Influence of corticosteroids on prolactin release from anterior pituitary cell aggregates cultured in serum-free medium. Differential effects on dopamine-induced inhibition, post-dopamine rebound and stimulation by TRH, vasoactive intestinal peptide (VIP), angiotensin II and isoproterenol

Dispersed rat anterior pituitary cells were allowed to reassociate into spherical aggregates by gyrotory shaking in serum-free chemically defined culture medium. When aggregates were superfused after being cultured for 5 days in this medium, stimulation of PRL release by TRH, VIP, angiotensin II and the beta-adrenergic agonist isoproterenol was comparable to that of aggregates cultured in serum-supplemented culture medium. Addition to the serum-free medium of 80 nM dexamethasone (Dex) resulted in a significant enhancement of the stimulation of PRL release by TRH, VIP and angiotensin II but not of the stimulation of PRL release by isoproterenol. Dex also failed to influence the inhibition of PRL release by 10 min exposure to 10 nM dopamine (DA). However, Dex significantly enhanced the post-DA rebound secretion of PRL. After 3 weeks in culture Dex provoked a similar potentiation of the response to angiotensin as at 5 days in culture but it abolished almost completely the stimulatory effect of isoproterenol. It is concluded that pituitary cell aggregates cultured in defined serum-free medium are a reliable system to study the multifactorial control of PRL release. The data show that peptidergic, dopaminergic and beta-adrenergic control at the pituitary level is differentially modulated by corticosteroids.     

Adrenaline involvement in the presynaptic beta-adrenoceptor-mediated mechanism of dopamine release from slices of the rat hypothalamus

Slices of rat hypothalamus were superfused and endogenous release of dopamine (DA) was measured by high performance liquid chromatography combined with electrochemical detection. The K+ (20 mM)-evoked release in the presence of tetrodotoxin was Ca2+-dependent. The evoked release was facilitated by a beta-agonist, isoproterenol and this effect was completely abolished by a beta-antagonist, 1-propranolol. Isoproterenol also concentration-dependently facilitated the electrically (at 5Hz) evoked release of DA. The pretreatment with 1-propranolol, beta 1-antagonist, atenolol and beta 2-antagonist, butoxamine shifted the concentration-effect curve of isoproterenol to the right. On the other hand, beta 1-agonist, tazolol, beta 2-agonist, salbutamol and low concentration (10(-9) M) of adrenaline also facilitated the release. 1-Propranolol alone reduced the electrically (at 2 Hz) evoked release, and this effect was completely abolished when the adrenaline content in the brain was drastically reduced by use of a potent PNMT inhibitor, DCMB. These findings suggest that in the rat hypothalamus adrenaline released from adrenaline-containing nerve terminals probably modulates DA release via presynaptic beta 1- and beta 2-adrenoceptors on DA nerve terminals.     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.     

Regulation of prolactin release by angiotensin

In superfused anterior pituitary cell aggregates, prolactin release is stimulated by angiotensin II (AII) in a concentration-dependent fashion between 0.1 and 10 nM. When studied in aggregates prepared from pituitary cell populations separated according to size by unit gravity sedimentation, the PRL response to AII was weak in a population enriched in lactotrophs but deprived of gonadotrophs. In other separated populations, the response increased with the proportional number of gonadotrophs. The response also increased when lactotrophs were co-aggregated with an enriched population of gonadotrophs. It is proposed that the PRL response to AII is augmented by an intercellular messenger system presumably operating between gonadotrophs and lactotrophs.     

