Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland.

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K(+) (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K(+) currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (beta(1)gamma(2)) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K(+) currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.

Estradiol enhances excitatory gamma-aminobutyric [corrected] acid-mediated calcium signaling in neonatal hypothalamic neurons.

Contrary to the situation in adulthood, gamma-aminobutyric [corrected] acid (GABA)(A) receptor activation during early brain development depolarizes neurons sufficiently to open L-type voltage-gated Ca(2+) channels. Because GABA is excitatory during the sensitive period of steroid-mediated brain sexual differentiation, we investigated whether estradiol modulates excitatory GABA during this period, by examining two parameters: 1) magnitude of GABA-induced calcium transients; and 2) developmental duration of excitatory GABA. Dissociated hypothalamic neurons from embryonic-day-15 rat embryos were loaded with the Ca(2+) indicator, fura-2, and transient rises in [Ca(2+)](i) (Ca(2+) transient) were measured after application of 10 microM muscimol, a GABA(A) receptor agonist. Cells were treated with 10(-10) M estradiol or vehicle from 0-3 days in vitro (DIV) and imaged on 4 DIV, whereas others were treated from 3-6 DIV and imaged on 7 DIV. The mean amplitude of Ca(2+) transients after muscimol administration were 68% and 61% higher in estradiol-treated neurons on 4 DIV and 7 DIV, respectively, relative to controls. Consistent with GABA becoming inhibitory in mature neurons, 50% fewer control neurons responded on DIV 7, relative to DIV 4. However, estradiol treatment maintained excitatory GABA on DIV 7 (72% in estradiol-treated vs. 35% in control). This is the first report of hormonal modulation of excitatory GABA, and it suggests that estradiol may mediate sexual differentiation by enhancing GABA-induced increases in intracellular Ca(2+).

Dopamine withdrawal elicits prolonged calcium rise to support prolactin rebound release.

Dopamine (DA) acts directly on pituitary lactotropes to inhibit the release of PRL. Removal of DA elicits a pronounced transient rise in PRL release to values exceeding pre-DA rates (PRL rebound). Electrophysiological studies have shown that lactotropes exhibit a period of increased Ca2+ action potential activity after DA withdrawal, leading to the proposal that enhanced Ca2+ influx during this period may support the rebound secretion of PRL. In the present studies, we investigated the effect of DA application and removal on the cytosolic free calcium concentration ([Ca2+]i) monitored by fura-2 in single rat lactotropes. Unchallenged lactotropes fell into two functionally distinct groups: those with stable [Ca2+]i that was not acutely sensitive to extracellular Ca2+, and those with spontaneous fluctuations in [Ca2+]i that were dependent upon influx of external Ca2+. There was striking variability in the [Ca2+]i patterns of the latter group, ranging from irregular, low amplitude fluctuations to rhythmic, repetitive oscillations with definable rise and decay kinetics. Application of DA resulted in a rapid decrease in [Ca2+]i concomitant with the cessation of these spontaneous [Ca2+]i fluctuations. After DA removal, these cells resumed oscillatory [Ca2+]i activities similar to those observed before DA application. In quiescent lactotropes, acute application of DA exerted no effect on resting [Ca2+]i, but quiescent cells could be activated to produce [Ca2+]i fluctuations by the application and withdrawal of DA. Again, the character of the induced [Ca2+] activity showed significant cell to cell variation. In contrast, the pattern of [Ca2+]i fluctuations was remarkably characteristic in a given cell in response to repeated challenges. A composite [Ca2+]i profile of 13 cells paralleled the PRL secretory rebound after application and removal of DA. The oscillatory rise in [Ca2+]i is functionally linked to the rebound release of PRL after DA removal, as both were immediately abolished by blockade of Ca2+ influx. These data demonstrate that the rebound secretion of PRL is dependent upon enhanced influx of extracellular Ca2+ after cells recover from DA-induced hyperpolarization and support the hypothesis that a population of inactivated Ca2+ channels has been recruited in response to application and withdrawal of DA.

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 µm(2)). 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.

Stimulation of prolactin release by dopamine withdrawal: role of membrane hyperpolarization.

