Effect of Posterior Pituitary Denervation (PPD) on Prolactin (PRL) and α-Melanocyte-Stimulating Hormone (α-MSH) Secretion of Lactating Rats

Brain Research Bulletin, Vol. 43, No. 3, pp. 313–319, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00

PII S0361-9230(97)00011-7

Effect of Posterior Pituitary Denervation (PPD) on Prolactin (PRL) and a-Melanocyte-Stimulating Hormone (a-MSH) Secretion of Lactating Rats MIKLO´S VECSERNYE´S,* KRISZTINA KREMPELS,† BE´LA E. TO´TH,† JA´NOS JULESZ,* GA´BOR B. MAKARA‡ AND GYO¨RGY M. NAGY† 1 *Endocrine Unit of 1st Department of Medicine, Albert Szent-Gyo¨rgyi Medical University, Szeged, Hungary, †Neuroendocrine Research Laboratory, Department of Human Morphology and Developmental Biology, Semmelweis University Medical School, Budapest, Hungary, and ‡Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary [Received 5 September 1996; accepted 12 December 1996] ABSTRACT: Previous data have clearly suggested that the posterior pituitary (PP), consisting of neural lobe (NL) and intermediate lobe (IL), has a role in the control of anterior pituitary PRL secretion. However, basic aspects of this regulatory mechanism like (1), the role of an intact hypothalamic innervation of the PP as well as (2) the site of production of previously found PRL releasing substance(s) have not yet been characterized. Denervation of the PP (PPD) is an effective method for having a selective lesion of the innervation of PP, indeed, PPD results in a disappearance of neurosecretory materials from NL and tyrosine hydroxylase (TH) immunoreactivity from IL, leaving blood supply of all three lobes intact. Blood samples were taken from freely moving sham and PP-denervated lactating rats before and after 4-h separation from their pups and during the suckling stimulus. PPD blocks separation-induced depletion but only attenuates suckling induced release of PRL. Furthermore, it doubles plasma level of a-MSH during the entire sampling period, which has been used as a marker for in vivo secretory activity of IL cells. Lack of the separation-induced depression in plasma PRL of PPD animals can be partially restored by normalizing the diabetes insipidus with treatment of a vasopressin analogue, 1desamino-8-D-arginine-vasopressin (dDAVP). In contrast, dDAVP, neither alone nor in combination with oxytocin (OXY), can change PPD-induced elevation of plasma a-MSH as well as attenuation of PRL response induced by suckling. It is concluded that: (1) contribution of the THDA system parallel to the confirmed role in the regulation of a-MSH seems to be crucial for the depletion of plasma PRL induced by separation but not for the elevation due to suckling stimulus, (2) intact hypothalamic innervations of both NL and IL, regulating water intake and the secretion of a-MSH, respectively, are necessary for normal secretory responses of AL during lactation, (3) as well as for the presence of PRF activity in PP, (4) which does not solely responsible for suckling-induced PRL release. Therefore, an interplay between several substances produced by NIL of the pituitary gland must have been responsible for the intact regulation of PRL secretion during lactation. Q 1997 Elsevier Science Inc.

KEY WORDS: Posterior pituitary denervation, Prolactin, a-MSH, Lactating rats.

