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J D Neill

Publications and source records attributed to J D Neill.

At least 73 records · Page 4Linked to original sources
PubMed1987

Simultaneous measurement of hormone release and secretagogue binding by individual pituitary cells.

The quantitative relationship between receptor binding and hormone secretion at the single-cell level was investigated in the present study by combining a reverse hemolytic plaque assay for measurement of luteinizing hormone (LH) secretion from individual pituitary cells with an autoradiographic assay of 125I-labeled gonadotropin-releasing hormone (GnRH) agonist binding to the same cells. In the plaque assay, LH secretion induces complement-mediated lysis of the LH-antibody-coated erythrocytes around the gonadotropes, resulting in areas of lysis (plaques). LH release from individual gonadotropes was quantified by comparing radioimmunoassayable LH release to hemolytic area in similarly treated cohort groups of cells; plaque area was linearly related to the amount of LH secreted. Receptor autoradiography was performed using 125I-labeled GnRH-A (a superagonist analog of GnRH) both as the ligand and as the stimulant for LH release in the plaque assay; the developed silver grains appearing over cells in the center of plaques were measured microscopically. The grains appeared to represent specific and high-affinity receptors for GnRH because no pituitary cells other than gonadotropes bound the labeled ligand and grain development was progressively inhibited by coincubation with increasing doses of unlabeled GnRH-A. Despite high correlations between mean grain number and mean plaque area in dose-response curves, the correlation coefficients for these parameters were low (range 0.02-0.38) in the individual cells comprising these groups. We conclude that GnRH receptor number for any individual gonadotrope is a weak determinant of the amount of LH it can secrete; nevertheless, full occupancy of all its GnRH receptors is required for any gonadotrope to reach its full LH-secretory capacity. Apparently the levels of other factors comprising the steps along the secretory pathway determine the secretory capacity of an individual cell.

Animals↗
PubMed1986

Dopamine levels in the anterior pituitary gland monitored by in vivo electrochemistry.

The present study was designed to assess the contributions of the long portal vessel and neurointermediate lobe routes of dopamine delivery to the anterior pituitary gland. Dopamine levels were monitored in the anterior pituitary of anesthetized diestrus 1 rats using in vivo differential pulse voltammetry at a carbon paste electrode. Measurements were taken during control periods, following neurointermediate lobectomy and hypophysial stalk section or after inhibition of catecholamine synthesis and dopamine infusion. Neurointermediate lobectomy resulted in a slight rise in plasma prolactin and a significant fall in voltammetric current. Subsequent stalk section elevated prolactin significantly and further reduced the voltammetric output. Inhibiting catecholamine synthesis significantly elevated plasma prolactin and reduced the voltammetric current. Stepped infusions of dopamine then suppressed plasma prolactin and elevated the voltammetric output in a dose related manner. The final values of both parameters were not significantly different from pre-inhibition levels. These data provide direct estimates of the relative contributions of various vascular routes to dopamine in the anterior pituitary and support the sufficiency of dopamine as the physiological inhibitor of prolactin secretion.

Animals↗
PubMed1986

Detection and measurement of secretion from individual neuroendocrine cells using a reverse hemolytic plaque assay.

Recent advances in technology have dramatically increased the resolution with which we may examine many features of biological systems. Intracellular recording and tracer injection techniques allow one to study the function of individual neurons and later characterize the same cells morphologically. In situ hybridization techniques can give us information about messenger RNA levels in single cells. More established techniques such as immunocytochemistry and electron microscopy also provide information at the cellular and even subcellular level. With each of these technological advances we have learned more about the mechanisms underlying cell function. We are also beginning to appreciate the role of heterogeneity among cells in relation to the function of the whole organism. Application of the reverse hemolytic plaque assay to the study of hormone or neurotransmitter secretion should help clarify this role. This technique permits accurate quantitation of hormone secreted from a large number of cells. Thus while cells can be studied individually they can also be categorized into functional subpopulations. As discussed in this chapter, many other techniques may be applied on cells which have already been functionally defined with the plaque assay. This should result in a clearer understanding of the roles of secretagogue binding and internalization, activation of second messenger systems, protein synthesis, and the cytoskeleton in hormone secretion. In the plaque assays described in this chapter individual pituitary cells are isolated in culture free from possible interactive effects coming from other cells. While these interactions are no doubt critical to the understanding of the function of the organism as a whole they can result in totally uninterpretable results. In fact, when we have gained some understanding into the functioning of individual cells it should be possible using the plaque assay to study the interactions among cells in a controlled fashion.

