Acute effect of basic fibroblast growth factor on secretion of prolactin as assessed by the reverse hemolytic plaque assay

The effect of basic fibroblast growth factor (bFGF) on acute secretion of PRL by pituitary lactotrophs was examined under basal conditions and after treatment with TRH or dopamine. We used the reverse hemolytic plaque assay (RHPA) to determine the amount of PRL secreted per lactotroph and the percentage of pituitary cells secreting PRL. Young (2- to 3-month-old) female Sprague-Dawley rats were ovariectomized and 1 week later implanted with a Silastic capsule containing 180 micrograms/ml estradiol in sesame oil. Three days later, rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. In Exp I, time and dose responses to bFGF were determined using the RHPA. Basic FGF reduced (P less than 0.0001) the mean basal secretion of prolactin per lactotroph. The effect was similar at 30, 60, 120, and 240 min of incubation. The reduction in PRL was greatest at 3.36 x 10(-6) M, while lesser reductions were observed at 1.68 x 10(-6) and 5.60 x 10(-7) M. A dose of 3.36 x 10(-6) M (60 ng/ml) and an incubation time of 60 min were subsequently used in Exp II. In Exp II, we examined the effects of bFGF on TRH stimulation and dopamine inhibition of PRL secretion. PRL secretion was maximally stimulated (P less than 0.01) by 10(-7) M TRH. Basic FGF blocked the TRH-induced increase in PRL secretion. PRL secretion was maximally reduced (P less than 0.001) by 10(-5) M dopamine. Coincubation of bFGF with dopamine reduced (P less than 0.01) the mean plaque area to the same extent as dopamine alone. In each experimental situation changes in mean plaque area reflected a shift in the frequency distribution of the plaque area. Neither bFGF, TRH, dopamine, nor the combined treatments influenced the percentage of pituitary cells secreting PRL compared to basal conditions. We have demonstrated that 1) bFGF reduces the basal secretion of PRL in an acute manner; 2) bFGF blocks the TRH-induced increase in PRL; and 3) bFGF does not potentiate the inhibitory effect of dopamine on PRL secretion and, therefore, may act in part through the same inhibitory pathway as dopamine. We conclude from these data that bFGF, or related factors, could play a role in the regulation of PRL secretion.     

Characteristics of immunoglobulin secretion in man evaluated by a reverse hemolytic plaque assay

Summary The kinetics of Pokeweed mitogen-induced transformation of human B-lymphocytes into immunoglobulin-secreting cells were examined in a reverse hemolytic plaque assay using protein A-coated sheep red blood cells as indicator cells. Peak responses occurred consistently after 6–8 days of incubation. After determination of the optimal experimental conditions the RHPA was found to be a reliable tool to estimate ISC in vitro; the technique was also found to be applicable for experiments surveying the B-cell response of an individual over a period of months.The PWM-induced transformation of B cells was absolutely T-cell-dependent. Other substances known as typical T-cell mitogens were also tested for polyclonal B-cell activation and some of them showed significant responses. Further experiments have shown that co-cultivation of non-HLA identical cells did not increase the number of plaques in unstimulated cultures whereas the addition of PWM leads to the generation of ISC within the expected range. These findings open a wide field of application of the RHPA in experimental and clinical immunology.Supported by Deutsche Forschungsgemeinschaft (Ru 215/2)     

Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay

The reverse hemolytic plaque assay was used to study hormone release in vitro by seven clinically nonfunctioning human pituitary adenomas associated with no clinical or biochemical evidence of hormone excess. Four of seven tumors were oncocytomas, one a null cell adenoma, and two gonadotroph adenomas based on immunocytochemical and ultrastructural features. In all seven tumors, plaques were formed with antiserum against beta FSH; four produced plaques for beta LH, and five for glycoprotein hormone alpha-subunit. The percentage of plaque-forming cells and the mean size of plaques were smaller than those of clinically functioning adenomas studied for comparison (five GH- and/or PRL-producing adenomas). These results correlated with those of hormone release in tissue culture, immunocytochemistry on paraffin secretions of the tumors, and immunocytochemistry after reverse hemolytic plaque assay. We conclude that clinically nonfunctioning pituitary adenomas release small quantities of hormones, primarily gonadotropins, and that hormone release is attributable to only a small percentage of tumor cells.     

