Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Effect of beta-endorphin on endocrine function

It has been disclosed that beta-endorphin exerts marked effect on the secretion of ACTH, prolactin, corticosterone, aldosterone and somatotrophin formation in the pituitary but does not produce any effect on blood thyrotropin. Maximal rise in the concentration of the hormones was seen at the 20th minute, despite the fact that an elevation in the content of some hormones was recorded at the 5th minute following intravenous injection of beta-endorphin. At the 60th minute after beta-endorphin injection the content of prolactin, corticosterone and aldosterone in the blood dropped to the control level, while ACTH content remained significantly higher than in intact animals. One of the possible mechanisms underlying the action of beta-endorphin on the secretion of the hormones indicated might be the changes in the ratio of brain monoamines (a decrease in dopamine).     

Origin of prolactin cells in the pituitary autografts in the renal capsule of young male and female rats.

Origin of the prolactin cells in the pituitary grafts autotransplanted into the renal capsule was electron microscopically investigated in young male and female rats. Prolactin cells may mainly originate from the completely degranulated acidophils after grafting in both sexes. The comprehensive degranulation of acidophils may be essential condition for the development of prolactin cells. Formation of initial prolactin granules is related to the Golgi apparatus in 3 and 6 days in the males. Even prolonged transplantation for the duration of 10 and 20 days failed to develop the typical prolactin cells storing large polymorphic granules in the grafts in the males. In the females, however, the population of prolactin cells became higher (20-30%) and their granulation was more rapidly advanced than in the male. Nine and 16 days after grafting quite numerous typical prolactin cells, characterized by the pronounced vesiculation of ER stored large polymorphic granules in the females. Progressive vesiculation of ER in degranulated acidophils may be the second necessary condition for establishing the prolactin cells. Thus, the entire course of cell conversion of acidophils into prolactin cells was observed in this study. All the graft cells including prolactin cells finally turned into the cells losing the respective individuality (10 days in the males; 32 days in the females). This fine structural similarity was striking in the males. We could not, however, deny the possibility of origination of prolactin cells from the follicular cells or their adjoining undifferentiated cells.     

Trifluoperazine inhibits thyrotropin-releasing hormone-stimulated TSH secretion

In previous work changes of the thyrotropic secretion after administration of some substances affecting the calcium content in the cytosol were demonstrated. The object of the present investigation was to assess the hormonal response to the administration of trifluoperazine, a psychopharmaceutical preparation, the main mechanism of its action being the inactivation of the cytosol receptor for the calcium signal - calmodulin. The poor utilization of intracellular calcium of the secretory cell is then the factor which inhibits secretion proper. The thyrotropic secretory reserve (delta TSH) was assessed in the same subjects before and after trifluoperazine administration by the TRH test as the difference of values at rest and TRH-stimulated TSH levels during the 20th, 30th, 40th and 60th minute following intravenous administration of 200 micrograms TRH. It was revealed that this calmodulin antagonist administered for one week in amounts of 6-12 mg per day by mouth significantly inhibits the secretory response of TSH to TRH in healthy subjects during the 20th and 40th min. (P less than 0.05). The reproducibility of the TRH test repeated in a group of subjects not treated with trifluoperazine, however, under equal conditions and after the same time intervals as in the experiment with trifluoperazine was very satisfactory and thus physiological inhibition caused by repeated TRH administration could be ruled out. The inhibition of the secretory TSH response to TRH can be therefore considered the consequence of the direct effect of trifluoperazine on the thyrotropic secretory mechanism. Trifluoperazine significantly reduced serum calcium levels and raised phosphate levels, while it did not affect the blood levels of magnesium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of pituitary hormones (hCG, TSH, Pr, GH) on testosterone level and on the functional activity of the Leydig cell in rat fetuses

