Dispersed Rat Anterior Pituitary Cells Research Articles

Ca+2 dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone secretion: in vitro studies using continuously perifused dispersed rat anterior pituitary cells.

A continuously perifused dispersed rat anterior pituitary cell system was used to determine the importance of calcium (Ca+2) on the release of LH by GnRH. In response to continuous exposure to 10 nM GnRH, LH was released in a biphasic fashion; arbitrarily, phase I was defined as being the LH released during the initial 40 min and phase II as the subsequent release. Withdrawal of Ca+2 from the perifusion medium during phases I or II of LH release led to a rapid inhibition of the LH secretion. Cells were exposed to GnRH for 2.5 min, washed with medium for 30 min, and then reexposed to GnRH for 30 min. This sequence was repeated 1 h later under identical conditions in the presence of a Ca+2 blocking agent; D600 (20 or 100 microM). D600 inhibited both the 2.5- and the 30-min GnRH-stimulated LH release. The results were expressed as the ratio obtained by dividing the total LH released during the second GnRH exposure (either 2.5 or 30 min) by the total LH released during the respective initial GnRH exposure of same duration. For the cells perifused with 20 microM D600 the ratios +/- SE (D600 vs. control) were 0.48 +/- 0.06 vs. 1.28 +/- 0.13 (P = 0.0001) and 0.29 +/- 0.05 vs. 1.01 +/- 0.08 (P less than or equal to 0.0001) for the 2.5- and 30-min exposures, respectively. For the cells perifused with 100 microM D600 the ratios +/- SE (D600 vs. control) were 0.18 +/- 0.05 vs. 1.28 +/- 0.13 (P less than or equal to 0.00001) and 0.12 +/- 0.03 vs. 1.01 +/- 0.08 (P = 0.002) for the 2.5- and 30-min exposures, respectively, revealing an even more profound inhibitory effect of D600 on GnRH stimulated LH secretion. Our data both confirm previous reports that Ca+2 is involved in LH release and demonstrate that Ca+2 is an essential requirement during both phases of GnRH-stimulated LH release in perifused dispersed rat anterior pituitary cells.

The Effect of Single-Dose Radiation on Cell Survival and Growth Hormone Secretion by Rat Anterior Pituitary Cells

Cranial irradiation has been shown to impair growth hormone secretion in children. In this study a cell culture of dispersed rat anterior pituitary cells was exposed to single doses of radiation in the range of 100-1500 rad: Survival curves were obtained for the different anterior pituitary cell lines, and growth hormone secretion was measured in the tissue culture medium. Both survival and growth hormone secretion curves showed an initial shoulder in the range of 0-300 rad, followed by a decline between 300-750 rad. It is concluded that growth hormone secreting acidophilic pituicytes are sensitive to radiation at single doses greater than 300 rad.

Diphenylhydantoin inhibits the thyrotropin response to thyrotropin-releasing hormone in man and rat.

We studied the effect of diphenylhydantoin (DPH) on TRH-stimulated TSH secretion in four patients (three of whom had hypothyroidism; one treated with L-T4), in euthyroid rats, and in dispersed rat anterior pituitary cells in short term tissue culture. DPH treatment of four patients (100 mg, three times daily for 14 days) caused a 22% decrease in mean serum T4 (P < 0.06), a 31% decrease in mean serum free T4 (P < 0.01), and a 6% decrease in mean serum T3. Mean basal serum TSH was unchanged, but the integrated TSH response after TRH injection (500 μg, iv) decreased 28% (P < 0.06) and 51% (P < 0.01) after 7 and 14 days of DPH treatment, respectively. Changes in the integrated PRL response to TRH did not correlate with those in TSH.Injection of DPH into euthyroid rats (5 mg/100 g BW·day for 9 days) caused a significant decrease in mean serum T4 but no change in TSH release after TRH treatment. However, 1 h after a large dose of DPH (20 mg/100 g BW), there was a marked reduction (58%) in TRH-induced TSH releas...

Biphasic luteinizing hormone secretion in response to gonadotropin-releasing hormone during continuous perifusion of dispersed rat anterior pituitary cells: changes in total release and the phasic components during the estrous cycle.