Enumeration of circulating ferritin-secreting cells by a reverse hemolytic plaque assay

In the present study, we tried to detect ferritin-secreting cells (FSC) as plaque-forming cells using a reverse hemolytic plaque assay and thereby to evaluate some of the cellular aspects of ferritin dynamics associated with various hematological disorders. Healthy adult males had 433 FSC and adult females had 244 FSC in 1 X 10(5) peripheral blood mononuclear cells (PBM). The mean numbers of FSC in 1 X 10(5) lymphocyte-enriched fraction were 120 in males and 53 in females, and those in 1 X 10(5) monocyte-enriched fraction were 932 in males and 668 in females. These results indicate that monocytes contain about 8-13 times as many FSC as lymphocytes. Each fraction of PBM from patients with iron deficiency anemia contains only about half as many FSC as those of normal individuals. Whereas, in states of increased iron storage such as seen in sideroblastic anemia, aplastic anemia and myelofibrosis, the mean numbers of FSC are markedly increased to about 4-8 times in PBM, about 11-20 times in lymphocytes and about 3-7 times in monocytes, compared with each counterpart of the normal individuals.     

Effects of gonadal steroids on somatotroph function in the rat: analysis by the reverse hemolytic plaque assay

The mechanism by which gonadal steroids modulate GH secretion is not known. We have used the reverse hemolytic plaque assay to examine whether gonadal steroid-induced modulation of GH secretion is effected by changes in the population of somatotrophs and/or alterations in their secretory properties. Two groups of Sprague-Dawley rats were studied: group 1 (n = 6) comprised male (M), castrate (Cx), and testosterone-replaced castrate male (Cx + T) rats and group 2 (n = 5) consisted of male (M), female (F), and 17 beta-estradiol-replaced castrate male (Cx + E) rats. The number of plaque-forming cells (expressed as both absolute number and a percentage of all cells) was determined, and secretory status was assessed by measuring plaque areas in response to 0, 0.01, 0.1, 1, 10, and 100 nM GHRH. While mean basal GH plaque areas were similar among the treatment groups of group 1, the maximal GH plaque area was significantly decreased in Cx [16.8 +/- 2.4 vs. 26.4 +/- 3.9 X 10(6) microns2 (mean +/- SEM); P less than 0.05], but not in Cx + T (27.5 +/- 4.1 microns2) rats. The GHRH EC50 was unaffected by castration or T replacement. The percentage and absolute population of somatotrophs were reduced in Cx, but not in Cx + T, rats, while the numbers of lactotrophs remained unchanged in these treatment groups. For group 2, the mean peak GH plaque area was reduced in Cx + E (16.5 +/- 2.9 microns2; P less than 0.001) compared to that in M rats (36.2 +/- 2.3 microns2), but was not significantly different from that in F (13.0 +/- 1.5 microns2) rats. The EC50 was significantly (P less than 0.025) greater in Cx + E (10.9 +/- 2.3 nM) and F (7.9 +/- 1.6 nM) compared to M rats (2.8 +/- 0.7 nM). The absolute somatotroph and lactotroph populations were increased in Cx + E compared to M and F rats, as were the populations of other pituitary cell types. Testosterone enhances GH secretion by increasing the secretory capacity, but not the sensitivity, of somatotrophs to GHRH and by recruiting the function of a subpopulation of somatotrophs. Estradiol reduces the secretory capacity and sensitivity of somatotrophs to GHRH, but increases the population of somatotrophs, lactotrophs, and non-GH- and non-PRL-secreting cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Postnatal development of pituitary lactotropes in the rat measured by reverse hemolytic plaque assay