One day after the cessation of treatment the Leydig cells of the fetuses of pregnant rats, treated between the 11th and 15th or the 16th and 20th days of gestation, reacted to pituitary hormones. This finding indicates that both the receptors and the postreceptor mechanisms were in operative state. The effect of the thyrotropic hormone (TSH) overlaps the effect of related gonadotropic hormone (hCG), although this effect becomes smaller from the 21st day. The parameters investigated - the spectrocyto-fluorimetrically measured RNA-DNA ratio and the plasma testosterone level - ran generally in parallel. Similarly to the above-mentioned hormones, prolactin also increased the testosterone level (though to lesser degree than hCG and TSH did), however, while it increased the RNA level but at the age of 16 days, it decreased it the age of 21 days. Somatotropin (GH) also increased somewhat the testosterone level; however, the effects of the two related hormones (Pr and GH) fell far beyond the effect of either TSH or hCG.     

Studies of calcitonin cells and parathyroid glands of the Indian palm squirrel, Funambulus pennanti in response to experimental hypercalcaemia.

Long term hypercalcaemia was induced in F. pennanti by alternate day intramuscular injections of 50,000 IU of vitamin D2 and by giving them 1% CaCl2 solution prepared in tap water to drink. The controls were not injected with vitamin D2 and were given tap water. The serum calcium levels at various stages of the experiment (1-29 days) show increased values as compared with those of control animals. The calcitonin cells in the treated animals generally exhibit an increase in their number up to the 15th day. Mitotic figures are also encountered between the 7th and the 15th day of treatment. This exhibits the increase in the number of C cells. Constant calcium challenge results in increased quantities of secretory granules among these cells up to the 15th day and in degranulation from the 17th day onwards. It also causes degenerative changes in a certain number of C cells. The parathyroids exhibit atrophic changes (25 days onwards) due to chronic hypercalcaemia. For short term hypercalcaemia, animals were injected intravenously with 1 ml of 10% solution of calcium gluconate. The calcitonin cells do not exhibit any change during the first half hour but thereafter they exhibit progressive degranulation, resulting in marked degranulation after 5 hours of the injection. The parathyroids remain unaffected throughout the experiment and show no histological change.     

Hormonal regulation of prolactin storage in a clonal strain of rat pituitary tumor cells

GH4C1 cells (GH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. GH cells have been used to study hormone secretion, but they store relatively little prolactin compared to normal prolactin-secreting cells. They are not suitable, therefore, for studying some aspects of pituitary function. We have found that the amount of prolactin GH cells store can be regulated. When GH cells were plated at 10(6) cells/well and treated for six days with 180 nM insulin or 1 nM estradiol, there was a 60 percent increase in prolactin storage compared to control cells. Insulin and estradiol in combination acted synergistically to cause a 190 percent increase in prolactin storage. In contrast, they were additive in increasing extracellular prolactin; there was a 40 percent increase in extracellular prolactin after insulin, a 20 percent increase after estradiol, and a 50 percent increase after insulin plus estradiol. The increases in prolactin storage were always greater than the increases in extracellular prolactin. The increases in prolactin storage were dose-dependent and reached maximal levels after four days of treatment with 180 nM insulin plus 1 nM estradiol. Reducing the plating density to 10(3) cells/well increased the response to insulin and estradiol to nineteenfold. Epidermal growth factor (10 nM) acted synergistically with estradiol and insulin in combination to increase prolactin storage 27-fold. The insulin- and estradiol-induced increase in extracellular prolactin was caused by a specific increase in the rate of prolactin synthesis. The fractional increase in prolactin storage above the increase in prolactin production could not be explained by an increase in prolactin synthesis, an increase in intracellular transit time, or a change in the cell-cycle distribution of the population. Hormone storage can, therefore, be regulated independently from other processes which control hormone production. The prolactin stored in response to insulin and estradiol was releasable by potassium depolarization. Following depletion of intracellular prolactin by depolarization, the cells retained their increased capacity for prolactin storage. The ability to increase prolactin storage will make GH cells a more useful system in which to study pituitary function.     