In vivo studies in the human and both in vivo and in vitro studies in animals have shown that GnRH-stimulated LH release is a function of the gonadal steroid environment. Furthermore, when GnRH is given in a sustained manner, the pattern of LH release is biphasic. Using an in vitro technique in which rat anterior pituitary cells were dispersed and perifusion was begun within 2 h after removal of the gland from the donor animal, we tested the hypothesis that both the overall secretion and the relative sizes of the two phases of GnRH-stimulated LH release would vary in a reproducible manner during the estrous cycle. Sprague-Dawley rats (aged 60–70 days) were killed at 0800 h on diestrus day 1, diestrus day 2, proestrus, and estrus and at 1400 h on proestrus. After 4 h of perifusion, during which a stable baseline of LH release was established, the cells were challenged for 5 h with 10 nM GnRH, and secretory rates of LH release were calculated. Basal LH release did not vary among the experimental groups. LH ...

Dopaminergic control of the rat thyrotroph.

The established technique of dynamic perfusion of dispersed rat anterior pituitary cells was used to investigate the role of dopamine in the release of thyroid-stimulating hormone (TSH). The basal release of TSH was 124 +/- 22 ng/ml (mean +/- SEM) and this remained unchanged in the presence of dopamine (5 X 10(-6) M), while basal prolactin release was suppressed to 50% of control values. Perfusion with thyrotrophin-releasing hormone (TRH) in doses between 1.4 X 10(-10) and 22.4 X 10(-10) M produced a consistent and reproducible dose-response curve for TSH (n = 14) which was not significantly altered by the presence of dopamine (n = 5). Perfusion with bromocriptine (10(-6) M), a dopamine agonist, did not alter the basal or stimulated TSH release. No stimulation of TSH release was observed when the cells were challenged with 2-min pulses of domperidone, a dopamine antagonist (2 X 10(-9) to 2 X 10(-5) M). The inclusion of somatostatin-14 (3 X 10(-9) M) in the perfusate inhibited TRH-stimulated TSH release. Our results suggest that, in the rat, dopaminergic inhibition of TSH release does not occur at the level of the pituitary.

Effect of the peptide PHI-27 on prolactin release in vitro.

The present study demonstrates that PHI, a peptide belonging to the glucagon-secretin group and thus structurally similar to VIP, can release prolactin from dispersed rat anterior pituitary cells and also causes release of prolactin from hemipituitaries. PHI-like immunoreactivity has previously been demonstrated in a hypothalamic system with nerve endings in the median eminence, and, taken together, these findings suggest that PHI may represent a physiologic prolactin-releasing factor.

Beta-adrenergic stimulation of growth hormone release from perifused rat anterior pituitary cells.

Perifusion of dispersed rat anterior pituitary cells with pulses of the beta-adrenergic agonists isoproterenol and epinephrine resulted in a prompt, but brief, rise in growth hormone release. This effect was blocked by propranolol, a beta-adrenergic antagonist, but not phentolamine, an alpha-adrenergic antagonist. These results, in conjunction with other recent reports, suggest that epinephrine may act directly at the anterior pituitary gland as a physiological growth hormone-releasing factor.

Effects of culture age on PRL and GH responses to bromocriptine and somatostatin from primary cultures of rat anterior pituitary cells.

AbstractTo study the effects of time in culture on prolactin (PRL) and growth hormone (GH) responses to exogenous stimuli, bromocriptine (10−7M) or somatostatin (10−7M) were added to primary cultures of dispersed rat anterior pituitary cells (DC). Cells which had been in culture for 3, 6, or 9 days were then incubated for 6 hr and the media were radioimmunoassayed for PRL and GH. The inhibitory effects of bromocriptine on PRL release (76% decrease) and somatostatin on GH release (62% decrease) from cultured cells were maximal on Day 3 and decreased with age of the culture. In addition, the inhibitory effect of bromocriptine on GH release (32% decrease) was slight but maximal on Day 3 and decreased with time in culture. In contrast, somatostatin showed slight and stable inhibition of PRL release (26 to 29% decrease) at each of the three time periods. At the end of each incubation, the media were replaced with Ham's F-10 medium lacking bromocriptine or somatostatin and the cultures were incubated again for ...