Hypothalamic dopamine (DA) tonically inhibits prolactin (PRL) release from the anterior pituitary gland, whereas removal of DA markedly augments its release to values exceeding pre-DA rates. We investigated whether electrical events induced by DA contribute to this secretory rebound. In primary cultured lactotropes, spontaneous Ca(2+)-dependent spiking activity was enhanced after recovery from DA-induced hyperpolarization. Voltage clamp studies showed a rapidly and a slowly inactivating Ca2+ current that were both augmented by a hyperpolarizing conditioning potential. We measured PRL release from perifused cells exposed to DA to correlate the electrical with the secretory responses. DA inhibited PRL release by 67%, whereas PRL secretion increased three- to fourfold over basal release after washout of DA. Valinomycin, used to directly hyperpolarize the cell membrane, mimicked the actions of DA, inhibiting PRL release (65%) and, upon washout, augmenting PRL secretion. Blocking the DA- or valinomycin-induced hyperpolarization by elevating external K+ concentration blocked both the inhibition and rebound of PRL release. These novel results demonstrate that hyperpolarization of the lactotrope membrane by DA is critical for the development of PRL rebound after DA withdrawal. We hypothesize the mechanism involves the removal of inactivation from a population of Ca2+ channels, leading to enhanced Ca2+ influx and PRL release upon recovery of the resting membrane potential after DA removal.

Stimulation of prolactin release by dopamine withdrawal: role of calcium influx.

Withdrawal of dopamine (DA), a neurotransmitter that inhibits prolactin (PRL) release from the anterior pituitary, stimulates PRL release with transient (30- to 45-min) secretory rates that exceed those observed before application of DA ("PRL rebound"). Using patch-clamp methods on identified rat lactotropes, we have demonstrated that a period of increased Ca(2+)-spiking activity follows recovery from the DA-induced hyperpolarization. The present experiments used dissociated pituitary cells to identify the relative roles of adenosine 3',5'-cyclic monophosphate (cAMP), inositol phosphates, and the enhanced influx of Ca2+ in the rebound secretion of PRL. Rebound secretion of PRL after DA withdrawal was completely blocked by the Ca2+ channel blocker verapamil (20 microM), which also inhibited spontaneous Ca(2+)-spiking activity. DA-induced changes in cAMP levels could be completely dissociated from the PRL rebound. Production of inositol phosphates rose after DA withdrawal but was secondary to the influx of Ca2+. These data demonstrate that influx of extracellular Ca2+ through verapamil-sensitive channels is a critical step in inducing PRL release after DA withdrawal. This finding supports our theory that DA-induced hyperpolarization recruits previously inactivated Ca2+ channels and upon DA washout the enhanced influx of Ca2+ through these voltage-regulated channels supports the rebound release of PRL.

D2 dopamine receptor activation of potassium channels in identified rat lactotrophs: whole-cell and single-channel recording.

Dopamine (DA) is the major physiological regulator of prolactin secretion from the anterior pituitary, exerting a tonic inhibitory control that is mediated by D2 DA receptors. D2 receptors in both the anterior pituitary and CNS are thought to produce some of their inhibitory effects via a coupling to potassium (K+) channels to increase K+ conductance. Utilizing the reverse hemolytic plaque assay and patch-clamp techniques, we characterize the actions of DA on membrane potential and associated DA-activated whole-cell current, as well as the single K+ channels that underlie the response in primary rat lactotrophs. We demonstrate that DA (5 nM to 1 microM) or D2-selective agonists (RU24213 and quinpirole) evoke a hyperpolarization of membrane potential that was blocked by D2 antagonists and associated with an increased K+ conductance. Whole-cell current responses to ramp voltage commands revealed a DA-activated current whose reversal potential was near the calculated Nernst potential for K+, varied as a function of K+ concentration, exhibited some inward rectification, and was Ca2+ independent. The current was insensitive to tetraethylammonium (TEA; 10 mM), partially blocked by 4-aminopyridine (4-AP; 5 mM), and almost completely inhibited by quinine (100 microM). Cell-attached recordings in the presence of DA or a D2 agonist revealed the opening of a K+ channel that was not present in the absence of DA or when a D2 receptor antagonist was included with DA. Analysis of the single-channel current showed the current-voltage relationship to be linear at negative patch potentials and yielded a unitary conductance of 40.2 pS in the presence of 150 mM KCl. The channels were not blocked by TEA (10 mM), were slightly suppressed by 4-AP (5 mM), and were almost completely inhibited by quinine (100 microM). These experiments establish that in primary rat lactotrophs, DA acts at D2 receptors to activate the opening of single K+ channels, which results in an increase in K+ conductance and associated membrane hyperpolarization. This is the first characterization of single DA-activated K+ channels in an endocrine cell.