The effects of prostacyclin (PGI2) on prolactin secretion in vitro

Continuously superfused rat anterior pituitary cells were used to study the effects of exogenous prostaglandins (PGs) and thromboxanes (TXs) on the secretion of prolactin (PRL). No change in hormone release was observed upon superfusion with TXB2 (10(-5)M) or the TX synthesis inhibitor, imidazole (1.5 mM). PGs A2, B2, D2, E1, E2, F1 alpha, F2 alpha, and endoperoxide analogs, U-44069 and U-46619, also had no effect on PRL secretion (all at 10(-5)M). In contrast 10(-5)M PGI2 was repeatedly found to stimulate PRL release to a level at least 125% above control, while producing no apparent change in the amount of hormone secreted in response to TRH. Somatostatin (SRIF), at a dose of 10(-6)M, maximally inhibited TRH-induced PRL output, but failed to alter the PRL response to PGI2. These studies indicate that PGI2 may have a direct effect on the anterior pituitary to modify PRL secretion.     

Differential response of adenylate cyclase to beta-adrenergic agonists in white adipocyte membranes from lean and obese (ob/ob) mice

The quality of the beta-adrenergic response, as measured by the activation of adenylate cyclase, was found to differ in adipocyte membranes from lean and obese mice. In the tissue from lean mice, the response was similar to that in rat adipose tissue and could, by analogy, be classified as beta 1-receptor response. In the tissue preparations from the obese mice, the rank order of potency of the three classical agonists (isoproterenol, epinephrine, and norepinephrine) was more typical of a beta 2-receptor response.     

Effect of the bombesin receptor blockers [Leu13, psi CH2NH-Leu14]bombesin and N-pivaloyl GRP(20-25) alkylamide (L 686,095-001C002) on basal and neuromedin C-stimulated PRL and GH release in pituitary cell aggregates.

Perifusion of rat anterior pituitary cell aggregates, cultured in estrogen-supplemented serum-free medium with 1 nM of the bombesin (BBN)-like peptide, neuromedin C (NMC), significantly stimulates GH and PRL release. This effect is dose-dependently inhibited by the BBN receptor blocker L 686,095-001C002 [an N-pivaloyl-gastrin-releasing-peptide(20-25) alkylamide]. The IC50 was 0.20 nM in the case of the GH response and 0.16 nM in the case of the PRL response. The antagonist has no effect on basal PRL or GH release. [Leu13, psi CH2NH-Leu14]BBN (psi BBN) displays an IC50 of 0.41 microM for inhibiting the GH response and 0.36 microM for inhibiting the PRL response to NMC. At a concentration of 0.5 microM or 5 microM, however, the latter antagonist stimulates PRL and GH release when perifused alone. This stimulatory effect is dose dependent, augments when aggregates are cultured in 1 nM E2 (as is the case for NMC) and is abolished by 2 nM L 686,095-001C002. It is concluded that L 686,095-001C002 is a potent and pure antagonist of pituitary BBN receptors mediating PRL and GH release, whereas psi BBN is a relatively weak antagonist with considerable partial agonist activity.     

Effect of TNF-alpha on prolactin secretion from rat anterior pituitary and dopamine release from the hypothalamus: comparison with the effect of interleukin-1 beta.

The effects of human recombinant interleukin-1 beta and -6 and tumor necrosis factor-alpha (TNF-alpha) on the releases of PRL and dopamine were examined using monolayer cultures of rat pituitary cells and hypothalamic cells. The release of PRL from rat pituitary cells in 30 min was increased about 2-fold (p less than 0.05) by 10(5) U/l interleukin-1 beta, 10(5) U/l interleukin-6 or 100 micrograms/l TNF-alpha. TNF-alpha at 100 micrograms/l significantly increased PRL release within 5 min incubation and this effect continued throughout the next 30 min of incubation. Incubation for 5 min with TNF-alpha caused dose-dependent stimulation of PRL release. These cytokines did not modulate [3H]-dopamine release from primary cultures of hypothalamic cells. These results suggest that these cytokines stimulate PRL release directly at the pituitary gland, without modifying the release of dopamine from the hypothalamus.     