FLUORESCENCE ACTIVATED CELL SORTING OF FUNCTIONAL ANTERIOR PITUITARY CELLS

The fluorescence activated cell sorter (FACS) offers a new approach to purification and analysis of pituitary cell types. Using dispersed rat anterior pituitary cells 29.5 ± 3.2% of pituitary cells can be recovered from the FACS of which up to 93% are viable. More importantly, these cells actively adhere to cell culture dishes and respond to both the gonadotropin releasing hormone (GnRH) by increasing LH secretion and to dopamine by decreasing prolactin secretion. Using the narrow angle light scatter discriminator combined with the detection of LH beta subunit antibody conjugated to highly fluorescent microspheres, immunocytochemically identified gonadotropes have been enriched from the normal control range of 7.4 ± 1.4% to 52.3 ± 11% of sorted cells (n = 4). This enhancement was also confirmed by measuring the ratio of LH to prolactin in the cell homogenates of control and sorted groups. This ratio rose from 8 to 650, 26 to 35, and 55 to 170 in three independent experiments. The ability of the fluorescen...

Angiotensin II stimulates the release of ACTH from dispersed rat anterior pituitary cells.

In a rat anterior pituitary dispersed cell system, angiotensin II was found to stimulate the release of ACTH at concentrations ranging from 100 pmol/l to 1 mumol/l, with a maximal response being given by 10 nmol/l. The angiotensin II antagonist, saralasin, was able to block completely at a concentration of 1 mumol/l the stimulation of ACTH induced by 10 nmol/l angiotensin II, but had no effect on the basal release. The log dose-response curve for ACTH release by angiotensin II was shifted to the right in a parallel fashion by saralasin 10 nmol/l, suggesting competitive antagonism. The stimulation of ACTH release by a rat stalk-median eminence extract or by arginine vasopressin was unaffected by saralasin. The data are interpreted as suggesting that rat corticotrophs contain angiotensin II receptors, and that these may be involved in ACTH release in response to hypovolaemic stress.

GH secretion by cultured rat pituitary cells under radiotherapy

Radiotherapy to the CNS has been shown to impair GH secretion by children. In this study a tissue culture model of dispersed rat anterior pituitary cells was used. The cells were irradiated through a cobalt machine in a single or fractionated doses, in the range of 100-1500 rads. A dose response cell survival curve revealed T-50 at 300 rads 48 hrs. after treatment. Survival of control, non-irraduated cells was 75% at that time. GH secretion into the culture medium on the 7th and 11th day after irradiation showed a dose response increase at 100-500 rads with a decline in secretion under higher doses. Treatment of cells during transport and irradiation with 0.5nM estradiol produced a significant increase in GH secretion 11 days later. These preliminary data indicate that: 1. Cultured rat anterior pituitary cells are a valid model for studies on irradiation effect on somatotrophs. 2. Radiotherapy enhanced GH secretion at low doses and impair it at high doses. 3. Estradiol treatment of the cultured cells during irradiation may improve GH secretion.

Response of luteinizing hormone from columns of dispersed rat pituitary cells to a highly potent analogue of luteinizing hormone releasing hormone.

The response of LH from a perfused column of dispersed rat anterior pituitary cells to LH releasing hormone (LH-RH) and the analogue, D-Ser(But)6-desGly10-Proethylamide9-LH-RH (Hoe 766), was investigated. Dose-response curves showed non-parallelism between LH-RH and the analogue. but it was evident that the analogue was considerably more potent. After a single pulse of LH-RH, LH output returned to basal values in 8 min; this was prolonged to 20 min in the case of the analogue. During this 20 min the cells were refractory to pulses of LH-RH but pulses of the analogue maintained output of LH. During constant-dose perfusion with either synthetic LH-RH or the analogue, output of LH rapidly reached a peak and then gradually fell over several hours to approach baseline values. However, a pulse of 50 mmol potassium chloride/l was still able to release LH at this time. The data are consistent with the view that this analogue of LH-RH is highly potent and is strongly bound by the LH-RH receptor. Furthermore, since it desensitizes the LH-RH receptor, it appears that continued turnover of either LH-RH or the analogue at the receptor is necessary for output of LH to be maintained.