Changes in the kinetics of [3H]dopamine release from median eminence and striatal synaptosomes during aging.

The release of preaccumulated tritium-labeled dopamine [( 3H]DA) was examined in isolated nerve terminals (synaptosomes) prepared from the median eminence (ME) and corpus striatum (CS) of young (2-3 months), middle-aged (11-12 months), and old (19-21 months) male rats. Fractional release of [3H]DA was measured over 1- to 10-sec time intervals under basal (5 mM K+) and depolarizing (75 mM K+) conditions in the presence of calcium. No differences in the rate of basal efflux between the age groups were observed in either ME or CS preparations. Fast-phase evoked [3H]DA release (0-1 sec) from CS synaptosomes was unchanged from young to middle-aged, but was decreased in old preparations. These data demonstrate that the nigrostriatal nerve terminal has a diminished ability to respond fully to depolarizing stimuli in advanced age. Mean serum PRL levels in old rats were 2.3-fold greater than those in both young and middle-aged rats, while serum LH levels were decreased 2.0-fold in middle-aged and old compared with those in young rats. The fact that LH levels were already decreased in middle-aged rats while PRL levels had not yet increased suggests that decreased gonadotropin titers in old rats do not result from the coincident hyperprolactinemia. In ME synaptosomes, depolarization-induced [3H]DA release was decreased at all time points in middle-aged preparations compared to that in young preparations. The reduced fractional release from the middle-aged ME synaptosomes was due to a depressed rate of release during the initial second of depolarization. Evoked release from ME terminals of old rats was comparable to that measured in the young group. Thus, there occurred an age-related biphasic change in the initial rate of evoked DA release from ME synaptosomes. Diminished response of ME dopaminergic terminals to depolarizing stimuli during middle age may be important in the later development of hyperprolactinemia in aging male rats. The increased PRL available for feedback on the tuberoinfundlbular dopaminergic neurons may, in turn, be associated with the apparent recovery of evoked [3H]DA release from ME synaptosomes of old rats.

Selective effects of hyperprolactinemia on in vitro dopamine release from median eminence synaptosomes.

Prolactin is thought to exert an autoregulatory, negative feedback effect on its own secretion via stimulation of the tuberoinfundibular dopaminergic (TIDA) neurons. To investigate possible mechanisms involved in this feedback, the effects of experimentally induced hyperprolactinemia on the release of 3H-dopamine (3H-DA) were studied in nerve terminals (synaptosomes) isolated from rat median eminence (ME), the TIDA neuronal projection field. Synaptosomes were prepared from adult male rats treated with ovine prolactin (oPRL) or the vehicle for 48 hr. Synaptosomes were incubated in 0.1 microM 3H-DA at 30 degrees C until steady-state conditions were achieved, and then release of the preaccumulated transmitter was measured over 1-20 sec time intervals under basal and depolarizing conditions. Release of 3H-DA elicited by depolarization of the terminals was significantly greater in ME synaptosomes prepared from oPRL-treated animals as compared with preparations from controls. This effect of the hyperprolactinemia appeared to be specific to the TIDA neurons since oPRL treatment did not result in increased evoked release of 3H-DA from terminals prepared from the mesolimbic, tuberohypophyseal, or nigrostriatal dopaminergic neurons. Basal efflux in all preparations was not changed from controls. The increased evoked release in oPRL-treated ME occurred when depolarization was induced either with high external [KCl] or veratridine. The enhanced 3H-DA efflux was evident during depolarization over a wide range of external calcium concentrations (0.01-3.0 mM), in the presence of 20 nM Ni2+ to block Ca2+ influx through voltage-gated channels, or when all external Ca2+ had been chelated, indicating that this effect of oPRL involves DA released through a mechanism independent of external calcium.

Rapid release of [3H]dopamine from median eminence and striatal synaptosomes.

