Search

Oxytocin (OT) is one of the secretagogues for stress-induced ACTH release. OT-induced ACTH release is reported to be mediated by the vasopressin V1b receptor in the rat pituitary gland, which contains both OT and V1b receptors. We examined OT-induced ACTH release using primary cultures of anterior pituitary cells from wild-type (V1bR+/+) and V1b receptor knockout (V1bR-/-) mice. OT stimulated similar levels of ACTH release from pituitary cells of V1bR+/+ and V1bR-/- mice. OT-induced ACTH release was significantly inhibited by the selective V1b receptor antagonist SSR149415 and the OT receptor antagonist CL-14-26 in V1bR+/+ mice. In addition, cotreatment with SSR149415 at 10(-6) m and CL-14-26 at 10(-6) m inhibited OT-induced ACTH release to the control level inV1bR+/+ mice. In V1bR-/- mice, OT-induced ACTH release was significantly inhibited by CL-14-26 at 10(-8) m and completely inhibited at 10(-7)m. These results indicate that OT induces the ACTH response via OT and V1b receptors inV1bR+/+ mice but via only OT receptors in V1bR-/- mice. The gene expression level of the OT receptor was significantly higher in the anterior pituitary gland of V1bR-/- mice than in that of V1bR+/+ mice, suggesting that the OT receptor is up-regulated to compensate for ACTH release under conditions of V1b receptor deficiency.

Perinatal glucocorticoid (GC) treatment is increasingly associated with long-term disturbances in hypothalamo-pituitary-adrenocortical function. In the male rat, such treatment induces profound molecular, morphological and functional changes in the anterior pituitary gland at adulthood. To determine whether these effects are sex-specific, we have examined the effects of perinatal dexamethasone treatment on the female pituitary gland, focusing on (i) the integrity of the annexin 1 (ANXA1) dependent regulatory effects of GCs on adrenocorticotrophic hormone (ACTH) release and (ii) corticotroph and folliculo-stellate (FS) cell morphology. Dexamethasone was given to pregnant (gestational days 16-19) or lactating (days 1-7 post partum) rats via the drinking water (1 microg/ml); controls received normal drinking water. Pituitary tissue from the female offspring was examined ex vivo at adulthood (60-90 days). Both treatment regimes reduced the intracellular and cell surface ANXA1 expression, as determined by western blot analysis and quantitative immunogold electron microscopic histochemistry. In addition, they compromised the ability of dexamethasone to suppress the evoked release of ACTH from the excised tissue in vitro, a process which requires the translocation of ANXA1 from the cytoplasm to the cell surface of FS cells. Although neither treatment regime affected the number of FS cells or corticotrophs, both altered the subcellular morphology of these cells. Thus, prenatal dexamethasone treatment increased while neonatal treatment decreased FS cell size and cytoplasmic area. By contrast, corticotroph size was unaffected by either treatment, as also was the size of the secretory granules. Corticotroph granule density and margination were, however, increased markedly by the prenatal treatment, while the neonatal treatment had no effect on granule density but decreased granule margination. Thus, perinatal dexamethasone treatment exerts long-term effects on the female pituitary gland, altering gene expression, cell morphology and the ANXA1-dependent GC regulation of ACTH secretion. The changes are similar but not identical to those reported in the male.