Adrenocorticotropic Hormone↗
PubMed1986

Antibodies to the binding site of the receptor for luteinizing hormone-releasing hormone (LHRH): generation with a synthetic decapeptide encoded by an RNA complementary to LHRH mRNA.

A molecular recognition code has been hypothesized to exist in which ligands and their binding sites are encoded on complementary segments of genomic DNA. We have tested this hypothesis by generating a rabbit antibody to a synthetic decapeptide (complementary peptide) encoded by an RNA complementary to the mRNA for luteinizing hormone-releasing hormone (LHRH) and determining whether this antibody recognizes the LHRH receptor. When the antibody was used for immunoperoxidase staining of enzymatically dispersed rat anterior pituitary cells, only those that contained and secreted luteinizing hormone (i.e., the gonadotropes) were recognized. This staining could be abolished by preincubation with the complementary peptide or with an LHRH agonist, suggesting that the antibody is specific to the complementary peptide and is directed at the binding site of the receptor. Further evidence that the antibody recognizes the LHRH receptor was obtained in immunoblot experiments on solubilized receptors from pituitary glands. Immunoperoxidase staining with the antibody revealed two bands at 60 kDa and 51 kDa, which are values similar to those previously obtained for the LHRH receptor in photoaffinity-labeling experiments. The staining of these bands was inhibited by preincubation with the complementary peptide or an LHRH agonist. The antibody as well as the complementary peptide to LHRH also suppressed LHRH-stimulated luteinizing hormone release in a quantitative reverse hemolytic plaque assay, presumably by binding to the LHRH receptor and by binding LHRH, respectively. These findings suggest that the synthetic decapeptide whose sequence is specified by the complementary RNA to LHRH mRNA is sufficiently similar to an LHRH binding site that the peptide not only binds LHRH but was also recognized by the immune system as such a site. These findings provide strong support for the hypothesis that recognition molecules are encoded by complementary segments of genomic DNA.

Amino Acid Sequence↗
PubMed1986

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.

Animals↗
PubMed1985

Oxytocin attenuates TRH-induced TSH release from rat pituitary cells.

The observation that suckling evokes a modest rise in serum TSH when compared with that of prolactin is inconsistent with the hypothesis that TRH serves as a hypophysiotropic mediator of this response. In the present study we attempted to provide an explanation for this discrepancy by determining whether any of a growing number of putative prolactin releasing factors could alter pituitary responsiveness to TRH. Anterior pituitaries from lactating (day 14) rats were monodispersed with trypsin, cultured for 2 days, and then incubated in the presence of medium alone or medium containing TRH, dopamine, or a combination of these secretagogues. Companion sets of cultures were incubated concurrently with either beta-endorphin, neurotensin, oxytocin, serotonin, vasoactive intestinal polypeptide, or lysine vasopressin. As expected, TRH stimulated and dopamine suppressed prolactin release. None of the substances tested except oxytocin had a significant effect on pituitary cell responsiveness to TRH or dopamine. Oxytocin had no effect on prolactin secretion when tested alone or in combination with TRH and dopamine. TRH alone stimulated TSH release by these cultures, while oxytocin and dopamine were ineffective by themselves. However, TSH secretion by cultures treated simultaneously with TRH and oxytocin could be suppressed to approximately half of that released by cells incubated with TRH alone. These results demonstrate that oxytocin attenuates TRH-induced TSH release by a direct action on pituitary cells without affecting the prolactin response. This selectivity of responsiveness imparted by oxytocin might contribute to the blunted release of TSH after suckling.

Animals↗
PubMed1984

Brief decreases in dopamine result in surges of prolactin secretion in monkeys.

We have reported previously that the amount of dopamine in hypophysial stalk blood was sufficient to account for tonic hypothalamic inhibition of prolactin secretion in rhesus monkeys. In the present study we determined the effect on prolactin secretion of decreases in dopamine. When hypophysial stalk-transected, estrogen-treated monkeys were infused with dopamine at a rate (0.1 microgram X kg-1 X min-1) to achieve peripheral plasma concentrations similar to those previously measured in stalk blood, their elevated serum prolactin levels fell to base line (from 47.6 +/- 4.8 to 9.7 +/- 2.7 ng/ml, mean +/- SE, n = 4). Brief interruptions of the dopamine infusions lasting for 2.5, 5.0, or 7.5 min evoked rapid increases in serum prolactin concentrations (to 27.5 +/- 8.3, 52.9 +/- 3.6, and 58.9 +/- 8.1 ng/ml, respectively, at 10 min). Maximal prolactin levels were attained within 10 to 20 min after dopamine removal and serum prolactin remained elevated for an additional 35 to 150 min. Uninterrupted dopamine infusions of stalk-transected, estrogen-treated monkeys (n = 3) led to continuously suppressed prolactin values. These results demonstrate that brief decreases in dopamine lead to major increases in prolactin secretion. Thus, if a brief decrease in hypothalamic dopamine secretion occurs after a suckling stimulus in monkeys, as it does in rats, a decrease in dopamine secretion alone may account, in part, for suckling-induced prolactin secretion in monkeys.