Developmental changes and sexual differences in the lactotrope population were studied with a reverse hemolytic plaque assay for detection and measurement of PRL secretion from individual cells in conjunction with PRL immunocytochemistry (ICC). Pituitary glands from both sexes at different ages were monodispersed with 0.1% trypsin. Freshly dispersed cells were incubated in Cunningham chambers for measurement of the fraction of plaque-forming cells and the size of plaques formed, or attached to glass slides for measurement of the fraction of cells staining for PRL by ICC. The percentage of plaque-forming cells in both sexes gradually increased with age from about 5% of the total anterior pituitary cell population at 5 days of age to the adult levels of about 54% in the females at proestrus and about 37% in the males. Sexual differences in the percentage of plaque-forming cells were consistently seen at 40 days of age and thereafter. The mean size of plaques in both sexes also increased with age from about 1,100 microns 2 at 5 days of age to a peak level of about 15,600 microns 2 in the females at proestrus and about 6,700 microns 2 in the males at 60 days of age; then fell to the adult levels of about 5,500 microns 2 in the females at proestrus and about 3,200 microns 2 in males. Sexual differences first appeared at 40 days of age and were estrogen-dependent. The results from ICC closely matched those from the plaque assay, except at 5 days of age and in the adult males. At 5 days, the fraction of cells staining for PRL was twice that of cells forming plaques. In adult male rats there were about 47% immunostained PRL cells but only about 39% plaque-forming cells. This difference, however, disappeared after estrogen treatment. These results demonstrate that sexual differences in the lactotrope population develop around puberty in rats and appear to be estrogen dependent.     

A cellular basis for growth hormone deficiency in the dwarf rat: analysis of growth hormone and prolactin release by reverse hemolytic plaque assay

The purpose of this study was to elucidate the cellular basis for growth hormone deficiency in Lewis-derived dwarf rats. To this end, we used reverse hemolytic plaque assays to evaluate hormone release. This allowed us to determine the proportional abundance of GH- and PRL- secreting cells and estimate the relative amount of hormone secreted by individual pituitary cells from dwarf rats and their somatically normal counterparts. The percentage of all pituitary cells that released GH (formed plaques) in pituitary dispersions was substantially lower for dwarfs when compared with normals in the absence (3.8 +/- 2.2% vs. 43.4 +/- 2.2%) or presence (6.6 +/- 3.8% vs. 45.7 +/- 1.4%; n = 3) of GRF. In addition, GH secretors from dwarfs released less hormone (formed smaller plaques) than their normal counterparts under both basal and stimulated conditions. An analysis of the relative number of GH cells that formed plaques of various sizes was accomplished by constructing a frequency distribution. Dwarf GH secretors formed more small plaques and proportionately fewer larger plaques than normals under both basal and stimulated conditions. For comparison, we also quantified the proportions of PRL secretors and found that they were actually more abundant in dwarfs than normals in the absence (52.4 +/- 4.6% vs. 33.7 +/- 3.7%) or presence (53.4 +/- 4.9% vs. 33.8 +/- 4.1%; n = 4) of TRH. Treatment with this secretagogue consistently increased mean PRL-plaque area for both groups. Our findings demonstrate that dwarf rats are severely deficient in the proportion of GH secretors. In addition, the few GH secretors present in the dwarf pituitary were less responsive to GRF than normals. In contrast, PRL cells in dwarfs appear to be functionally similar to those of their normal counterparts. The reciprocal relationship in the proportions of GH and PRL secretors in dwarfs provides a rather unique model for investigating the functional relationship between these cell types.     

Basic fibroblast growth factor inhibits basal and stimulated relaxin secretion by cultured porcine luteal cells: analysis by reverse hemolytic plaque assay.