Corticotrope or ACTH cells were immunohistochemically identified in the pituitary pars distalis (PD) of viscacha by using the polyclonal antiserum against ACTH (1-24). The localization, distribution, shape, percentage immunopositive area, and major cellular and nuclear diameters of these cells were analyzed by image analysis in adult male viscachas captured in their natural habitat during the year and after the chronic administration of melatonin. The same parameters were analyzed in immature male and adult female viscachas. ACTH cells in adult males were mainly localized in the dorsal and cephalic regions of PD. They were isolated, forming small groups and in contact with follicular structures and blood vessels. They were pleomorphic, with some of them being polygonal, oval, round, and others, stellate with cytoplasmic extensions. The percentage immunopositive area and the major cellular diameter showed seasonal variations with lower values during June and July (early winter). A decrease in the percentage immunopositive area was observed after the administration of melatonin in adult male animals. ACTH cells of immature animals differed from the adults' cells in their distribution, shape, pattern of immunolabeling, and percentage immunopositive area. These parameters in adult females did not vary in relation to adult males at the same time of the year although. However, the cells in females were smaller in size during April-May. In pregnant viscachas (June-July), these parameters did not show significant differences with the results of non-pregnant females (April-May). This suggests that the environmental stressors do not exert the same influence on the hypothalamo-pituitary-adrenal axis in adult male and female viscachas, probably due to the physiological changes caused by pregnancy. Our results in adult male viscacha demonstrate that the morphology of the ACTH cells varies according to the different seasonal conditions, thus participating in the adaptation process of this rodent to the environment. The elevated levels of melatonin during winter months might inhibit the synthesis of ACTH, probably when affecting some secretagogue of this pituitary hormone. Moreover, the morphological variations observed between adult and immature male viscachas and between both sexes suggest that the steroid gonadal hormones might act on the development and activity of ACTH cells.

The rise in cytosolic free Ca2+ concentration ([Ca2+]i) is the major trigger for secretion of ACTH from pituitary corticotropes. To better understand the shaping of the Ca2+ signal in corticotropes, we investigated the mechanisms regulating the depolarization-triggered Ca2+ signal using patch-clamp techniques and indo-1 fluorometry. The rate of cytosolic Ca2+ clearance was unaffected by inhibitors of Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase (PMCA), slightly slowed by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, but dramatically slowed by mitochondrial uncouplers or inhibitor of mitochondrial uniporter. Measurements with rhod-2 revealed that depolarization-triggered increase in mitochondrial Ca2+ concentration. Thus, mitochondria have a dominant role in cytosolic Ca2+ clearance. Using the Mn2+ quench technique, we found the presence of a continuous basal Ca2+ influx in corticotropes. This basal Ca2+ influx was balanced by the combined actions of mitochondrial uniporter and PMCA and SERCA pumps. Inhibition of the mitochondrial uniporter or PMCA or SERCA pumps elevated basal [Ca2+]i. Using membrane capacitance measurement, we found that the change in the shape of the depolarization-triggered Ca2+ signal after mitochondrial inhibition was associated with enhancement of the exocytotic response. Thus, mitochondria have a dominant role in the regulation of Ca2+ signal and exocytosis in corticotropes.

Silver foxes from a commercial population (farm bred or unselected for behavior control) and from populations selected for tame behavior and enhanced aggressiveness towards man have been investigated. Plasma cortisol and adrenocorticotropic hormone (ACTH) levels, pituitary ACTH levels, POMC gene expression in the anterior pituitary, and corticotropin-releasing factor (CRF) gene expression in the hypothalamus were assessed. The results indicate that the males from the tame-behavior group have lower plasma cortisol and ACTH levels and POMC gene expression in the anterior pituitary in response to capture and handling in comparison with unselected ones. Foxes from the aggressive behavior group also have lower POMC expression, although plasma cortisol and ACTH levels remain the same as in unselected ones. The three groups of animals show no significant changes in the ACTH level in the pituitary and CRF expression in the hypothalamus.

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.