STIMULATION OF RELEASE OF LUTEINIZING HORMONE FROM CULTURED PITUITARY CELLS BY 2- AND 4-HYDROXYLATED OESTROGENS

We have examined the effect of the catechol oestrogens 2-hydroxyoestradiol (2-OHE2), 4-hydroxyoestradiol (4-OHE2) and 2-hydroxyoestrone (2-OHE1) and their corresponding primary oestrogens on secretion of LH and FSH by enzymatically dispersed rat anterior pituitary cells in monolayer culture. Basal LH levels in the medium were significantly higher than in control wells when cells were exposed to 10(-8) M-oestradiol-17 beta for 40 h: oestrone and all three catechol oestrogens (in the same doses) also stimulated basal LH concentrations to levels quantitatively similar to those seem after oestradiol treatment. The same effects were observed when steroids were given at 10(-9) mol/l. Oestradiol, 2-OHE2, and 4-OHE2 but not 2-OHE1 increased pituitary responsiveness to LH releasing hormone (LH-RH) (given in a range of doses from 10(-11) to 10(-6) mol/l). The responses of cells treated with 2-OHE2 and 4-OHE2 were similar, though less than the response seen after treatment with oestradiol. This contrasts with the very different oestrogenic effects of 2- and 4-OHE2 previously observed in vivo. Neither oestradiol nor the catechol oestrogens had any effect on basal or LH-RH-stimulated FSH release.

GABA and muscimol inhibit the release of prolactin from dispersed rat anterior pituitary cells.

Prolactin release from columns of dispersed rat anterior pituitary cells was inhibited by GABA and the GABA agonist muscimol. The log dose-response curves for GABA and muscimol appeared to be parallel, but were not parallel to that for dopamine. Two GABA antagonists, bicuculline and picrotoxin, blocked the action of both GABA and muscimol, but the dopamine antagonists metoclopramide and perphenazine did not. The data suggest that there are specific GABA receptors on pituitary lactotrophs.

Metergoline and cyproheptadine suppress prolactin release by a non-5-hydroxytryptaminergic, non-dopaminergic mechanism.

A dispersed rat anterior pituitary cell system has been used to investigate the effects of cyproheptadine and metergoline on prolactin secretion. Both drugs were potent inhibitors of prolactin secretion. However, the inhibition was not antagonized by either 5-hydroxytryptamine or a variety of dopamine antagonists. We conclude that both drugs act through mechanisms that are neither 5-hydroxytryptaminergic nor dopaminergic.

The release of biologically and immunologically reactive somatostatin from perifused hypothalamic fragments.

The purpose of this investigation was to 1) develop a hypothalamic perifusion system which would allow measurement of spontaneous basal somatostatin (SRIF) release, 2) compare the immunological and biological activities of released SRIF, and 3) study the effect of membrane depolarization, extracellular calcium, and several neurotransmitters on SRIF release. Release was greater at the beginning of the perifusion and decayed with time for 90 min, after which it stabilized and remained constant for 3 h, the period used to determine the mean rates of release. Basal release was 20.2 pg/fragment x 10 min. Membrane depolarization with 55 mK K+ increased SRIF release 30 to 4-fold in a calcium-dependent manner. The immunoreactivity and biological activity of SRIF concentrated by affinity chromatography of hypothalamic perifusates were compared to those of synthetic SRIF and rat hypothalamic extract. Biological activity was assessed by the inhibition of radioimmunoassayable rat GH released from cultured dispersed rat anterior pituitary cells. Hypothalamic fragments were exposed to several neurotransmitters as well as other substances known to influence rat GH secretion. Our results may be summarized as follows. 1) The perifused medial basal hypothalamus releases immunoactive and bioactive SRIF at a constant basal rate. 2) Membrane depolarization with high potassium stimulates a small releasable pool of SRIF in a calcium-dependent manner. 3) Affinity chromatography is an alternative technique to collect and concentrate SRIF released from tissues. 4) Common neurotransmitter agents did not modify SRIF release from the medial basal hypothalami under the present conditions.

Role of Ca2+ and cyclic nucleotides in the control of prostaglandin E production in the rat anterior pituitary gland

In an attempt to relate changes in the intracellular concentration of prostaglandin E to the secretion process, two agents known to increase cyclic nucleotide concentrations and hormone release were added to dispersed rat anterior pituitary cells. They caused increases in teh intracellular prostaglandin E concentrations. Increasing the K+ concentration in the medium (which stimulates hormone release) caused a rapid rise in prostaglandin E concentrations. The addition of the Ca2'onophore A23187 had a similar effect. The effects of changes in the K+ and Ca2+concentrations and the addition of EDTA were measured on the redistribution of radioactivity in pituitary glands prelabelled with [3H]arachidonic acid. Elevated K+ concentrations stimulated the transfer of label to prostaglandins and free arachidonic acid, suggesting an increased phospholipase A activity. On the other hand, the absence of extracellular CaCl2 and the addition of EDTA had the opposite effect, which could be cancelled by the addition of sufficient amounts. of CaCl2. It is concluded that the addition of agents that increase membrane permeability to bivalent cations probably results in an influx of Ca2+ and this appears to result in increased phospholipase A activity, which in turn leads to an increase in prostaglandin production.