The role of the basic fibroblast growth factor (basic FGF) in the control of secretion of the ovarian protein hormone relaxin (RLX) by porcine large luteal cells (LLCs) was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled sheep erythrocytes. In the presence of complement and porcine relaxin antiserum, a zone of hemolysis (a plaque) developed around a RLX-releasing LLC. The rate of plaque development in time-course experiments was used as an index of the rate of RLX secretion. Monolayers were bathed in medium containing graded concentrations of basic FGF in the presence or absence of a stimulatory secretagogue [0.01 microM prostaglandin E2 (PGE2)]. Exposure of luteal cell-containing monolayers to basic FGF resulted in a significant reduction (P less than 0.05) in the rate of RLX-induced plaque formation, evidence of an inhibitory effect of basic FGF on the rate of basal RLX secretion. This suppressive effect was variable in onset (1-3 h of incubation) and dose related. Minimally and maximally effective doses were about 10 and 30 ng/ml basic FGF, respectively. Higher doses of basic FGF (20 and 30 ng/ml) entirely suppressed RLX from a substantial subset of LLCs (10-20% of all LLCs), an indication of a differentially sensitive subpopulation. Addition of basic FGF (30 ng/ml) to monolayes also treated with PGE2 resulted in a significant (P less than 0.05) attenuation of the stimulatory effect of PGE2 on RLX secretion, evidence that these agents functionally interact in the modulation of RLX. We conclude that these results taken in association with the prior demonstration of the presence of basic FGF in luteal tissue suggest that basic FGF serves as a local inhibitory mechanism that regulates RLX secretion. Furthermore, the ability of basic FGF to counteract the effect of PGE2 implies that intraluteal stimulatory/inhibitory agents may act in concert to achieve fine control of RLX secretion. The observation of a preferentially responsive subpopulation is consistent with the possibility that basic FGF is implicated in heterogenous RLX secretion. Nevertheless, the physiological role(s) of basic FGF in the control of RLX secretion and the interrelationships of basic FGF with other local and systemic secretagogues remain to be clearly defined.     

Functional maturation of somatotropes in fetal rat pituitaries: analysis by reverse hemolytic plaque assay

Immunocytochemistry (ICC) and a reverse hemolytic plaque assay for GH were used to investigate the temporal relationships between the initiation of hormone storage and release by developing somatotropes and the onset of responsiveness of these cells to stimulatory and inhibitory secretagogues. Anterior pituitaries obtained from rats on days 18-21 of fetal development (pups were generally delivered on fetal day 22, which is equivalent to day 0 of neonatal life) were monodispersed with trypsin, cultured for 24 h, and then subjected to reverse hemolytic plaque assay and/or ICC for GH. GH-containing cells (determined by ICC) were extremely rare (less than 1%) in cultures derived from day 18 fetuses, but accounted for 22.4%, 25.2%, and 24.5% of all cells in cultures from day 19-21 fetuses, respectively. The proportion of GH-releasing cells, as determined in a long term (120-min incubation with antibody) plaque assay, was less than 1%, 22.4%, and 22.9% for days 18, 20, and 21, respectively, but only 13.6% for day 19 cells. Thus, many pituitary cells from day 19 fetuses contained, but did not release, GH. While GH-releasing factor (1-44) (1 X 10(-7) M) had no effect on the percentage of GH plaque-forming cells in long term incubations, it enhanced (by approximately the same degree in day 19-21 groups) the percentage of cells that formed plaques and the size of the plaques in short term (45-min) incubations with antibody. Somatostatin (1 X 10(-7) M) exerted inhibitory effects on these variables when tested in long term incubations, and age of the donor rats did not influence pituitary responsiveness to this secretagogue. These results suggest that the capacities of fetal somatotropes to store GH and release it under basal and regulated conditions are attained, in large part, within an extremely narrow time frame between days 18 and 19 of fetal development.     