Immunocytochemical identification and ontogeny of adenohypophyseal cells in a cave fish,Phreatichthys andruzzii (Cypriniformes: Cyprinidae)

The morphogenesis of the pituitary gland and the chronological appearance of adenohypophyseal cells were investigated for the first time in the Somalian cave fish Phreatichthys andruzzii by immunocytochemistry. The adult adenohypophysis contained: a rostral pars distalis, with prolactin (PRL) cells arranged in follicles and adrenocorticotropic (ACTH) cells, a proximal pars distalis with somatotropic (GH), β‐thyrotropic (TSH), β‐gonadotropic type I (FSH) and type II (LH) cells and a pars intermedia with α‐somatolactin (SL), α‐melanotropic (MSH) and β‐endorphin (END) cells. All regions were deeply penetrated by neurohypophyseal branches. At hatching (24 h post‐fertilization) the pituitary was an oval cell mass, close to the ventral margin of diencephalon. The first immunoreactive cells appeared as follows: PRL at 0·5 days after hatching (dah), GH and SL at 1·5 dah, END at 2 dah, TSH, ACTH and MSH at 2·5 dah, FSH at 28 dah and LH at 90 dah. The neurohypophysis appeared at 5 dah and branched extensively inside the adenohypophysis at 130 dah, but there was no boundary between rostral pars distalis and proximal pars distalis at this stage. The potential indices of prolactin and growth hormone production increased until 28 and 60 dah, respectively. The potential index of growth hormone production correlated positively with total length. Activity of PRL and GH cells, measured as ratio of cell area to nucleus area, was significantly higher in juveniles than in larvae.     

The identification of the thyrotropic cells in the adenohypophysis of the cichlid fish Cichlasoma biocellatum and the role of these cells and of the thyroid in osmoregulation

Summary In order to identify the thyrotropic cells in the pituitary gland of Cichlasoma biocellatum, adult animals were treated with thyroxine. Periodic acid Schiff (PAS)- and aldehyde fuchsin (AF)-positive, and Alcian blue (AB)-negative basophils in the meso-adenohypophysis as well as the thyroid were strongly inactivated by such treatment. Since other cell types in the adenohypophysis were not affected, it is concluded that the PAS-positive and AB-negative basophils in the meso-adenohypophysis are the thyrotrops. In normal animals the thyrotrops and the thyroid are very active. Propylthiouracil did not cause a further activation of TSH cells. Keeping the animals in 25% sea-water caused a strong inactivation of the prolactin cells, the thyrotrops and the thyroid. In deionized water the prolactin cells were stimulated, but the thyroid was less active than in controls. Injections of ovine prolactin did not enhance the activity of the thyroid in salt water animals. It is suggested that in Cichlasoma biocellatum prolactin does not have a direct stimulatory effect on the thyroid.Thanks are due to Prof. Dr. P. G. W. J. van Oordt for his continual interest and valuable suggestions, and to Dr. L. Boomgaart for correcting the english. We are indebted also to Messrs. H. van Kooten and E. van der Vlist for making the photographs. The National Institutes of Health, Bethesda, Maryland (U.S.A.) kindly presented the ovine prolactin.     

The influence of prolactin (PRL) on progesterone secretion by porcine luteal cells in vitro

Porcine luteal cells were obtained from corpora lutea on the 5th, 13th and 17th days of the estrous cycle. The cells were suspended at a concentration of 5 × 10⁴ cells/ml in Eagle's medium with 2% human serum albumin. These cells were incubated with or without 0.01, 0.1, 1 or 10 μg/ml porcine prolactin. The amount of progesterone in cultures was estimated by a radio-immunological method after 30 min, 3 h and 6 h of culturing.Luteal cells obtained on the 5th day of the estrous cycle and incubated without prolactin secreted 71.24 ± 21.91 ng progesterone/ml of medium, whereas under the influence of prolactin at 0.01, 0.1, 1 and 10 μg/ml, 39.06 ± 13.33, 44.31 ± 12.69, 44.88 ± 16.85 and 51.62 ± 15.01 ng progesterone/ml (P<0.01) were secreted. Luteal cells from the 13th day of the estrous cycle incubated without prolactin secreted on average 70.72 ± 9.21 ng progesterone/ml of medium, whereas under the influence of different prolactin doses 50.75 ± 8.52, 46.54 ± 7.13, 43.30 ± 6.78 and 41.68 ± 7.21 ng progesterone/ml (P<0.01) were secreted.Prolactin did not change progesterone secretion by luteal cells obtained on the 17th day of the estrous cycle. An influence of the incubation time on progesterone secretion by these cells was observed: after 30 min of incubation the cells secreted 8.83 ± 2.95 ng/ml, after 3 h 8.12 ± 2.57 ng/ml and after 6 h 6.86 ± 1.91 ng/ml, irrespective of the amount of PRL added.The results suggest that prolactin plays a role in the luteolysis of the corpus luteum.     