INTRODUCTION During the last few years evidences have been accumulated that the neurointermediate lobe (NIL) of the pituitary gland plays a role in the regulation of PRL secretion of the anterior lobe (AL), in particular during lactation. The absence of sucklinginduced PRL release after removal of the NIL has provided one of the strongest evidences [24] for developing a hypothesis that the NIL might contain a potent PRL releasing factor (PRF). While most of the known peptides produced and released by the neural lobe (NL) like oxytocin (OXY) [20,31,32], vasopressin (AVP) [31] and its associated glycopeptide [30], as well as by the intermediate lobe (IL), like b-endorfin ( b-END) [3,32,34] and a-melanocyte stimulating hormone ( a-MSH) [11], which are collectively missing after posterior pituitary (PP) lobectomy, have already been suggested to play some role in the regulation of PRL release but their uniqueness and physiological significance holds many contraversions. Our previous studies have indicated that osmoregulation and the regulation of PRL secretion of an animal are closely coupled. Disturbances in the water and electrolyte regulation at the level of the NL severely alters PRL secretion of the AL in lactating rats [25,28,31]. Surgical removal of the NIL abolishes the rise in plasma PRL induced by suckling [24] and at the same time causes diabetes insipidus. In addition, there is no suckling-induced hormone response in Brattleboro homozygous mothers suffering diabetes insipidus without AVP treatment [28]. Moreover, increasing the salinity of the animals (acute and/or subchronic dehydration) dramatically reduces PRL secretion from pituitary lactotropes [25]. This dehydration-induced depletion of

1 Request for reprints should be addressed to Gyo¨rgy M. Nagy, The Florida State University, Department of Biological Science Biomedical Research Faculty, Tallahassee, FL 32306-4075.

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314 plasma PRL is supposedly mediated through an osmosensitive hypothalamic dopaminergic mechanism called the tuberohypophyseal dopaminergic (THDA) system, which is also known to tonically inhibit a-MSH and b-END secretion from melanotropes of IL [10,13,35,38]. Furthermore, crude extracts of the NIL have been reported to stimulate PRL release in vitro [14,15]. It has also been shown that the PRF activity disappears from the NIL after pituitary stalk section [16], suggesting that a PRF might be produced by hypothalamic neurons that project to and terminate in the NIL. However, PRF activity can be found after a week of NIL cell culture [19], indicating that a PRF is synthesized by cells of the NIL. This obvious contradiction suggests the existence and a possible role of more than one substance in the NIL, which can stimulate PRL secretion. These data clearly show that a functionally intact NIL is required for normal mammotrope function but its own and/or integrated role in the regulation of PRL secretion remains to be elucidated. Up to the present, an unconsidered fact is that PP lobectomy as well as pituitary stalk section destroys or at least disturbs the normal blood flow of the AL. PPD, developed by G. B. Makara [21], is an effective method for having a selective lesion of the innervation of the NIL leaving the blood supply of all three lobes of the pituitary gland intact. While, it interrupts axonal transport to the NL [8,17,21] and presumably innervation of the IL as well. In contrast, the neural control of AL function is supposed to be normal [8,17,21]. Therefore, PPD has been used (1) to examine whether the intact innervation of the PP is important in PRL responses due to separation as well as suckling stimulus, and (2) to determine whether the PRL releasing activity (PRF) found in intact NIL is still present and capable to release PRL when it is extracted from a denervated PP. MATERIALS AND METHODS Animals and Treatments Primiparous lactating rats (bred in our animal facilities from Sprague–Dawley stock, obtained from Hannover, Germany) were used at days 7–11 of lactation. Litter size was standardized to eight from the second day postpartum. The animals were housed in an air-conditioned room (temperature 21–237C) with alternating 14-h light and 10-h darkness. They received food and tap water or 1-desamino-8-D-arginine vasopressin (dDAVP, 2 mg/l, Adiuretin-SD, SPOFA, Prague, Czech Republic) containing water, as it is indicated in the results, ad lib. Daily water consumption (ml) and weight (g) of the mothers and litters were measured at 24-h intervals. One group of dDAVP treated mothers received in addition two subcutaneous (SC) injections of OXY (2 IU/kg b.wt., Richter Gedeon Pharmaceutical Company, Budapest, Hungary) 24 and 1 h before suckling stimulus. Cannulation and Blood Sampling Two or 3 days prior to test, a cannula was implanted into the right jugular vein (silicon tubing connecting to a polyethylene extension, i.d. 0.50 mm, e.d. 0.90 mm, Dow Corning Corp., Midland, MI, and Becton Dickinson Parsipparny, NY). Dams were separated from their litters for a period of 4 h before suckling stimulus. Separation and suckling-induced changes in PRL and a-MSH release were tested on 2 consecutive days. Blood samples (300–500 mM) for hormone analysis were obtained before and at the end of the 4-h separation period. Further blood samples were taken 15, 30, and 60 min after suckling stimulus. Posterior Pituitary Denervation (PPD) For denervation of the PP, an L-shaped rotating wire knife with the blade 1107 to the shaft was used as previously described