Complement action on secretory cells identified by the reverse hemolytic plaque assay: modified assay eliminates exposure of secretory cells to complement

The application of a hemolytic plaque assay to antigen-secreting endocrine cells has brought about great advances in the study of regulation of hormone secretion. The reverse hemolytic plaque assay (RHPA) has enabled quantitation of secretion at the single cell level with simultaneous analysis of the population response. Moreover it has allowed unambiguous identification of specific cell types in mixed cell populations while maintaining the viability of the cells for further physiological experiments. Concern has arisen, however, regarding potential complement attack on those cells of interest, causing sublytic permeabilization leading to altered physiological function. To test this possibility, prolactin release from dispersed anterior pituitary cells was quantitated in two protocols of the RHPA. Cells were exposed to complement either subsequent to the termination of antiserum incubation or simultaneously with antiserum incubation, during which time hormone release is being detected. The presence of complement during antiserum incubation resulted in significant increases in mean plaque area as compared to the separate incubation procedure (13 709 ± 698vs 9251 ± 547 μm²). Analysis of the population profile of plaques indicated that the increased mean plaque area reflected a rightward shift in the frequency distribution of plaque size. The general increase in hormone release in the antibody/complement group is consistent with a predicted permeabilizing action of the complement on the secretory cells. To avoid this potentially damaging effect of complement on secretory cells to be used in subsequent physiological experiments, we have developed a modification of the RHPA in which the secretory cells are unequivocally identified without being exposed to complement.     

Analysis of mammosomatotropic cells in normal and neoplastic human pituitary tissues by the reverse hemolytic plaque assay and immunocytochemistry

The reverse hemolytic plaque assay (RHPA) was used to study hormone release from cultured normal and neoplastic human pituitary cells. The RHPA revealed a lower percentage of GH- and PRL-producing cells in normal and neoplastic pituitaries compared to the percentage of these cells revealed by immunocytochemical (ICC) staining for GH and PRL. Normal pituitary tissues as well as some PRL- or GH-producing adenomas contained large numbers of mammosomatotropic (MS) cells when analyzed by RHPA, combined RHPA-ICC, and ultrastructural immunohistochemistry with immunogold labeling. The percentage of GH and PRL cells in normal pituitaries ranged from 37-51% and 30-60%, respectively, by RHPA, while the percentage of MS cells ranged from 29-49%. The percentage of GH and PRL cells in normal pituitaries estimated by ICC ranged from 53-65% and 32-55%, respectively, while the percentage of MS cells estimated with this technique ranged from 26-50%. Double labeling with the immunogold technique detected GH and PRL in the same cells and within the same granules in both normal and neoplastic pituitary cells. These results indicate that MS cells are present in normal human pituitaries as well as in some pituitary adenomas, and in some pituitaries these two hormones are stored within the same secretory granules.     

Use of the reverse hemolytic plaque assay to study the regulation of anterior lobe adrenocorticotropin (ACTH) secretion by ACTH-releasing factor, arginine vasopressin, angiotensin II, and glucocorticoids