CNS - endocrine pancreas system. IV. Evidence for the existence of a direct hypothalamic-vagal descending pathway

Influence of TRH, T3 and cGMP on the volume of thyrotropic cells nuclei in rat pituitary in vitro has been examined. A significant increase of the volume of thyrotropic cells nuclei in the culture exposed to TRH, in another one exposed to cGMP and in the third one exposed to both TRH and cGMP together have been observed. The mean volume of cells nuclei within the group exposed to T3 was significantly lower, when compared to the control. T3 inhibited the TRH-induced increase of the volume of thyrotrophs nuclei. The obtained results suggest that the role of cGMP and T3 in the regulation of the thyrotropic cell function are independent and rather opposed. Since TRH increases the level of cAMP in the anterior pituitary, additive effect of TRH and exogenous cGMP support the assumption that both the cyclic nucleotides within the thyrotropic cell act in the same direction.     

Immunohistochemical Study of Adenohypophysial Cells During Embryonic Development in the Reptile Chalcides chalcides (Squamata, Scincidae)

The immunohistochemical avidin-biotin complex method was used to study hormone-producing cells in the adenohypophysis of the skink Chalcides chalcides during embryonic development. In Chalcides, the formation of Rathke's pouch was evident between stages 28 and 30 of embryonic development. The adenohypophysial cells begin to differentiate before the morphological development of the gland was complete. At stage 29, few corticotropic cells were present only in the dorsal face of Rathke's pouch. No other immunoreactive cell type was revealed at this stage. At stage 32, the hypophysis had developed to a great extent though it was not yet elongated in a cephalic-caudal direction. At this stage, the corticotropic cells appeared more numerous and well differentiated in the rostral pars distalis and in the pars intermedia. Melanotropic, somatotropic and gonadotropic cells appeared simultaneously, with the same distributions as in the adult skink. At stage 34, the first thyrotropic cells appeared in the pars distalis but also in the pars intermedia, whereas rare prolactin cells were observed only at stage 35 in the medial pars distalis. Between stages 36 and 38, the gland was developed in the cephalic-caudal direction and all the cell types were completely differentiated with an evident increase in the number of prolactin cells. In embryos close to birth (stages 39-40), the hypophysis and the adenohypophysial cells were already similar to those of the adult animal.     

Hypothalamic neurosecretion and metamorphosis in Xenopus laevis

Summary Tadpoles of Xenopus laevis were treated with propylthiouracil from the second half of prometamorphosis. Sagittal sections of the head region were stained a.o. with pseudoisocyanine. The goitrogen caused a degranulation of neurosecretory cells in the dorsal part of the preoptic region of the hypothalamus, suppressed the development of ventral neurosecretory cells and of the outer zone of the median eminence, stimulated the thyrotropic cells in the adenohypophysis, caused a hypertrophy of the thyroids, and impaired metamorphosis. Returning the animals to tap water had reciprocal effects and restored the normal activity of the hypothalamus, adenohypophysis and thyroid glands. It is concluded that thyroid hormones exert a morphogenetic influence upon hypothalamic centres and the outer zone of the median eminence and that a negative feed back relation exists between the thyroids on the one hand and the dorsal neurosecretory cells and the thyrotropic cells on the other.The author thanks Prof. Dr. P. G. W. J. van Oordt for his active interest and helpful advice, and Miss Tineke Aafjes for technical assistance.     

