced a complete reversal of the Nb2BA and RIA relationship. Differences between the estimates of PRL by the two assays were not altered in females in estrus, ovariectomized females or bromocriptine-treated females. Similarly, no changes were noted in either males or females following ether administration. It is interesting to note that all the treatments which significantly and consistently altered the circulating forms of PRL have been shown to act at least partially through a dopaminergic system. However, the possibility that the BA-RIA discrepancies found in this work were not due to secretory processes but to peripheral (blood or tissue) modifications of the PRL molecule has not been ruled out. It is noteworthy that the pattern of PRL secretion in males in several of the experimental groups obviously differed from that of the females receiving the same treatment. Similarly, the levels of PRL obtained for males in the various treatment groups were abnormally elevated. It is possible that this observation was due, at least in part, to nonspecific stress and the degree of previous adaptation of the rats to handling. This was apparent in the present experiment, as the basal PRL concentrations obtained from females of the different treatment groups, which were handled daily to obtain vaginal smears, were at acceptable levels. Basal PRL concentrations from males, on the other hand, which were scarcely handled before experimentation, were elevated. Although several authors have previously reported alterations in the relationship of PRL estimates by bioassay and RIA, the cumbersome bioassays used precluded a systematic approach with large numbers of animals. The general conclusion of such studies was that, although bioassay values were greater than the estimates by RIA, the acceptable correlation between the two methods validated the RIA as an evaluating tool. In the present study we have taken advantage of the availability of a new PRL bioassay (Nb2BA) which
shares with RIA the reproducibility, precision and ability to run a larger number of samples per assay. The screening thus performed allowed us to detect not only Nb,ZBA-RIA discrepancies, but, more importantly, that those discrepancies are stimulus-dependent. In fact, depending on the stimulus employed, the Nb2BA/RIA ratio can be greater than, equal to or less than 1, and was completely reversed in some experiments. The high precision and reproducibility of the Nb2BA allows us to rule out analytical imprecision as the cause of the divergences. However, the exact meaning and intimate mechanisms subserving them are at present far from clearly understood. The fact that some divergences have been identified must not obscure the finding that on the whole the differences were modest and that a high correlation was generally found between BA and RIA estimates of PRL. Thus, the continued use of RIAs for measuring serum PRL in physiological and experimental studies appears justified. Acknowledgements This research was supported by grants from the Medical Research Council of Canada and USPHS HD-07843-11. F.F.C. is the recipient of a Canadian Medical Research Council Fellowship. We thank Mrs. J. Mueller for typing the manuscript. References Asawaroengchai, 301-308.
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