A reverse hemolytic plaque assay (RHPA) for ACTH was developed to study the responses of anterior lobe corticotropes to secretagogues-CRF, arginine vasopressin (AVP), angiotensin II (A-II), or to a 6- to 24-h pretreatment with corticosterone. Tests showed that optimal plaque formation was obtained after 3-4 h with 1:100-1:200 anti ACTH-(25-39) and 1:20-1:50 complement. Under optimal basal conditions, 6.6% of pituitary cells from normal male rats formed plaques. The addition of 50-90 micrograms/ml ACTH to the anti-ACTH for 48 h before its use in the RHPA resulted in a decrease in percentage of ACTH plaques to levels not different from those obtained with preimmune serum, or when complement was omitted from the assay (0.9-1.3%). There was a gradual increase in percentage of ACTH plaques to 9.8% of the population after exposure to increasing doses of CRF (0.1-10 nM). These same high percentages of ACTH plaques could also be obtained by the addition of 1 nM AVP or A-II with the lowest doses of CRF (100-500 pM). Exposure to 1 nM AVP alone resulted in no significant increases in percentages above basal values. Average plaque areas were increased to maximal levels of three to four X basal with increasing doses of CRF. Finally, when cells were pretreated with 100 nM corticosterone for up to 24 h, the percentage of plaques formed under basal conditions was reduced by 60% (2.6%) and it did not increase after exposure to 1 nM CRF. The data from the RHPA correlate well with previous studies of corticotropes. Since ACTH cells normally represent 10% of the anterior lobe cell population, the RHPA shows that a subset of corticotropes (6.6%) is actively secreting under basal conditions tested in this study. The remaining 3% can be stimulated to form plaques by high doses of CRF (greater than 1 nM) or low doses of CRF (100 pM) and 1 nM AVP or A-II. Glucocorticoids reduce the percentage of ACTH plaques formed under basal or stimulated conditions and allow no CRF-stimulated increase in plaque area. This correlates with recent reports that show glucocorticoids inhibit proopiomelanocortin messenger RNA synthesis and lower the number of pituitary CRF receptors.     

Analysis of relaxin release from cultured porcine luteal cells by reverse hemolytic plaque assay: influence of gestational age and prostaglandin F2 alpha

Relaxin release from dispersed luteal cells was detected by a reverse hemolytic plaque assay to determine the influence of gestational age on basal relaxin secretion. Monodispersed luteal cells were derived by collagenase treatment of corpora lutea obtained from pigs in early (days 19-26), mid-(days 47-62), and late (days 80-99) gestation. The rate of plaque development under nonstimulated conditions progressively accelerated as gestation advanced, as did the rate of increase in plaque size. These results unequivocally demonstrate that basal relaxin release increases with advancing gestation. However, only about 50% of large luteal cells released relaxin at all stages of pregnancy examined up to day 100. These data indicated not only that basal relaxin release increases during pregnancy, but also that considerable heterogeneity exists with respect to relaxin output by individual cells. In contrast, both the basal rate of relaxin release and the percentage of cells committed to relaxin release declined significantly when luteal cells derived from preparturient sows (days 107-112 of gestation) were examined. Exposure of cultured luteal cells to prostaglandin F2 alpha (10(-8) and 10(-6) M) resulted in a rapid stimulation of relaxin secretion, but this agent did not recruit additional cells into the secretory pool. These data are consistent with the idea that autonomous changes and the action of secretagogues may combine at different times to achieve overall regulation of relaxin release by the corpus luteum. The significance of nonrelaxin-releasing luteal cells remains to be determined.     

Subpopulations of lactotropes detected with the reverse hemolytic plaque assay show differential responsiveness to dopamine

Cultured adenohypophysial cells secreting PRL were detected with a reverse hemolytic plaque assay. In this assay, PRL secretion from a pituitary cell results in hemolysis of cocultured protein A-coupled ovine erythrocytes in the presence of PRL antiserum and complement, so that a zone of hemolysis (a plaque) surrounds each lactotrope. The extent of hemolysis was related to the amount of PRL secreted by each lactotrope: batches of cohort cells incubated under similar conditions either in petri dishes for measurement of PRL secretion by RIA or in Cunningham chambers for measurement of plaque area revealed a significant relationship between secreted PRL and plaque area (r = 0.97; regression coefficient = 0.0007 pg/micron2). Measurement of plaque area on lactotropes derived from proestrous rats revealed a bimodal frequency distribution that was composed of cells forming small plaques (35% of the total lactotrope population) and others forming large plaques (65%). Treatment with 10(-7) M dopamine appeared to preferentially inhibit the large plaques; they decreased to 42% of the total with corresponding increases in the number of small plaques, but the total number of secretory lactotropes did not change. At 10(-5) M dopamine, large plaques virtually disappeared (only 9% remained), and small plaques appeared in increased numbers, but the number of secretory lactotropes decreased by about one third. These results suggest that the reverse hemolytic plaque assay can be used to quantify PRL secretion by individual lactotropes, that lactotropes from proestrous rats exist as two secretory subpopulations, and that dopamine may preferentially suppress the subpopulation secreting large amounts of PRL.     