[21]. Briefly, the rats were anesthetized with Hexobarbital Sodium (50 mg/kg b.wt., IP.) then fixed in a David Kopf (Tunage, CA) apparatus in the position 5 mm nose down. The optimal site for compression was at 5.0 to 5.5 mm posterior to the bregma, at a point where the stalk joins to the NL. The knife was lowered in the midline through the sagittal sinus to 9 mm below the level of the top of the skull. The blade was turned to /607 to either side and was further lowered 2 mm until the base of the skull was reached. After bringing back to the midline the knife was removed from the brain. This procedure interrupts the nerve fibers in the stalk by pressing them against the bone. For sham operation, the knife was lowered 5 mm beneath the surface of the skull. Criteria for inclusion of animals in this study were practically the same than in all previous studies where PPD have been performed [21]. Briefly, after decapitation, both the stalk-median eminence and the pituitary gland were always inspected and any changes in size or color of these tissues were recorded. Based on the increased water intake (above 100 ml/24 h) and the autopsy, it was possible to predict the success of the operation. Subsequent histological study of both, the hypothalamus and the pituitary gland helped us to make the final judgement of possible hypothalamic injuries or infarction of the AL. Thirty-one out of 63 operated animals could be used. Selection of NILs from PPD animals for PRF extraction was based upon the apparent diabetes insipidus, inspection, and histological study of the hypothalamus. Preparation of NIL Extracts NILs have been obtained from intact as well as PP denervated lactating rats. Extraction procedure was as originally described by Hyde et al. [15]. Briefly, pools of two NILs were extracted in 0.5 ml 0.1 N perchloric acid at 47C using a sonicator (Cole– Palmer Instr. Co. Chicago, IL, model CP50). The homogenates were freeze thawed, resonicated, and centrifuged at 10,000 1 g for 5 min. The supernatants were lyophilized and reconstituted in medium (DMEM containing 0.1% BSA) just before performing the reverse hemolytic plaque assay (RHPA). The concentrations of NIL extracts were expressed as tissue equivalents. One equivalent is comparable to 1 mg wet weight of NIL. Within one experiment, tissue extracts from the same batch, but between experiments, different batches of NIL were used. Reverse Hemolytic Plaque Assay (RHPA) The RHPA has been described previously in detail [30,31]. Briefly, anterior pituitary glands of lactating rats were removed aseptically then dispersed with 0.1% trypsin in Minimum Essential Medium (MEM). Immediately after dispersion, the cells were washed with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 0.1% BSA and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). These monodispersed cells were counted and plated onto 35-mm plastic culture dishes (Falcon 3001) at a density of 5 1 10 5 cells/2 ml culture medium (0.1% BSA-DMEM containing 10% heat inactivated horse serum) and cultured for 48 h at 377C a water saturated atmosphere of 95% air–5% CO2 . On the morning of the experiment the cells were recovered from the culture dishes by a brief retrypsinization (0.025% trypsin in MEM for 10 min) and washed with 0.1% BSA-DMEM than subjected to the RHPA for PRL. The cells (30,000/ml at final dilution) were mixed with protein A-coupled ovine erythrocytes and aliquots (70–100 ml) were infused into poly-L-lysine-coated Cunningham chambers to form a monolayer (45 min preincubation). The cells were incubated with PRL antiserum (1:100 dilution) at 377C for 1 h followed by 30-min treatment with guinea pig complement (1:50 dilution). Either

