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R D Kineman and L S Frawley

Abstract

Considerable information regarding the regulation of GH and prolactin (PRL) release has been generated using pituitary cell lines as model systems. Inasmuch as these cultures have been derived from single cells by clonal selection it has frequently been assumed that they are composed of homogeneous populations of hormone secretors. However, experience with GH3 cells clearly demonstrates that such is not the case, since this GH- and PRL-producing line is comprised of a mixture of cells that are bihormonal, secrete only GH, or secrete neither hormone. Interestingly, the relative amount of these phenotypic subpopulations is not fixed, but can be altered by treatment with established regulators of GH and PRL secretion. This potential for secretory heterogeneity and phenotypic plasticity prompted us to examine the cellular composition of other commonly used GH- and/or PRL-secreting cell lines under control and treatment conditions. First, GH4C1, GH1, GC, MMQ and P0 cells were maintained according to established media and culture protocols and the relative abundance of GH and PRL secretors was assessed by reverse haemolytic plaque assays. As shown previously for GH3 cells, two cell lines were found to be functionally heterogeneous. Specifically, GH4C1 and GH1 were comprised of mixed populations of GH (25·9±1·1% and 51·3±6·5% (s.e.m.) respectively) and PRL (44·8±3·7 and 66·1 ±4·1% respectively) secretors. However, MMQ and GC cell cultures were relatively homogeneous with respect to hormone secretion in that the MMQ cells released PRL (72·2 ± 4·9%) but not GH, while GC cells released GH (93·6 ±1·4%) but not PRL. Similarly, P0 cell cultures contained predominantly GH-secreting cells (96·4±1·2%), but a few cells were detected that released PRL (1·1 ±0·1%). We then tested whether cortisol or oestradiol-17β (OE2) could alter the secretory composition of cell lines identified as reasonably homogeneous (GC, MMQ and P0) by treating cultures with steroids at doses ranging from 0·001 to 1000 nmol/l for up to 144 h. The presence of steroids in GC and MMQ lines did not affect the percentage of GH and PRL secretors. However, treatment of P0 cells with 0·001 to 1000 nmol OE2/l for 24, 48 and 144 h resulted in a dramatic increase in PRL cells when compared with controls. These increases occurred without alterations in GH percentages and in the absence of significant cell proliferation. In fact, OE2 suppressed the proliferation of P0 cells to 20% of the control rate (P<0·05), suggesting that OE2 induced pre-existing GH-secreting cells to release both GH and PRL. Taken together, these results clearly indicate that clonal origin does not guarantee homogeneity of function, and alteration in the culture environment can have profound effects on the secretory cell composition of acidophilic cell lines. These considerations have important implications for the interpretation of results generated with GH- and/or PRL-secreting cell lines.

Journal of Endocrinology (1994) 140, 455–463

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R. D. Kineman, W. J. Faught and L. S. Frawley

ABSTRACT

The ontogeny of GH- and prolactin-releasing cells in the developing bovine pituitary was evaluated by reverse haemolytic plaque assays which allows for the detection of hormone release from individual pituitary cells in culture. With this approach, we observed that GH-releasing cells ontogenically preceded prolactin-releasing cells. In fact, GH secretors were observed as early as 59 days of gestational age while cells that released prolactin were not identified until 98 days. The amounts of both GH- and prolactinreleasing cells increased with time to reach more than 50% and 20% of all pituitary cells near term (term ∼280 days) respectively. Interestingly, the first cells shown to release prolactin also released GH (i.e. were mammosomatotropes). This temporal and functional relationship between GH and prolactin secretors provides suggestive evidence that GH-secreting cells act as the progenitor cells for prolactin secretors via a functional intermediate, the mammosomatotrope.