Stimulation of spontaneous and dopamine-inhibited prolactin release from anterior pituitary reaggregate cell cultures by angiotensin peptides

In superfused anterior pituitary reaggregate cell cultures angiotensin II (AII) stimulated both spontaneous and dopamine-inhibited prolactin (PRL) release from subnanomolar concentrations. Angiotensin I (AI) and angiotensin III (AIII) also stimulated PRL release. The magnitude and rate of response to AI was equal to or only slightly lower than that to AII. However, the angiotensin converting enzyme (ACE) inhibitors captopril and teprotide (1 microM) completely abolished the PRL response to 0.1 nM AI and strongly reduced that to 1 nM AI. The intrinsic activity of AIII was lower than that of AII but could be enhanced by adding 2 microM of the aminopeptidase inhibitor amastatin to the superfusion medium. After withdrawal of AIII, PRL secretion rate rapidly returned to baseline levels, whereas after withdrawal of AI or AII, secretion fell to a level remaining significantly higher than basal release. The present findings indicate that stimulation of PRL release by AI is weak unless it is converted into AII by ACE and that aminopeptidase may be important in determining the magnitude and termination of the PRL response. Furthermore, the active peptides induce a different pattern of response.     

Inhibition of prolactin secretion by gastrin releasing peptide (GRP) in the rat

Synthetic gastrin releasing peptide (GRP) injected intraventricularly (1 microgram/rat), but not intravenously, suppressed rat prolactin (PRL) release induced by a Met-enkephalin analog, FK33-824 (10 micrograms/100 g body wt., iv). GRP also blunted PRL release induced by a dopamine antagonist, domperidone (1 microgram/100 g body wt., iv). In contrast, GRP did not suppress elevated plasma PRL levels sustained by a large dose of domperidone (10 micrograms/100 g body wt., iv). GRP (10(-5) M) had no effect on PRL release from superfused pituitary cells in vitro. These results suggest that GRP inhibits PRL secretion in the rat by acting through the brain to stimulate the dopaminergic mechanism.     

Effects of VIP, TRH, GABA and dopamine on prolactin release from superfused rat anterior pituitary cells

Effects of VIP, TRH, dopamine and GABA on the secretion of prolactin (PRL) from rat pituitary cells were studied in vitro with a sensitive superfusion method. Dispersed anterior pituitary cells were placed on a Sephadex G-25 column and continuously eluted with KRBG buffer. Infusion of TRH (10(-11) - 10(-8)M) and VIP (10(-9) - 10(-6)M) resulted in a dose-related increase in PRL release. LHRH (10(-8) - 10(-5)M) had no effect on PRL release. On the other hand, infusion of dopamine (10(-9) - 10(-6)M) and GABA (10(-8) - 10(-4)M) suppressed not only the basal PRL release from dispersed pituitary cells but also the PRL response to TRH and VIP. The potency of TRH to stimulate PRL release is greater than that of VIP, and the potency of dopamine to inhibit PRL secretion is stronger than that of GABA on a molar basis. These results indicate that TRH and VIP have a stimulating role whereas dopamine and GABA have an inhibitory role in the regulation of PRL secretion at the pituitary level in the rat.     

The effects of prostacyclin PGI2 on prolactin secretion in vitro

Continuously superfused rat anterior pituitary cells were used to study the effects of exogenous prostaglandins (PGs) and thromboxanes (TXz) on the secretion of prolactin (PRL). No change in hormone release was observed upon superfusion with TXB₂ (10⁻⁵M) or the TX synthesis inhibitor, imidazole (1.5 mM). PGs A2, B2, d2, e1, e2, f1α, F2α, and endoperoxide analogs, U-44069 and U-46619, also had no effect on PRL secretion (all at 10⁻⁵M), In contrast 10⁻⁵M PGI2 was repeteadly found to stimulate PRL release to a level at least 125% above control, while producing no apparent change in the amount of hormone secreted in response to TRH. Somatostatin (SRIF), at a dose of 10⁻M, maximally inhibited TRH-induces PRL output, but failed to alter the PRL response to PGI₂. These studies indicate that PGI₂ may have a direct effect on the anterior pituitary to modify PRL secretion.     