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NIL DENERVATION–PRL AND SECRETION dopamine (DA, 10 06M), NIL extracts (0.5 eq from intact as well as PP denervated mothers) were added alone or in combination during the entire 1-h incubation period. The number of plaque forming cells were counted and the plaque areas were measured with a calibrated ocular reticule. Two hundred cells or plaques (100 on each of duplicate slide), from each of three to four completely separate experiments, were performed to determine the percentage of plaque forming cells (number of secretory mammotropes) and the mean plaque area (mean PRL secreted/mammotropes). Total secretion index (percentage of plaque forming cells 1 mean plaque area), which represents integrated hormone secretion, was also calculated. Histology and Immunohistochemistry Six to 8 days after PPD or sham surgery, animals were decapitated and their hypothalami and pituitaries were removed and fixed in 4% paraformaldehyde containing 0.1% phosphate buffer. Tissues were embedded in paraffin and serially sectioned in the coronal plane. Alternate sections were processed for staining of the neurosecretory material using Go¨mo¨ri method [9] and for tyrosine hydroxylase (TH) immunostaining using the ABC method (primary antibody TE101-TH, LOT # 2124, 1:2.000 dilution, Eugene Tech International, Inc.). Hormone Determinations The plasma was separated from each blood sample and stored at 0207C until analyzed for hormones. Plasma PRL was measured by RIA using the materials kindly provided by the National Hormone and Pituitary Program (Rockville, MD). Plasma level of a-MSH was determined by using a sensitive RIA developed by Vecsemye´s and Julesz [37]. All samples were measured in duplicate and from a particular experiment in the same assay. The intra-, and interassay coefficient of variations were below 10%. Statistical Analysis All results are expressed as mean { SEM. Statistical analysis of the data was performed using the ANOVA and Dunnett’s multiple range post test. RESULTS Effect of PPD on Neurosecretory Material in the NL and Tyrosine Hydroxylase (TH) Immunoreactivities in the IL Five to 7 days after PPD no stainable neurosecretory material can be detected in the shrinking NL (Fig. 1B). In controls, both the IL and NL contains a dense plexus of TH immunoreactive fibers. A more visible staining in the IL than in the NL clearly shows the characteristic network like pattern of dopaminergic fibers and varicosities (Fig. 1C). PPD results in a complete disappearance of TH immunoreactivity (Fig. 1D). The AL is completely nonreactive for TH in both sham-operated and PPD animals. Effect of PPD on Water Consumption The 6-day profile of water consumption is depicted on Fig. 2 detected in rats after sham operation or PPD. Daily water intake is doubled in PPD mothers 24 h after the surgery then it remains constant and significantly higher than in the sham-operated animals until the beginning of dDAVP treatment on day 4. One day after drinking dDAVP containing solution, water consumption returned to the level of the sham-operated animals. Water intake of both intact (data not shown) and sham-operated