Journal of Endocrinology (1992) 134, 91–96

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R D Kineman, T W Gettys and L S Frawley

Abstract

It is clear that dopamine (DA) at high concentrations (>100 nmol/l) inhibits the release of prolactin (PRL). Paradoxically, this monoamine at low concentrations (<10 nmol/l) has also been shown to augment PRL secretion. One possible explanation for these divergent effects is that DA binds receptors capable of interacting with multiple G protein subtypes that recruit opposing intracellular signaling pathways within lactotropes. To identify G proteins which couple DA receptor activation to PRL secretion, we have selectively immunoneutralized the activity of Giα3 and G in primary cultures of rat pituitaries and subsequently tested the ability of these cultures to respond to high and low dose DA. Specifically, permeabilized pituitary cell cultures from random-cycling female rats were treated with control immunoglobulins (IgGs; 50 μg/ml) purified from preimmune serum (PII) or IgGs directed against the C-terminal portion of Giα3 or G. After immunoneutralization of these G proteins, cells were challenged with 10 or 1000 nmol Da/l and the relative amount of PRL released was assessed by reverse hemolytic plaque assay. Results were expressed as % of basal values and compared. Under control conditions (PII), 1000 nmol DA/l inhibited (61·4 ±7·6% of basal values; mean ± s.e.m.) while 10 nmol DA/l augmented (120·0 ± 7·0%) PRL release in five separate experiments. Treatment of cells with anti-Giα3 attenuated the inhibitory effect of high dose DA (87·3 ± 14·5%). However, elimination of Giα3 activity did not significantly alter the PRL stimulatory effect of 10 nmol DA/l (121·0 ± 5·2%). Interestingly, immunoneutralization of G resulted in a reciprocal shift in the activity of the lower dose of DA from stimulatory to inhibitory (69·7 ± 7·3%) while combined treatment of anti-Giα3 and anti-G abrogated the responsiveness of pituitary cell cultures to either DA treatment (1000 nmol/l, 70·7 ± 12·5% and 10 nmol/l, 87·5 ± 21·4%). These data reveal that ligand-activated DA receptors can interact with both Giα3 and G. Elimination of the stimulatory component (G) favors the DA receptor activation of the inhibitory pathway (Giα3) suggesting a competition between negative and positive intracellular signaling mechanisms in normal lactotropes. In addition to DA treatment, we also challenged permeabilized pituitary cells with 100 nmol thyrotropin-releasing hormone (TRH)/1 as a positive control for secretory integrity. As anticipated, TRH stimulated PRL release to 188·0±31·0% of basal values under control conditions. Unexpectedly, immunoneutralization of G completely blocked the ability of TRH to induce PRL release (101·8 ± 12·0% This neutralizing effect was specific to G in that blockade of Giα3 activity had no significant effect on TRH-stimulated PRL release (166·2 ± 13·1%). These data are the first to support a direct role of G in TRH signal transduction within PRL-secreting cells.

Journal of Endocrinology (1996) 148, 447–455

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E. Ellerkmann, R. D. Kineman, T. E. Porter and L. S. Frawley

ABSTRACT

We have previously reported that hypophysial neurointermediate lobe peptides, di-acetylated α-melanocyte-stimulating hormone (di-ac-α-MSH) and N-acetylated β-endorphin (N-ac-β-END), can acutely increase the relative number of prolactin-secreting cells in anterior pituitary cell cultures from ovariectomized rats. Inasmuch as the des-acetylated forms of these peptides (des-ac-α-MSH and β-END) were not effective in this regard, we concluded that acetylation was an absolute requirement for manifestation of the recruitment response. The aim of the present study was to determine whether these des-acetylated variants could antagonize the mammotrope-recruiting activity of their acetylated congeners. Treatment of anterior pituitary cell cultures with di-ac-α-MSH and N-ac-β-END increased the relative amount of prolactin secretors above control values. Interestingly, des-acetylated variants of α-MSH and β-END blocked the mammotrope-recruitment activity of their respective acetylated forms. In addition, β-END antagonized the mammotrope-recruitment activity of di-ac-α-MSH while des-ac-α-MSH did not attenuate the stimulatory effect of N-ac-β-END. Given that mammotropes maintained in vivo are exposed to all these peptides, it is possible that these acetylated and non-acetylated congeners may act in an opposing manner to regulate dynamic prolactin release.