Release of preaccumulated, tritium-labeled dopamine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1-20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+ resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+- and the veratridine-stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+-induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]o and the [Ca2+]o dose-response curves, consistent with actions of Mg2+ on screening surface membrane charges and blocking voltage-dependent Ca2+ channels. In all studies, steady-state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.

Distribution of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylglycol (DOPEG) in microdissected brain structures and the pituitary gland: metabolite changes in the median eminence in response to hyperprolactinemia and suckling.

Dopamine (DA), norepinephrine (NE), epinephrine (E), 3,4-dihydroxyphenylglycol (DOPEG) and dihydroxyphenylacetic acid (DOPAC) were determined simultaneously by a radioenzymatic, thin-layer chromatographic assay able to detect 1-10 pg of the parent compounds and 80-120 pg of their metabolites. A localization study of these compounds in 20 micro-dissected hypothalamic and limbic structures and the anterior and posterior pituitary glands of male rats was completed. DOPAC was detectable in 14 of 22 structures with the lowest DOPAC/DA ratio being found in the caudate nucleus (7.1%) and the highest in the medial aspect of the ventromedial nucleus of the hypothalamus (422.0%). There was a higher DOPAC/DA ratio in the lateral (21.5%) than in the medial (11.3%) portion of the median eminence suggesting that a greater portion of released DA in the medial median eminence enters the portal circulation. DOPEG was detectable in 6 of 22 structures with DOPEG/NE ratios ranging from 8% (interstitial nucleus of the stria terminalis, ventral aspect) to 32% (medial median eminence). A poor correlation exists between DOPAC and DA concentrations in the various brain regions while there was a stronger relationship between DOPEG and NE concentrations. Male rats were rendered hyper-prolactinemic for 48 hours with injections of ovine prolactin (oPRL) every 8 hours (4 mg/kg body weight sc). In such rats there was a suppression of endogenous rat PRL (rPRL) secretion, the DOPAC/DA ratio increased 2.2-fold in the medial (MEm) and 1.9-fold in the lateral median eminence (MEl), and the DA concentration in the anterior pituitary also increased 2.6-fold. In 10 day postpartum lactating rats, suckling produced marked increases in serum rPRL but no change in DOPAC/DA ratios in the ME or in the DA concentration in the anterior pituitary. The data reveal a wide range of DOPAC/DA ratios (7-422%) in brain regions containing cell bodies, axons and terminals of the different dopaminergic neuronal tracts in brain and pituitary. Considering the DOPAC/DA ratios in the MEm and MEl, it is suggested that a large perturbation of dopaminergic transmission produces a significant ratio change while a smaller perturbation is not detected by this index of neuronal metabolism.

Suckling-induced prolactin release is suppressed by naloxone and simulated by beta-endorphin.

The role that opiate peptides play in suckling-induced prolactin (PRL) release was examined in 10-day postpartum lactating rats. The opiate receptor antagonist naloxone (NAL) suppressed suckling-induced PRL release in a dose-dependent manner and a large dose abolished the response. These results suggest either that opiate neurons are situated in the neuronal pathway mediating this neuroendocrine response, or alternatively, that opiate neurons are situated such that they can modulate neuronal transmission in this pathway. It is suggested that NAL blocks a tonic, inhibitory beta-endorphinergic input to the tuberoinfundibular dopaminergic (TIDA) neurons, hence, NAL administration in effect stimulates the TIDA neurons and in this way overrides the suckling response. Intravenous, bolus administration of beta-endorphin (beta-END) produced a PRL response that was similar to the suckling response in terms of latency of onset and duration while the magnitude of the beta-END-induced response was 2-fold greater than that produced by the suckling stimulus. NAL abolished beta-END-induced PRL release at a much lower dose than that required to inhibit suckling-induced PRL release. This suggests that the neural mediation of the suckling response involves a mechanism in addition to the one inhibited by opiate receptor blockade.

Suckling decreases dopamine turnover in both medial and lateral aspects of the median eminence in the rat.