The properties of the hyperpolarization-activated inward cation current (Ih) in mouse anterior pituitary, AtT20 D16:16 corticotropes was characterized by whole cell patch clamp recording. In response to hyperpolarizing steps a large, slowly activating, voltage-dependent inward current was activated with a half maximal activation voltage (V0.5) of -96.2+/-3.1 mV with a time constant of 168+/-13 msec determined at -140 mV at room temperature. Ih had a reversal potential of -35.5+/-1.0 mV and -23.3+/-1.4 mV using 5 mM and 25 mM extracellular K+, respectively, with a relative permeability ratio for Na+ and K+ of 0.24. The current was completely blocked by 2 mM extracellular CsCl and partially blocked by ZD7288 (100 microM) but was unaffected by TEA (10 mM) or Ba2+ (1 mM). RT-PCR analysis revealed robust expression of HCN1, but not HCN2 or HCN3, subunits of hyperpolarization-activated cation channels. The endogenous Ih current was weakly activated by cAMP but robustly inhibited by the cAMP antagonist, Rp-8-CPT-cAMPS. Activation or suppression of protein kinase C activity had no significant effect on the Ih current. The data suggest that in AtT20 D16:16 corticotropes Ih is tonically regulated by the cAMP-signaling cascade and may serve to limit excessive hyperpolarization.

The spatial and proportional representation of the various cell groups in the anterior pituitary is fairly constant, although it may differ between sexes. Recognizable changes occur in a number of physiological and pathological situations. The relative roles of hormones and growth factors in these processes are not fully elucidated, nor are their kinetics. In this paper, published work on basal proliferation, growth factor expression and the growth of specific cell types is reviewed. In addition, we present new data to indicate that the maximum level of proliferation in the anterior pituitary of the male Sprague-Dawley rat occurs around 28 days. We have also demonstrated a circadian rhythm of mitosis in the adult male, with a peak around 1100 h. Cell kinetic analysis suggests a duration for G2 of about 2 hours, and for S phase of 10 1/2 to 11 hours. Finally, we provide data which confirm that the expansion of the corticotroph population after bilateral adrenalectomy is partly the result of an early proliferative response in both corticotrophs and other pituitary cells. Our data also suggest that a further expansion takes place which may reflect differentiation of a population other than committed corticotrophs.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to increase plasma ACTH concentrations in male Sprague-Dawley rats and in male rat primary anterior pituitary cell cultures. The present study examined whether the anterior pituitary effects observed after TCDD exposure are mediated via the Ah receptor (AhR). Primary anterior pituitary cell cultures were prepared from normal 180- to 220-g male rats and the cultures treated with alpha-naphthoflavone (ANF), an antagonist; beta-naphthoflavone (BNF), an agonist; BNF + TCDD; 3,3',4,4',5-pentachlorobiphenyl (PCB), which is known to bind to the AhR; and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB), which does not bind the AhR. Support for the TCDD-AhR-mediated increases in ACTH concentrations is provided by the following observations: (1) ANF inhibited both the 1.3- to 2-fold TCDD-induced increase in basal medium and intracellular ACTH concentrations and the 30% TCDD-induced decrease in medium ACTH levels and the 1.2-fold increase in intracellular ACTH levels in corticotropin-releasing hormone (CRH)-stimulated cells, (2) BNF increased basal medium (1.7-fold) and intracellular (1.3-fold) ACTH concentrations, (3) BNF + TCDD demonstrated additivity by increasing basal medium (2.4-fold) and intracellular (1.7-fold) ACTH concentrations, (4) PCB increased basal medium (1.8- to 2.1-fold) and intracellular (1.3- to 1.8-fold) ACTH concentrations and inhibited medium ACTH secretion in CRH stimulated cells by 24-43%, and (5) HCB did not effect basal or CRH stimulated medium and intracellular ACTH concentrations. From this study it appears that TCDD-induced changes in ACTH secretion and synthesis by cultured anterior pituitary cells is mediated through the Ah receptor.

Contained in this report is a review of available data on pituitary cytokines in domestic species of agricultural importance. The concept is advanced that the pituitary gland is essential to appropriate generation of host defense mechanisms and thus should be considered among other tissues contributing to innate immunity. The functions of these intrapituitary cytokines, principally IL-6, are discussed in the context of potential regulation of the pituitary-adrenal axis (ACTH secretion) via intrapituitary PGE2 generation during the acute-phase response to infectious/inflammatory stimuli. Data from other species are cited as appropriate for comparative purposes and elaboration of proposed mechanisms. However, the scope of the review is not intended to comprehensively cover the vast literature on proinflammatory cytokines and prostaglandins generated peripherally and centrally during host responses to inflammatory stimuli.