Chlorpromazine, haloperidol, metoclopramide and domperidone release prolactin through dopamine antagonism at low concentrations but paradoxically inhibit prolactin release at high concentrations.

1. The effects of chlorpromazine, haloperidol, metoclopramide and domperidone on the release of prolactin from perfused columns of dispersed rat anterior pituitary cells were studied. 2. Chlorpromazine, haloperidol, metoclopramide and domperidone antagonized the dopamine-mediated inhibition of prolactin release at low concentrations. 3. Each dopamine antagonist displaced the dose-response curve for dopamine-induced suppression of prolactin release to the right in a parallel manner. 4. At higher concentrations, the four drugs became less effective as dopamine antagonists. 5. At high concentrations in the absence of dopamine, chlorpromazine, haloperidol, metoclopramide and domperidone paradoxically suppressed prolactin secretion by an unknown mechanism.

Relationship of receptor affinity to the modulation of thyroid hormone nuclear receptor levels and growth hormone synthesis by L-triiodothyronine and iodothyronine analogues in cultured GH1 cells.

We have previously demonstrated that L-triiodothyronine (L-T3) induces an increase in growth hormone synthesis and messenger RNA in cultured GH1 cells, a rat pituitary cell line. In addition to regulating the growth hormone response, L-T3 elicits a time- and dose-dependent reduction in the level of its nuclear receptor, which is a direct function of the occupancy of the receptor binding site. In this study we have compared the relative affinity of L-T3, triiodothyroacetic acid, D-triiodothyronine (D-T3), and L-thyroxine (L-T4) for the receptor with the induction of the growth hormone synthesis and the ability of these compounds to elicit a reduction in thyroid hormone nuclear receptor levels. Triiodothyroacetic acid and D-T3 were specifically examined because the biologic effect of these compounds in the intact rat is significantly lower than predicted by their affinity for the receptor using isolated rat liver nuclei in vitro. In intact cells each compound demonstrated an excellent relationship between the relative receptor affinity, the induction of growth hormone production, and the concentration-dependent reduction in nuclear receptor levels. With the exception of D-T3, the relative affinity of iodothyronine was identical for the receptor using intact cells in serum-free media, or isolated GH1 cell nuclei in vitro. The apparent receptor affinity of D-T3 with intact cells was 5.5-fold lower than with isolated nuclei, which suggests a decrease in cell entry of D-T3 relative to the other iodothyronines. Quantitation of the [125I]iodothyronine associated with the receptor in GH1 cells after a 36-h incubation with L-125I-T4 was 90% L-T4 and 10% L-T3, which indicates that the major effect of L-T4 in GH1 cells is a result of intrinsic L-T4 activity. Studies with dispersed rat anterior pituitary cells demonstrated that L-T3 induces growth hormone synthesis and elicits a reduction in nuclear receptor levels in the same fashion as GH1 cells. The observation that thyroid hormone influences dispersed rat pituitary cells in a fashion qualitatively similar to GH1 cells may have implications for the growth hormone response of the somatotroph cell in vivo to different thyroidal states.

Immunoreactive and biologically active somatostatin-like material in rat retina.

Somatostatin-like activity, as determined by radioimmunoassay and bioassay, is present in HCl extracts of rat retina. The concentrations of immunoreactive somatostatin are 612 +/- 43 (mean +/- SEM)pg per whole retina of 0.621 +/- 0.044 pg/microgram of protein in retinas from rats killed by decapitation, values which are not significantly different from those in retinas from rats killed by ether inhalation. The immunoreactive somatostatin was partially purified and concentrated by immunoaffinity chromatography. Both the crude retinal extracts and the immunoaffinity-purified immunoreactive somatostatin inhibited, in a dose-related manner, the release of rat growth hormone from dispersed rat anterior pituitary cells in culture. When the immunoaffinity-purified immunoreactive somotostatin was subjected to gel filtration chromatography, 96% of the recovered somatostatin eluted as a peak corresponding in position to that of synthetic somatostatin. Retinas from a group of rats with hereditary degeneration of the photoreceptor cells and another group of rats studied 1 year after transection of the optic nerves demonstrated an increased concentration of immunoreactive somatostatin compared to controls.