Beta 1-adrenoceptors in rat hepatoma. Desensitization by isoproterenol and phorbol-myristate-acetate

The beta-adrenergic responsiveness of hepatocytes obtained from hypothyroid rats and of a transplantable hepatoma cell line (AS-30D) were studied by measuring the accumulation of cyclic AMP. The potency order for agonists in hepatocytes was: isoproterenol greater than epinephrine much greater than norepinephrine whereas in the hepatoma cells the potency order was: isoproterenol greater than norepinephrine greater than or equal to epinephrine. The effect of isoproterenol was antagonized in hepatocytes by low concentrations of ICI 118551 and only partially by concentrations of atenolol as high as 100 microM. In hepatoma cells the effect of isoproterenol was inhibited by both antagonists with the potency order atenolol greater than ICI 118551. These data indicate that in hepatocytes the effect is mediated by beta 2-adrenoceptors whereas in hepatoma cells it is through beta 1-adrenoceptors. Preincubation of hepatoma cells with isoproterenol or phorbol-myristate-acetate diminished the subsequent beta-adrenergic responsiveness of the cells. Interestingly, when both isoproterenol and phorbol-myristate-acetate were present during the preincubation the beta-adrenergic desensitization observed was bigger than that induced by any of these agents alone.     

The (--)[3H]dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites

In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)[3H]dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)[3H]dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)[3H]dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)[3H]dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)[3H]dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r = 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)[3H]dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.     

Further evidence that peptide histidine isoleucine (PHI) may function as a prolactin releasing factor in rats

Intraventricular administration of peptide histidine isoleucine (PHI) (200 ng, 1, 5 and 10 micrograms/rat) resulted in a significant and dose-related increase in plasma prolactin (PRL) levels in urethane-anesthetized rats and in conscious rats with intraatrial and intraventricular catheters. Intravenous injection of PHI (10 micrograms/rat) also raised plasma PRL levels in these animals. In in vitro studies, PRL release from superfused rat anterior pituitary cells was stimulated by PHI (10(-9), 10(-8) and 10(-7) M) in a dose-related manner. The stimulating effect of PHI (10(-7)M) on PRL release in vitro was as potent as that of vasoactive intestinal polypeptide (VIP) (10(-7) M) and was observed even in the presence of dopamine (10(-7) M). These results suggest that PHI plays a stimulating role in regulating PRL secretion by acting, at least in part, directly on the pituitary in the rat.     

Effect of various catecholamine antagonists on prolactin secretion in conscious male rabbits

To clarify physiological roles of catecholaminergic systems in the control of rabbit prolactin (PRL) release, the effect of various catecholamine receptor antagonists on plasma PRL levels was examined in conscious, freely moving male rabbits. An intravenous (iv) injection of yohimbin (2.5 mg/kg body wt), an alpha 2-adrenoreceptor antagonist, but not prazosin (2 mg/kg body wt), an alpha 1-adrenergic receptor antagonist, resulted in a significant elevation of plasma PRL. Conversely, propranolol (2.5 mg/kg body wt, iv), a nonselective beta-adrenoreceptor antagonist, and metoprolol (2.6 mg/kg body wt, iv), a beta 1-adrenergic antagonist, slightly but significantly suppressed basal levels of plasma PRL. On the other hand, haloperidol (0.5 mg/kg body wt, iv), pimozide (0.3 mg/kg body wt, iv), sulpiride (5 mg/kg body wt, iv), chlorpromazine (3 mg/kg body wt, iv), and YM-09151-2 (0.2 mg/kg body wt, iv), all dopamine receptor antagonists caused a significant increase in plasma PRL. These results suggest that dopaminergic and alpha 2-adrenergic mechanisms exert a tonic inhibitory role and beta-adrenergic mechanisms, probably beta 1, a tonic stimulatory role in the regulation of PRL release in the rabbit.     