315 mothers has shown a slight but gradual increase during the 6-day observation period. Effect of PPD on Separation-Induced Depletion and SucklingInduced Elevation of Plasma PRL and Changes After Pretreatment With dDAVP Alone or in Combination With OXY Removal of the pups from the cage results in a marked decrease in plasma PRL concentrations of intact mothers but no change can be detected in PPD rats (Fig. 3A). When the mothers are reunited with their litters, there is an immediate increase in plasma PRL of intact but a significantly attenuated PRL response of PPD rats. Similar differences can be detected on two consecutive days (data are not shown). Suckling-induced elevation of plasma PRL is still significantly attenuated compared to sham operated controls (Fig. 3B) after treatment of PPD mothers with dDAVP alone or in combination with OXY. When basal values of PRL detected at 0900 h have been compared, dDAVP treatment of PPD animals significantly elevates plasma PRL levels (Fig. 3B) and normalizes depletion of plasma PRL due to separation, as it is also found in intact animals. The same treatment in combination with OXY shows no elevation in basal hormone concentrations as well as no reduction in PRL levels due to separation (Fig. 3B). Effect of PPD on the Plasma Levels of a-MSH and Changes After Pretreatment With dDAVP Alone or in Combination With OXY Plasma a-MSH levels of sham operated mothers have been neither significantly changed during the period of separation (from 0900 h to 1300 h) nor in response to 15, 30, and 60 min suckling stimulus (Fig. 3A). One week after PPD, plasma levels of a-MSH are doubled during the entire sampling period (Fig. 3A). Neither dDAVP treatment alone nor in combination with OXY can influence basal level of a-MSH (Fig. 3B). Although, the pattern of plasma a-MSH during the suckling stimulus varies between groups of PPD and PPD / dDAVP or PPD / dDAVP / OXY, these differences are statisticaly not significant. Effect of NIL Extract Obtained From Intact or PP Denervated Lactating Rats on PRL Secretion of Mammotropes In Vitro To examine the effect of PPD on the PRL releasing activity of NIL extracts, primary cultures of anterior lobe cells obtained from intact lactating rats were treated with DA and perchloric acid extracts of intact as well as PP denervated NIL of mothers. Hormone release was measured by the RHPA. This hormone specific assay permits the microscopic demonstration of changes in PRL release of individual mammotropes (number of plaque forming cells) and the plaque area is linearly related to the amount of hormone secreted. Under certain conditions (depending upon the time of incubation) total plaque area that represents integrated hormone secretion, can be calculated by multiplying % of plaque forming cells and mean plaque area. As shown on Table 1, AL cells respond to DA treatment with a reduction of both the proportion of all cells being able to release PRL (39%) and the total amount of hormone released by mammotropes (49%). We found a concentration-dependent stimulation of PRL secretion of anterior pituitary cells obtained from intact mothers challenged by NIL extracts of lactating or male rats (control Å 687.3 { 26.2, 0.1 eq Å 837.8 { 36.4 or 868.8 { 26.7 and 0.5 eq Å 1281.7 { 19.7 or 1175.9 { 32.1 mm2 1 10 03 , respectively), confirming earlier reports [14,15]. NIL extracts from PPD rats have no stimulatory effect on PRL secretion (% of PRL cells and total secretion index are even less than in controls) (Table 1).

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FIG. 1. The effect of PPD on the neurosecretory material in the NL and tyrosine hydroxylase (TH) immunoreactivity in the IL. Photomicrographs of the pituitary gland in frontal sections of a sham (A and C) and a PPD animal (B and D) stained for neurosecretory material (A and B) or for TH immunoreactivity (C and D). Note the absence of neurosecretory materials and the atrophy in the NL. (B) There is a total disappearance of TH immunoreactivity from both the IL and NL (D). Bars represent 200 mm.

DISCUSSION Our histological observation showing that neurosecretory materials disappeared after PPD confirms the results of earlier studies [8,17,21] that the surgical compression of the stalk against the bone for a few seconds is sufficient to destroy the fibers to NL (Fig. 1). An immediate and sustained increase in the water intake of PP denervated animals well represents the functional deficit in vasopressin secretion caused by this surgery (Fig. 2). Besides affecting the neurosecretory system of the NL, this surgical procedure also results in a disappearance of tyrosine hydroxylase (TH) immunoreactivities from both the NL and IL (Fig. 1D compared to C). Dopaminergic neurons found in the rostral portion of the arcuate nucleus [4,6,7,13] and the periventricular regions [10,18] project to and terminate in the PP. The best characterized function of this so-called tuberohypophyseal dopaminergic (THDA) system is the tonic inhibition of a-MSH release from the IL [ 35,38 ] . Our observation, that disruption of dopaminergic innervation ( Fig. 1C and D ) of the IL is accompanied by highly elevated plasma levels of a-MSH ( Fig. 3A and B ) is in agreement with this role and also shows the