Individual parathyroid cells exhibit cyclic secretion of parathyroid hormone and chromogranin-A (as measured by a novel sequential hemolytic plaque assay).

PTH and chromogranin-A (CgA) are the two major proteins secreted from the parathyroid gland. We investigated the secretory patterns of CgA and PTH using a sequential reverse hemolytic plaque assay (RHPA). The RHPA allows detection of hormone secretion from individual cells after a secretory stimulus. For the sequential RHPA, bovine parathyroid cells were mixed with protein-A-conjugated ovine erythrocytes (oRBC). Antiserum, either anti-PTH or anti-CgA, was added under optimal secretory conditions. The addition of complement caused lysis of oRBC surrounding hormone-secreting cells. In this stage (stage 1), individual cells were identified and indexed as secreting or nonsecreting cells. For stage 2, a new lawn of oRBC was established, and a second RHPA was performed on the same population of cells, allowing for the detection of secretory patterns. In the single stage RHPA, about three fourths of the cells formed CgA plaques compared to only about half that formed PTH plaques, suggesting that CgA and PTH are not always cosecreted. In the sequential RHPA, of the cells that did not secrete CgA or PTH in stage 1, up to half secreted in stage 2. Of those cells that secreted in stage 1, up to one fourth did not secrete in stage 2. These results indicate that the parathyroid cells "cycled" between secretory and nonsecretory phases. Our experimental design precluded our obtaining unequivocal data on whether CgA is an autocrine/paracrine regulator of PTH secretion.     

Sertoli cells in culture are heterogeneous with respect to transferrin release: analysis by reverse hemolytic plaque assay

It has been reported that not all Sertoli cells store the same product or respond morphologically to secretagogue stimulation. The following studies were performed to determine whether functional differences among these cells are also present with respect to the secretion of a product. Sertoli cells obtained from 18- to 20-day-old rats were cultured for 3 days and then subjected to reverse hemolytic plaque assays for transferrin (TF). Release of TF could be detected from only 62.7 +/- 0.47% (mean +/- SE; n = 4 experiments) of all cells in culture. Results obtained from immunocytochemical staining of different batches of cells from the same dispersions agreed quite closely with these plaque assay values, indicating that not all Sertoli cells in culture contain or secrete TF. Differences in the basal rate of TF release were observed among these secretors, as evidenced by a gradual appearance of plaques over an 8-h period. Addition of FSH, cAMP, or isoproterenol to the assay incubation mixture resulted in an acceleration in the rate of plaque formation. Although approximately twice as many secretors could be identified after 0.5 and 1 h of incubation in the presence of these agents than in their absence, it still required at least 4 h for the remainder of the TF cells to form plaques. This would indicate that only a portion of all TF secretors respond acutely to these modulators. Thus, our observations that not all Sertoli cells secrete TF, and those that do release this substance respond differently to at least three stimulatory agents demonstrate clearly that Sertoli cells are heterogeneous with respect to TF release. Moreover, these findings raise the possibility that differences in the functional capacity of individual cells may be an important factor contributing to the modulation of Sertoli cell secretion.     