Dopaminergic Inhibition of Prolactin Release and Calcium Influx Induced by Neurotensin in Anterior Pituitary Is Independent of Cyclic AMP System

The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated by the order of potency of agonists in inhibiting PRL release and calcium influx (DA greater than epinephrine greater than norepinephrine much greater than isoproterenol); by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and by the high degree of stereoselectivity of sulpiride. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of beta-adrenergic receptors in the rat vas deferens using [3H]-dihydroalprenolol binding

The beta-adrenergic antagonist, [3H]-dihydroalprenolol ([3H] DHA), binds to membranes prepared from the rat vas deferens in a specific and saturable manner. Scatchard and Hill plot analysis indicates a single class of binding sites with no evidence of cooperative interactions. The specific binding sites have a high affinity (Kd = 0.3 nM) and a maximal occupancy estimated to be 460 fmoles [3H]-DHA bound/g wet tissue weight. Beta-adrenergic agonists and/or antagonists inhibit [3H]-DHA binding to rat vas deferens membranes in a stereospecific manner and with a relative order of potency expected for beta-adrenergic receptors of the beta2 subtype. The receptor affinities of various beta-adrenergic antagonists in the rat vas deferens determined using inhibition of [3H]-DHA binding correlated with their receptor affinities determined physiologically using antagonism of isoproterenol-induced inhibition of neurogenic contractions in-vitro.     

μ-opioid receptor-mediated inhibition of the release of radiolabelled noradrenaline and acetylcholine from rat amygdala slices

Presynaptic modulation by opioids of electrically-evoked neurotransmitter release from superfused rat amygdala slices prelabelled with [³H]noradrenaline (NA) and [¹⁴C]choline was examined. Both [³H]NA and [¹⁴C]acetylcholine release were strongly inhibited by morphine, the mixed δ/μ-receptor agonist [ -Ala², -Leu⁵]enkephalin (DADLE) and the highly selective μ-agonist [ -Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), whereas the highly selective δ-agonist [ -Pen², -Pen⁵]enkephalin and the κ-agonist bremazocine were without effect. The inhibitory effects were potently antagonized by naloxone but not by the selective δ-receptor antagonist fentanylisothiocyanate. When the selective uptake inhibitor desipramine was used to prevent uptake of [³H]NA into noradrenergic nerve terminals, but sparing the uptake into dopaminergic nerve terminals, the electrically evoked release of tritium was strongly inhibited by bremazocine but not by DADLE or DAMGO.

The data indicate, that in the amygdala transmitter release from dopaminergic nerve fibres is inhibited only via activation of κ-receptors, whereas transmitter release from noradrenergic and cholinergic nerve fibers is subjected to inhibition by opioids via activation of μ-receptors only. Regional differences and similarities of modulation of neurotransmitter release by opioids in the rat brain are briefly discussed.     

Stimulation of prolactin secretion from turkey anterior pituitary cells in culture

Prolactin (PRL) secretion by monolayer cultures of turkey anterior pituitary cells was significantly increased (up to 44-fold) by vasoactive intestinal peptide (VIP), arginine vasotocin (AVT), and by an extract of turkey hypothalami (HE). Several other neuropeptides (including thyrotropin-releasing hormone) and neurotransmitters were ineffective in influencing PRL secretion at doses up to 10(-6) M. The dynamic PRL response to HE and VIP was studied using superfused pituitary cells attached to microcarrier beads. HE, administered in 30-min pulses, resulted in a significant, dose-related increase in PRL secretion from a basal secretion rate of 2.32 ng/min/10(7) cells to a peak secretion rate of 127.13 ng/min/10(7) cells at the highest dose of HE tested (1 mg tissue-equivalent weight/ml). VIP significantly increased PRL secretion at all doses studied (from 10(-10) to 10(-6) M), with 10(-8) M VIP producing a response similar to that observed with 1 mg/ml HE. A highly significant (P less than 0.001) linear relationship was demonstrated between the log-dose of VIP administered and peak PRL secretion rate. These studies suggest that VIP, but not TRH, may be a physiological stimulus for PRL release in the turkey.