Animals↗
PubMed1984

A reverse hemolytic plaque assay for microscopic visualization of growth hormone release from individual cells: evidence for somatotrope heterogeneity.

Growth hormone (GH) secreting cells direct complement-mediated plaque formation (clear zones of hemolysis surrounding the somatotropes) in mixed pituitary cell cultures incubated as a monolayer with protein-A coupled ovine erythrocytes (oRBC) in the presence of GH antiserum. Plaques were maximal in number after 4 h; growth hormone-releasing hormone (GHRH) and somatostatin increased and decreased, respectively, the rate of formation of plaques and their final sizes. Approximately 30% of all pituitary cells formed GH plaques and a similar fraction stained for GH using peroxidase-antiperoxidase immunocytochemistry (ICC). The plaque areas of individual somatotropes (reflecting the amount of GH released) covered a 20-fold range from the smallest to the largest in the 3 treatment groups. Somatostatin-treated and untreated cells formed frequency distributions of plaque sizes that were unimodal. In contrast, GHRH produced a bimodal frequency distribution suggestive of a sub-population of somatotropes preferentially responsive to this secretagogue. This new assay coupled with other morphological and biochemical techniques that can be applied to single cells will permit further analysis of these sub-populations of somatotropes.

Animals↗
PubMed1984

Biphasic effects of estrogen on gonadotropin-releasing hormone-induced luteinizing hormone release in monolayer cultures of rat and monkey pituitary cells.

The negative feedback effect of estrogen on LH secretion has been difficult to demonstrate in monolayer cultures of rat pituitary cells. The purpose of the present study was to establish the experimental conditions required for manifestation of this response and, in the process, to develop models for investigating the actions of steroids on pituitary cells. Both dynamic and static incubation systems were used. For perifusion experiments, trypsin-dispersed pituitary cells from rats at random stages of the estrous cycle were attached to glass coverslips with poly-L-lysine, incubated for 48 h, and then mounted in Sykes-Moore chambers. In each of these experiments, two chambers were perifused concurrently: one with medium containing 1.8 X 10(-10) M 17 beta-estradiol and the other with medium alone. GnRH (4.2 X 10(-9) M) was coinfused for 5 min out of every hour, and samples of perifusate were collected as 5-min fractions for assay of LH. Estrogen treatment significantly (P less than 0.01) suppressed LH release in response to the first five GnRH pulses compared to the control value. The inhibition was most pronounced early in the perifusion, but had disappeared by 6 h. These results demonstrate that estradiol exerts a potent but transient inhibition of GnRH-induced LH release in monolayer cultures of rat pituitary cells. In a subsequent set of experiments, we modified a static incubation system to assess sequentially the biphasic effects of estrogen on LH release by the same group of cells. Cultures of rat pituitary cells that had been established 42 h previously were treated simultaneously for 3 h with 17 beta-estradiol (3.7 X 10(-10) M) and various concentrations of GnRH (5 X 10(-10) to 1 X 10(-7) M) to measure the inhibitory effects of the steroid on LH secretion. This experiment was repeated on the same cells after 27 h of steroid exposure to estimate the facilitory actions of estrogen on LH release. The negative feedback of estrogen was demonstrable in static cultures of rat pituitaries provided that the period of estrogen exposure and duration of incubation were brief. Moreover, the results indicate that the same groups of cells can be used on consecutive days to investigate the inhibitory and stimulatory effects of estrogen on LH secretion. Experiments with cultures of monkey pituitary cells yielded similar results. Taken together, these findings indicate that cultured pituitary cells are responsive to the biphasic actions of estradiol and demonstrate the utility of two model systems for investigating these phenomena.

Animals↗
PubMed1984

Detection of LH release from individual pituitary cells by the reverse hemolytic plaque assay: estrogen increases the fraction of gonadotropes responding to GnRH.