The effect of suckling on dopamine (DA) turnover was studied in the medial and lateral aspects of the median eminence. In 10-day postpartum lactating rats suckling decreased DA turnover 2.2-fold in the medial and 2.1-fold in the lateral median eminence. Norepinephrine turnover did not differ in suckled and non-suckled rats. These results are consistent with the hypothesis that decreased DA release is a component of the neuroendocrine reflex mediating suckling-induced prolactin release. The results further indicate that dopaminergic neurons distributing to both medial and lateral aspects of the median eminence are involved in the suckling response.

Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster.

The changes in serum gonadotrophins in male hamsters following one injection of 15 micrograms luteinizing hormone releasing hormone (LHRH) (Group A) were compared with those following the last injection of LHRH in animals receiving an injection approximately every 12 hr for 4 days (Group B) or 12 days (Group C). Peak follicle stimulating hormone (FSH) levels (ng/ml) were 1776 +/- 218 (Group A), 2904 +/- 346 (Group B), and 4336 +/- 449 (Group C). Peak luteinizing hormone (LH) values (ng/ml) were 1352 +/- 80 (Group A), 410 +/- 12 (Group B), and 498 +/- 53 (Group C). Serum FSH:LH ratios, calculated from the concentrations measured 16 hr after the last LHRH injections, were higher in Groups B and C than in Group A. Similar injections of LHRH (100 ng or 15 micrograms/injection) for 6 days elevated the serum FSH:LH ratio in intact males. Five such LHRH injections (100 ng/injection) blunted the rise in serum LH in orchidectomized hamsters. Direct effects of LHRH on gonadotrophin secretory dynamics or altered brain-pituitary-testicular interactions may alter the ratio of FSH to LH in the hamster.

Autofeedback effects of prolactin on basal, suckling-induced, and proestrous secretion of prolactin.

Subcutaneous injections of ovine prolactin (oPRL, 4 mg/kg) were utilized to study the negative feedback effect of PRL on its own secretion in lactating and 4-day estrous cycling female rats. Basal PRL secretion in the 10-day postpartum lactating rat is not suppressed by acute or 48 hr of exposure to oPRL. In contrast, basal PRL secretion on the morning of proestrus is inhibited following 48 hr of exposure to oPRL. Thus, the lactating rat appears to be unresponsive to the negative feedback action of PRL compared with the adult female rat in regards to basal PRL secretion. The suckling-induced PRL response is partially suppressed by exposure to oPRL in 10-day postpartum lactating rats. Considering the areas under the PRL response curves, the suckling-induced release is blunted by 6 (decreased 49%), 12 (decreased 46%), and 48 (decreased 77%) hr of oPRL exposure compared to controls. In contrast, in cycling animals, 48 hr of exposure to elevated oPRL levels dramatically abolished the proestrous PRL surge. Elevated PRL levels, by a direct action on the brain and/or indirectly by altering ovarian function, inhibit or override the hypothalamic mechanism(s) mediating the proestrous PRL surge while having a lesser effect on the mechanism(s) mediating the suckling-induced PRL response. Taken together, the present data point to probable differences in the mechanism(s) mediating basal, suckling-induced, and the proestrous secretion of prolactin.

Immunohistochemical visualizations of prolactin, growth hormone, and a substance resembling placental lactogen in the in situ and ectopic pituitary in the hamster.

The effects of transplanting neonatal adenohypophyseal tissue into the hamster cheek pouch on the presence of intracellular materials related to PRL in the allografts were examined using immunohistochemistry. In situ pituitary and placental tissue were used as control. Cells with antirat PRL-reactive sites were scarce in the grafts, and radioimmunoassayable serum PRL was not detectable in hypophysectomized hosts with grafts. Antirat GH- and antihuman placental lactogen (hPL)-reactive sites were visualized in the grafts and in situ pituitary tissue. Intracellular material in hamster placental tissue was visualized with anti-hPL only. Results of various immunohistochemical procedures using in situ pituitary tissue and antirat GH and anti-hPL antisera indicated that three cell types could exist: 1) a cell type visualized with only antirat GH, 2) a cell type visualized with only anti-hPL, and 3) a cell type, the most frequently observed, visualized with both antisera. The physiological significance of this intracellular product, which resembles hPL and perhaps hamster PL immunologically, in hamster adenohypophyseal cells is unknown. Additionally, our data indicate that the ectopic site for pituitary transplantation in the hamster may influence the cell types present in the grafts.