functional insufficiency of the IL due to denervation. Furthermore, PPD leaves both hypothalamic-median eminence and certain pituitary functions intact [ 8,17,21,22 ] without causing changes in TH immunostaining of the median eminence ( data not shown ) or permanent ischemic damage in tissues of the pituitary gland ( Fig. 1 ) . It is well-documented that separation induces a rapid and pronounced depression in plasma PRL levels of the mothers [2,29]. It has been shown that a dopaminergic mechanism is responsible for this inhibition of PRL release [29]. Involvement of DA in the regulation of PRL secretion is not in doubt [5,31] but the relative contribution of the two dopaminergic systems, tuberoinfundibular (TIDA) that projects to the median eminence and THDA that project to the NIL, in the control of PRL secretion has not been established. Indirect evidences have suggested that THDA neurons, besides their tonic inhibitory role in regulating hormone secretion of IL cells, rather participate in the tonic inhibition of PRL release as well, than in the dynamic events during the first few minutes of suckling stimulus [31]. In agreement with this notion, plasma level of PRL remains elevated by the end of

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FIG. 2. Daily water consumption (ml) of mothers following PPD (n Å 11) before and after dDAVP treatment or sham operation (n Å 11) over the period after surgery. Values represent the mean { SEM.

the 4-h separation period and a-MSH level is also significantly increased in PP denervated animals compared to the sham-operated mothers (Fig. 3A and B). At the same time, sucklinginduced changes in the plasma level of a-MSH cannot be detected in either intact or PP denervated rats. Parallel changes in plasma concentrations of these two hormones after a selective disruption of dopaminergic innervation (i.e., THDA innervation) of the PP strongly support the tonic inhibitory role of THDA neurons in the function of mammotropes as well as melanotropes. Our previous data have also indicated that a-MSH may be a

317 ‘‘responsiveness factor’’ that is responsible for the change in responsiveness of PRL cells to DA detected after 10 min suckling stimulus [12,26]. Therefore, as an alternate, our present results can also support the notion that an increased secretory activity of IL cells, which manifests in an elevation of a-MSH release, may change the responsiveness of mammotropes to the inhibitory control of DA coming from TIDA terminals through the long portal vessels. To distinguish between these possible explanations needs to be further investigated. During the last decade, evidences have been accumulated about the role of NIL in the overall control of PRL secretion [5,14,19,24,31]. One of the strongest and the most frequently used arguments is that surgical removal of the NIL acutely and completely blocks suckling-induced release of PRL [24]. One thing is almost certain, while PP lobectomy results in a loss of the NIL, at the same time it destroys or at least disturbs the sequence of blood perfusion of the AL arriving from the NIL via the short portal vessels into and being drained towards the cavernous sinus from the hypothalamo-hypophysial unit [1,23,36]. In contrast, besides a complete denervation of the NIL, PPD is accompanied by an undisturbed blood flow of the pituitary gland and results in an elevated level of plasma PRL after 4-h separation and only an attenuation in PRL responses due to suckling stimulus (Fig. 3A). Moreover, similar to PP lobectomy, PPD causes diabetes insipidus. At the same time, an attenuation of separation induced depression and suckling-induced elevation in plasma PRL could also be detected in mother rats suffering either severe or moderate diabetes insipidus like in homozygous Brattleboro [28] and/or AVP antisera treated [27] animals, respec-

FIG. 3. The effect of PPD with (B) and without (A) dDAVP treatment alone or in combination with OXY on the suckling-induced release of PRL and on the plasma levels of aMSH. Mothers were separated from their pups at 0900 h and replaced at 1300 h for 1 h suckling. PPD was performed 5–7 days prior to test. Each value is the mean { SEM. * p ú 0.01 (sham vs. PPD, PPD / dDAVP, and PPD / dDAVP / OXY). The number of rats in each group is shown in parentheses. S: suckling stimulus.