Journal of Endocrinology (1993) 139, 295–300

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E Kim, S Sohn, M Lee, J Jung, R D Kineman and S Park

The impact of streptozotocin (STZ)-induced, insulinopenic diabetes on the GH axis of rats and mice differs from study to study, where this variation may be related to the induction scheme, severity of the diabetes and/or the genetic background of the animal model used. In order to begin differentiate between these possibilities, we compared the effects of two different STZ induction schemes on the GH axis of male Sprague–Dawley rats: (1) a single high-dose injection of STZ (HI STZ, 80 mg/kg, i.p.), which results in rapid chemical destruction of the pancreatic β-cells, and (2) multiple low-dose injections of STZ (LO STZ, 20 mg/kg for 5 consecutive days, i.p.), which results in a gradual, autoimmune destruction of β-cells. STZ-treated animals were killed after 3 weeks of hyperglycemia (>400 mg/dl), and in both paradigms circulating insulin levels were reduced to <40% of vehicle-treated controls. HI STZ-treated rats lost weight, while body weights of LO STZ-treated animals gradually increased over time, similar to vehicle-treated controls. As previously reported, HI STZ resulted in a decrease in circulating GH and IGF-I levels which was associated with a rise in hypothalamic neuropeptide Y (NPY) mRNA (355% of vehicle-treated controls) and a fall in GH-releasing hormone (GHRH) mRNA (45% of vehicle-treated controls) levels. Changes in hypothalamic neuropeptide expression were reflected by an increase in immunoreactive NPY within the arcuate and paraventricular nuclei and a decrease in GHRH immunoreactivity in the arcuate nucleus, as assessed by immunohistochemistry. Consistent with the decline in circulating GH and hypothalamic GHRH, pituitary GH mRNA levels of HI STZ-treated rats were 58% of controls. However, pituitary receptor mRNA levels for GHRH and ghrelin increased and those for somatostatin (sst2, sst3 and sst5) decreased following HI STZ treatment. The impact of LO STZ treatment on the GH axis differed from that observed following HI STZ treatment, despite comparable changes in circulating glucose and insulin. Specifically, LO STZ treatment did suppress circulating IGF-I levels to the same extent as HI STZ treatment; however, the impact on hypothalamic NPY mRNA levels was less dramatic (158% of vehicle-treated controls) where NPY immunoreactivity was increased only within the paraventricular nucleus. Also, there were no changes in circulating GH, hypothalamic GHRH or pituitary receptor expression following LO STZ treatment, with the exception that pituitary sst3 mRNA levels were suppressed compared with vehicle-treated controls. Taken together these results clearly demonstrate that insulinopenia, hyperglycemia and reduced circulating IGF-I levels are not the primary mediators of hypothalamic and pituitary changes in the GH axis of rats following HI STZ treatment. Changes in the GH axis of HI STZ-treated rats were accompanied by weight loss, and these changes are strikingly similar to those observed in the fasted rat, which suggests that factors associated with the catabolic state are critical in modifying the GH axis following STZ-induced diabetes.

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Ellen R Lubbers, Edward O List, Adam Jara, Lucila Sackman-Sala, Jose Cordoba-Chacon, Manuel D Gahete, Rhonda D Kineman, Ravneet Boparai, Andrzej Bartke, John J Kopchick and Darlene E Berryman

Adiponectin is positively correlated with longevity and negatively correlated with many obesity-related diseases. While there are several circulating forms of adiponectin, the high-molecular-weight (HMW) version has been suggested to have the predominant bioactivity. Adiponectin gene expression and cognate serum protein levels are of particular interest in mice with altered GH signaling as these mice exhibit extremes in obesity that are positively associated with insulin sensitivity and lifespan as opposed to the typical negative association of these factors. While a few studies have reported total adiponectin levels in young adult mice with altered GH signaling, much remains unresolved, including changes in adiponectin levels with advancing age, proportion of total adiponectin in the HMW form, adipose depot of origin, and differential effects of GH vs IGF1. Therefore, the purpose of this study was to address these issues using assorted mouse lines with altered GH signaling. Our results show that adiponectin is generally negatively associated with GH activity, regardless of age. Further, the amount of HMW adiponectin is consistently linked with the level of total adiponectin and not necessarily with previously reported lifespan or insulin sensitivity of these mice. Interestingly, circulating adiponectin levels correlated strongly with inguinal fat mass, implying that the effects of GH on adiponectin are depot specific. Interestingly, rbGH, but not IGF1, decreased circulating total and HMW adiponectin levels. Taken together, these results fill important gaps in the literature related to GH and adiponectin and question the frequently reported associations of total and HMW adiponectin with insulin sensitivity and longevity.