Immunohistochemical Identification of the Adenohypophysial Cells in the Indian Paddy Field Frog,Rana limnocharis

Cells of the pituitary pars distalis (PD) and pars intermedia (PI) in the frog Rana limnocharis have been identified by an unlabelled antibody enzyme method using antisera developed in rabbit against mammalian hypophysial hormones. On the basis of their immunoreactivity, six types of cells, viz. thyrotropic (TSH), gonadotropic (GTH), prolactin (PRL), growth hormone (GH), corticotropic (ACTH) and melanotropic (MSH), cells have been recognized. GTH and PRL cells are distributed throughout the PD. GH cells usually occur in the anterodorsal and central region of the gland. Immunoreactive TSH cells are fewer in number and are localized in the ventromedian region of the PD. Cells showing immunoreactivity to ACTH 1–24 antiserum are encountered in the rostroventral part of the PD. Cells of the PI also show immunoreactivity to ACTH 1–24 antiserum. PI cells cross-react with α-MSH antiserum at all dilutions up to 1: 50 000. However, when the same antiserum was used at dilutions up to 1: 20 000, the ACTH cells of the PD also showed cross-reactivity.     

Inhibition by progesterone of the lactogenic effect of prolactin in the pseudopregnant rabbit.

The effects of progesterone on lactose synthesis activity and changes in mammary gland cells were studied in pseudopregnant rabbits simultaneously treated with prolactin. The injection of progesterone alone on Days 15 and 17 of pseudopregnancy decreased the activity of lactose synthetase (LSA) and galactosyl transferase (GTA), while the administration of prolactin for 2-4 days increased their activities. Th e simultaneous administration of progesterone and prolactin decreased the increase in LSA observed with prolactin alone by 70% on the 4th day of treatment, and decreased GTA by 30%. Progesterone completely suppressed the polyribosome profile and the ratio of endoplasmic reticulum bound polyribosomes to free polyribosomes induced by prolactin. The increase in RNA content in the mammary gland induced by prolactin was also suppressed by progesterone. The results suggest that progesterone inhibits the lactogenic action of prolactin without interfe ring with its mammogenic role.     

Liver and muscle glycogen contents and blood glucose concentration after AET or MEA treatment of adult male mice.

A statistically significant decrease was found in the glycogen content expressed in mg per 1 g of both liver in groups of mice killed in the 60th and 90th minute, and skeletal muscles in those killed in the 30th, 60th, and 90th minute after MEA injection, as well as a statistically significantly reduced blood glucose concentration in a group of mice killed in the 30th minute after AET treatment.     

Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig

Summary Administration of ovine prolactin to castrated guinea pigs for 2 weeks induced hypertrophy of secretory cells in the lateral prostate when compared with the castrated controls. This was accompanied by an apparent increase in the number of profiles of granular endoplasmic reticulum and well developed Golgi complexes with dilated cisternae. An increase in the number of low-contrast electron-dense secretory granules was observed 4 weeks after prolactin treatment. In the seminal vesicle, dilatation and degranulation of granular endoplasmic reticulum and an apparent decrease in the number of secretory granules were observed 4 weeks after prolactin administration. Following castration and 2 weeks after prolactin treatment, thiamine pyrophosphatase (TPPase)-reaction product was mainly confined to 1–2 trans cisternae of the Golgi complexes in secretory cells of the lateral prostate and the seminal vesicle. In both glands, a reduction of TPPase activity was observed 2 weeks following prolactin administration, and the reaction product was totally absent after prolonged treatment for 4 weeks. The present study has provided morphological evidence that prolactin is capable of stimulating the secretory function of the lateral prostate while exerting some inhibitory effects on the seminal vesicle of the castrated guinea pig. In both glands, TPPase activity, and hence the process of glycosylation was inhibited after prolactin administration. The results from radioimmunoassay indicated that the action of prolactin on these glands could be a direct effect and not mediated through testosterone.     