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318 TABLE 1

ACKNOWLEDGEMENTS

EFFECT OF DOPAMINE (DA) AND PP EXTRACTS FROM INTACT (INT-PP) OR PP-DENERVATED (PPD-PP) LACTATING RATS ON PRL SECRETION OF MAMMOTROPES

This work was supported by the Hungarian National Research Fund (OTKA 13055 and 20916 to G.M.N., OTKA 2922 and 16827 to G.B.M. and F 13133 to B.E.T.), and Cultural and Scientific Fund of Ferencva´rosi ¨ nkorma´nyzat. Part of this study was presented at the 24th Annual MeetO ing of the Society for Neuroscience, Miami Beach, FL, 1994. We are grateful to Ms. M. Me´sza´ros for her excellent assistance with the animal studies, to Mrs. I. Salamon for her expert immunohystological assistence. Thanks are due to Mrs. M. Bala´zs and Mrs. K. Szentmiklo´si for their technical assistance of RIA.

Treatment

Control DA (1006 M) INT-PP (0.5 eq) PPD-PP (0.5 eq)

% of PRL Cells

36.5 { 22.3 { 40.1 { 28.5 {

0.4 0.5* 2.9 1.8

Total Secretion Index (mm2 1 1003)

893.2 { 455.7 { 1170.4 { 722.1 {

66.4 60.3* 104.1* 94.8

Anterior pituitaries of lactating rats were enzymatically dispersed. Secretory responses of mammotropes (number of plaques (% PL) Å number of secretory mammotropes, mean plaque are (MPA) Å mean PRL secreted/mammotropes) were measured in the reverse hemolytic plaque assay and total secretion index (TSI), which represents a mean total plaque area Å integrated hormone secretion, was calculated by multiplying %PL and MPA. Results are the mean { SEM of three independent experiments. Values that are significantly different (p õ 0.05) from control are denoted by an asterisk (*).

tively. However, in two of these animal models, correction of the diabetes insipidus by dDAVP treatment elevates basal as well as partially restores separation induced depletion of plasma PRL ([29], Fig. 3A and B). All these together further support the notion that thirst control and the regulation of PRL secretion are closely coupled. The possible site(s) (hypothalamic paraventricular or arcuate nuclei) of interaction(s) between these two regulatory mechanisms is/are presently unknown. An additional strong argument for the existence of a PRF in the PP is that crude extracts of the NIL can stimulate PRL release in vitro [5]. However, this releasing activity has not yet been characterized. Our present data provide an independent confirmation for the existence of a PRF activity in extracts of RP (Table 1). In addition, based upon the smallest measure possible paracrine influence in the RHPA, PRF activity of these extracts may be mediated directly through the mammotropes. One can easily conclude that the attenuated PRL response to suckling after PPD, observed in our in vivo experiments, is due to the missing PRF activity normally found in IL cells [19]. However, our data that NIL extracts from PPD rats have no stimulatory effect on PRL release (Table 1) only suggest that if this regulatory substance really exists, it can be synthesized either in the hypothalamus or in the IL cells. In this later case it can be produced and, therefore, operate properly when the hypothalamic innervation of the NIL is intact. All these together confirm that the NIL contains a physiologically significant PRF, but its exclusive role in the acute and dynamic changes of PRL secretion is uncertain. The possible contribution of the recently observed elevation in portal plasma concentrations of OXY and AVP after 1 week of PPD [22] in the mechanism of the attenuated PRL response to suckling is now under investigation. In conclusion, the relative contribution of THDA system terminating at the IL in the regulation of PRL secretion seems to be very important as it was predicted earlier. Although, the existence of a PRF activity [5,16,19] in intact PP extract has been confirmed but its disappearance after denervation of the NIL suggests that it may be produced by the hypothalamus or its secretion and proper function in the NIL is dependent on an intact hypothalamic–hypophysiotrophic influence. The missing interactions between three (dopaminergic, thirst, and PRF) regulatory systems may explain the observed disturbance of PRL release in NIL-denervated animals. However, their intimate relationship needs to be further clarified.

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