Gonadotropes that secrete LH were demonstrated microscopically among mixed anterior pituitary (AP) cells in culture with a reverse hemolytic plaque assay. LH released from a cell binds to adjacent RBCs bearing an LH Ab-protein A complex which results in a zone of complement-mediated hemolysis (a plaque) surrounding each gonadotrope. In untreated cultures a few, small plaques formed, but in the presence of hypothalamic GnRH (10(-7) M, a maximally stimulating dose) a 10-fold increase occurred in their number and size. Non-secretory gonadotropes were found in the pituitary of diestrous animals: 5-6% of all AP cells contained immunocytochemically detectable LH whereas only 2.5-3.0% formed plaques in the presence of GnRH (10(-7) M). Nearly all of the gonadotropes were secretory at proestrus. Estradiol treatment of diestrous cultures for 24 h increased the fraction of secretory gonadotropes to near the level observed in proestrous cultures. These results demonstrate the utility of the reverse hemolytic plaque assay for detection of LH secretion from individual gonadotropes, establish that not all cells containing LH can secrete it, and suggest that a previously unrecognized mode of estrogen action to evoke the preovulatory LH surge is mediated by increasing the fraction of secretory gonadotropes.

Animals↗
PubMed1984

Specificity of pseudorabies virus serotests.

Pigs experimentally inoculated with bovine herpesvirus-1 or equine herpesvirus-1 developed mild clinical disease signs. Regression of clinical disease was accompanied by development of specific virus-neutralizing antibodies. These antibodies did not react positively with pseudorabies antigens in the serum-virus neutralization test, an indirect radioimmunoassay, or a microimmunodiffusion test.

Animals↗
PubMed1983

Nursing induces a biphasic release of prolactin in rhesus monkeys.

The effect of nursing on serum PRL levels was studied in four conscious unrestrained rhesus monkeys (20--46 days postpartum) fitted previously with exteriorized cardiac catheters. On the day of an experiment, the infant was transferred to a separate room for 9.5 h. Blood samples for PRL assay were collected from the mother at frequent intervals for 1 h before and 3 h after returning the infant. In all instances, vigorous suckling of the nipple was observed within 10 min of reuniting mother and infant and resulted in an elevation of serum PRL from an initial value of 7.4 +/- 0.5 ng/ml (mean +/- SEM) to a peak at 90 min of 339.7 +/- 56.1 ng/ml. Analysis of secretory patterns from individual animals revealed that PRL release was biphasic. During phase I, which began within 10--50 min of returning the infants and lasted for 20--50 min, serum PRL concentrations increased at an average rate of 0.47 ng/ml . min. This was followed by phase II, lasting 20--40 min, during which serum PRL rose at a 20-fold greater rate (10.57 ng/ml . min) to sustained maximum levels. Whether this unusual pattern of PRL secretion occurs as a consequence of neuroendocrine interactions unique to this species or simply reflects the prolongation and partitioning of a secretory process common to other mammals remains to be determined.

Animals↗
PubMed1983

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?

Animals↗
PubMed1982

In situ voltammetric microelectrodes: application to the measurement of median eminence catecholamine release during simulated suckling.

Catechol-sensitive microelectrodes (10-30 microns) were developed and then used to study the dynamic regulatory role of the prolactin inhibiting factor, dopamine, under conditions of simulated suckling. Current flow resulting from the electrochemical oxidation of catecholamines at the microelectrode surface was linearly related to the concentration of catecholamines present in solution over the range of 5-100 microM. Endogenous catecholamine levels in the rat median eminence were readily detectable and the electrochemical signal corresponding to dopamine release responded in an appropriate manner to various pharmacologic manipulations. We then implanted carbon microelectrodes into the medial median eminence region among capillaries of the primary portal plexus of urethane anesthetized lactating rats. Catecholamine release into the extracellular fluid was electrochemically measured once each minute before, during and after electrical stimulation (15 Hz, 5-30 V, 15 min) of a surgically isolated mammary nerve trunk. This simulated suckling paradigm reliably evoked prolactin secretory episodes qualitatively similar to those observed during suckling of the nipples by the young. During the period of nerve stimulation, a transient (3-5 min) 65% decline in electrochemically detectable catecholamine release was observed. Following cessation of nerve stimulation an oscillatory pattern of catecholamine release was observed with an overall trend toward an increased level of release. This latter observation corresponds with previous reports of increased hypothalamic dopamine turnover during or following suckling and with the increased levels of dopamine measured in hypophysial portal blood following mammary nerve stimulation. The transient nature of the decline of catecholamine release during the nerve stimulation period may explain why a similar observation has not been forth-coming from experiments utilizing the stalk blood collection technique (unless the standard collection periods are considerably shortened). These observations lead us to reject the hypothesis of a mirror image relationship between stalk blood dopamine and peripheral prolactin levels. Instead, we suggest that a transient decline in dopamine secretion coincident with the onset of suckling acts to prepare the pituitary lactotrophs to respond to a prolactin releasing factor which then facilitates prolactin secretion.