Changes of cell morphology and prolactin secretion induced by 2-Br- alpha-ergocryptine, estradiol, and thyrotropin-releasing hormone in rat anterior pituitary cells in culture

The secretion of prolactin in cultured pituitary cells was studied in correlation with the cellular changes induced by stimulatory or inhibitory agents. The techniques used in this study were: radioimmunoassay, immunocytochemistry, scanning (SEM) as well as transmission (TEM) electron microscopy. Prolactin secretion was stimulated by 17 beta-estradiol (10 nM) as well as thyrotropin- releasing hormone (TRH) (3 nM) and inhibited by 2-Br-alpha-ergocryptine (CB-154) (1 muM). The total prolactin (release and cell content) increased between 2 and 8 d of estradiol treatment, indicating an increase of both synthesis and release of prolactin. This finding was in agreement with TEM observations because, in estradiol-treated prolactin cells, the Golgi saccules were distended and Golgi elements were increased, thus indicating increased synthetic activity of these cells. The addition of TRH over a 4-h period resulted in a significant degranulation of prolactin cells. In contrast, prolactin secretory granules became accumulated in the cells after CB-154 treatment for a period ranging from 4 to 24 h. In agreement, light microscope immunocytochemistry showed an increased reaction for prolactin after short-term (< 24 h) incubation with CB-154. Because prolactin cells represent approximately 70% of the glandular cell population as revealed by immunocytochemistry, it was then possible to observe the changes of cell surface by SEM. In most cells, estradiol and TRH led to an increase in the number and prominence of microvilli and blebs, whereas CB-154 treatment resulted in a slightly decreased number of microvilli and an increased occurrence of membrane foldings. This report thus provides morphological evidence for the stimulatory effects of estradiol and TRH, and the inhibitory effects of CB-154 on prolactin secretion in pituitary cells in primary culture. These data, moreover, show that acute changes in secretory activity of prolactin-secreting cells are accompanied by marked changes of their morphological characteristics.     

Osmotic regulation of prolactin secretion. Possible role of chloride

The role of osmotic pressure in the exocytosis of prolactin from rat pituitary tumor (GH) cells in culture was investigated. Reducing the osmotic strength of the medium from 300 mosm to 150 mosm by removal of NaCl did not alter basal secretion of prolactin but inhibited secretion stimulated by thyrotropin-releasing hormone (TRH) and forskolin. Both basal and stimulated secretion of prolactin were inhibited by increasing the osmotic strength of the medium with NaCl (IC50 at approximately 500 mosm). The stimulated release of hormone from GH-cells was independent of sodium and unaffected by replacement of sodium ion with tetramethylammonium or choline, or by addition of 500 nM tetrodotoxin. Secretagogue-stimulated release was, however, dependent upon chloride. Exchange of medium chloride with benzoate or isethionate significantly inhibited the stimulated release of prolactin (IC50 at approximately 60 mM exchange) regardless of the secretagogue utilized (phorbol ester, forskolin, depolarization plus BAY K8644, or TRH). Exchange of medium chloride with either isethionate or benzoate reduced cell volume by 10% compared to 60% for sucrose and mannitol, suggesting that inhibition of secretion by isethionate exchange was not a result of increased intracellular osmotic pressure. Complete exchange of medium chloride with isethionate did not alter equilibrium [3H]methyl-TRH binding, resting internal [Ca2+], or the [Ca2+]i response to depolarization and TRH as measured with intracellularly trapped Fura 2. Chloride removal did not change resting internal pH and recovery from an acid load as measured by the intracellular pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The stimulated secretion of prolactin was also inhibited by exchange of chloride with isethionate in normal pituitary cells in primary culture and the ability of normal cells to respond to the dopamine agonist bromocryptine was not affected by the exchange. These results suggest that exocytosis of prolactin from GH-cells and normal pituitary cells in culture is an osmotically driven process that is chloride-dependent. Stimulated release is more chloride-dependent than constitutive release. The inhibitory effect of isethionate substitution occurs after signal transduction and is distinct from the site of dopamine inhibition of prolactin release.