Capacity of individual somatotropes to release growth hormone varies according to sex: analysis by reverse hemolytic plaque assay

A reverse hemolytic plaque assay was used to investigate the mechanism(s) underlying sexual differences in GH release which evolve at puberty in rats. The percentages of GH-secreting cells in 24-h pituitary cultures from each sex were similar for pituitary donors up to 30 days of age (range = 38.9% to 41.7% of all cells in culture, n = 3 separate experiments) but decreased by day 50. The decrease was more striking for females (to 24.1 +/- 0.3% mean +/- SE) than for males (to 33.2 +/- 1.1%). However, owing to the greater increase in total pituitary cell number exhibited by female rats at this time, the absolute numbers of somatotropes recovered from male and female pituitaries were almost identical on 50 and 100 days of age. To assess the secretory capacities of individual somatotropes, we measured the sizes of plaques formed. In prepubertal rats (days 10-30), the plaque areas under basal conditions were comparable for males and females at each age studied, and treatment with GH-releasing factor increased plaque sizes to approximately the same degree (10-fold) for both sexes at each age. However, by day 100, plaques that formed under both basal and stimulated conditions were consistently larger (P less than 0.01) for male than for female donors. Taken together, our results demonstrate that sexual differences in GH release are attributable to the secretory capacities of individual somatotropes rather than to differences in the numbers of GH cells in pituitaries of male and female rats.     

Detection of hormone release from individual cells in mixed populations using a reverse hemolytic plaque assay

Prolactin (Prl) secreting cells in a mixed pituitary cell culture form microscopically-identifiable plaques (zones of hemolysis around the lactotropes) when incubated in a monolayer with staphylococcal protein-A-coated ovine erythrocytes in the presence of Prl antiserum and complement. Plaques form first at 15-30 min and are maximal in size and number at 2 h. Approximately 70% of the adenohypophyseal cells form plaques under these conditions. TRH increases, and dopamine decreases, the size and number of plaques at early times during incubation. This reverse hemolytic plaque assay probably can be used to detect any cell secretion for which an antibody is available. This technique, or a modified version of it in which sequential plaque assays are performed on identified cells--used together with immunocytochemistry, autoradiography or electron microscopy of those cells--should provide better answers to commonly asked questions about secretory systems: Do all or only a subset of cells containing the same hormone respond to a particular secretagogue? Can cells that contain two hormones release one of them preferentially?     

Angiotensinogen secretion by single rat pituitary cells: detection by a reverse haemolytic plaque assay and cell identification by immunocytochemistry.

A reverse haemolytic plaque assay (RHPA) for angiotensinogen was developed in rat hepatoma H4 cells and applied to investigate the possible secretion of angiotensinogen from rat pituitary cells in primary culture. Over a 24-hour incubation period in Cunningham chambers plaques with a mean area of 2,800 +/- 430 and 590 +/- 220 microns2/plaque (SD, n = 6) formed around all viable H4 cells and 2.8 +/- 0.59% of viable pituitary cells respectively. As a positive control PRL secretion from lactotrophs was routinely checked by the RHPA and shown to form plaques with a mean area of 4,050 +/- 1,850 microns2/plaque after a 4-hour incubation. By comparing plaque size in H4 cells with angiotensinogen release in cell culture, as quantified by radioimmunoassay, the secretion rate of angiotensinogen from pituitary cells was calculated as 22 +/- 8 ng/10(6) cells/24 h. Plaque-forming cells consisted of two morphologically distinct populations; 78% being small cells (less than 6 microns diameter) containing little cytoplasm and 22% were large (greater than 9 microns diameter) cells with an abundant cytoplasm. Immunocytochemical staining of pituitary cells after formation of plaques with anti-angiotensinogen, anti-LH and anti-PRL antiserum showed that the large plaque-forming cells were gonadotrophs and none were lactotrophs. All plaque-forming cells stained for angiotensinogen but only 44% of the viable cells which stained for angiotensinogen actually formed plaques. The possibility that cellular angiotensinogen was imported from extracellular sources was investigated by incubation of pituitary cells with pure 125I-angiotensinogen for periods up to 24 h. No uptake of the radiolabelled protein was found.(ABSTRACT TRUNCATED AT 250 WORDS)