Animals↗
PubMed1982

Gamma amino butyric acid (GABA) levels in hypophyseal stalk plasma of rats.

To determine the possible physiologic contribution of GABA to the tonic hypothalamic inhibition of adenohypophyseal prolactin secretion, we compared GABA levels in hypophyseal stalk plasma with those found in the peripheral circulation. Hypophyseal stalk blood was collected via a parapharyngeal approach from 8 urethane anesthetized diestrus rats. Peripheral blood was collected simultaneously from the external jugular vein of the same rat at a rate similar to hypophyseal stalk blood flow. Blood samples resulting from a single 4 hr collection per animal were centrifuged, and the plasma stored frozen before ethanol extraction and assay using a radioreceptor method. GABA levels in hypophyseal stalk plasma (909 +/- 171 pmol/ml; X +/- S.E.M.) were not significantly higher than levels in peripheral plasma (845 +/- 182; p greater than 0.05), indicating little or no secretion of GABA by the median eminence.

Animals↗
PubMed1982

Involvement of the neurointermediate lobe of the pituitary gland in the secretion of prolactin and luteinizing hormone in the rat.

The relationship between prolactin (PRL) secretion and the neurointermediate lobe (NIL) of the pituitary gland was investigated. Plasma PRL concentrations in rats bearing anterior pituitaries autografted with or without the NIL to the renal capsule were elevated to equal extents at 1 through 6 weeks after surgery (p greater than 0.10). PRL levels in ovariectomized rats in which the NIl had been removed surgically (NIL-X) or only visualized (NIL-C) were 3-7 ng/ml 4, 7, and 28 days after surgery (p greater than 0.10); however, they were slightly higher in NIL-X vs. NIL-C rats 14 days after surgery (p less than 0.05). Plasma luteinizing hormone (LH) concentrations in NIL-C rats increased by 36% from 2 to 4 weeks after surgery (p less than 0.5); this increase was not detected in NIL-X rats. PRL and LH surges were induced by estradiol implants in ovariectomized NIL-X and NIL-C rats; the profiles of the PRL surges were superimposable, although the magnitude of the LH surge was only 50% that in NIL-C rats (p less than 0.5). These results cast doubt on the importance of the NIL in the regulation of PRL secretion either via secreting hypophysiotropic hormones or via conducting anterior pituitary hormones directly to the median eminence. However, the NIL may have a physiologically important role in the regulation of LH secretion.

Animals↗
PubMed1982

The decrease in hypothalamic dopamine secretion induced by suckling: comparison of voltammetric and radioisotopic methods of measurement.

Previous in situ voltammetric microelectrode measurements of median eminence dopamine release during mammary nerve stimulation of anesthetized lactating rats revealed a transient (1-3 min) 70% decline of dopamine concentrations. This dopamine was believed to be destined for secretion into the hypophysial portal circulation, but direct experimental support for this supposition was lacking. Thus, in the present study, [3H]dopamine release into brief sequential samples of hypophysial portal blood was compared with dopamine release in the median eminence measured by voltammetry. Lactating female rats were urethane anesthetized, and the median eminence pituitary region was exposed. [3H]Tyrosine was injected into a jugular cannula (100 microCi) followed by continuous infusion (5 microCi/min). In a preliminary experiment, this regimen produced a steady state level of [3H]dopamine in the portal blood within 45 min. In subsequent experiments, portal blood was collected as sequential 3-min samples, and electrochemical sampling from a microelectrode placed in the median eminence occurred at 1-min intervals. Electrochemical current resulting from the oxidation of dopamine in the medial median eminence was unvarying throughout the 75-min experiment in control rats (n = 4) and during the 30-min control period preceding mammary nerve stimulation in the other group (n = 4). These results were parallel by [3H] dopamine levels in portal blood during the same periods of time. All animals showed simultaneous decreases in oxidation current and [3H]dopamine levels within 1-4 min after initiation of mammary nerve stimulation (respectively, 35 +/- 7% and 62.5 +/- 5.9%, mean +/- SEM). Significant increases in oxidation current, taking the form of brief 2- to 6-min pulses began within an average of 18.5 min after initiation of stimulation. Similar increases in [3H]dopamine levels in portal blood were also observed. These and earlier results demonstrate that mammary nerve stimulation (and by extension, suckling) induces a momentary, but profound, decrease in hypothalamic dopamine secretion which precedes or accompanies the rise in PRL secretion evoked by the same stimulus.

Animals↗