Labeling and immunoprecipitation experiments confirmed that three proopiomelanocortin (POMC)-derived peptides, the N-terminal peptide of POMC (NPP), joining peptide (JP) and adrenocorticotropic hormone (ACTH), were released by the bullfrog (Rana catesbeiana) anterior pituitary. The effects of these three peptides on luteinizing hormone (LH) release by bullfrog dispersed anterior pituitary cells were studied. NPP and ACTH, but not JP, enhanced LH release concentration-dependently. Approximately 6 hr elapsed before the gonadotrophs responded to NPP and ACTH by releasing LH, whereas their response to human GnRH (hGnRH) was faster, suggesting that the modes of action of these two peptides and hGnRH differ. These results raise the possibility that NPP and ACTH act as paracrine factors in the bullfrog pituitary to enhance LH release either directly or indirectly.

Corticotropin-releasing hormone (CRH) is an important regulator of adrenocorticotropin (ACTH) secretion from pituitary corticotroph cells. The intracellular signaling system that underlies this process involves modulation of voltage-sensitive Ca2+ channel activity, which leads to the generation of Ca2+ action potentials and influx of Ca2+. However, the mechanisms by which Ca2+ channel activity is modulated in corticotrophs are not currently known. We investigated this process in a Hodgkin-Huxley-type mathematical model of corticotroph plasma membrane electrical responses. We found that an increase in the L-type Ca2+ current was sufficient to generate action potentials from a previously resting state of the model. The increase in the L-type current could be elicited by either a shift in the voltage dependence of the current toward more negative potentials, or by an increase in the conductance of the current. Although either of these mechanisms is potentially responsible for the generation of action potentials, previous experimental evidence favors the former mechanism, with the magnitude of the shift required being consistent with the experimental findings. The model also shows that the T-type Ca2+ current plays a role in setting the excitability of the plasma membrane, but does not appear to contribute in a dynamic manner to action potential generation. Inhibition of a K+ conductance that is active at rest also affects the excitability of the plasma membrane.

The effects of a 7-day administration of neuromedin B (NMB) and/or (Tyr4, D-Phe12)-bombesin, an NMB-receptor antagonist (NMB-A) on the function of pituitary-adrenocortical axis were investigated in the rat. NMB raised the plasma concentration of aldosterone, without affecting that of ACTH or corticosterone; the simultaneous administration of NMB-A prevented the effect of NMB. Neither NMB nor NMB-A treatments induced significant changes in adenohypophysis and adrenal weights, nor in the average volume of zona glomerulosa and zona reticularis cells. NMB-A administration lowered the volume of zona fasciculata cells, an effect annulled by the concomitant NMB administration. Our results suggest that NMB specifically stimulates aldosterone secretion, and that endogenous NMB or NMB-like peptides exert a tonic stimulating action on the growth of zona fasciculata cells.

The alpha 2A-adrenergic receptor (alpha 2AAR), via its interaction with the pertussis toxin-sensitive Gi/G(o) class of G proteins, modulates multiple effector systems, including inhibition of adenylyl cyclase and Ca2+ channels and activation of K+ channels. Mutation of a membrane-embedded aspartate residue, highly conserved among G protein-coupled receptors, in the alpha 2AAR to asparagine (D79N alpha 2AAR) results in selective uncoupling of the receptor to K+ currents but retention of inhibition of cAMP production and of voltage-sensitive Ca2+ currents when expressed in AtT20 anterior pituitary cells in culture. It is known that attenuation of cAMP synthesis alone cannot account for alpha 2AAR suppression of stimulus-secretion coupling; thus, the D79N alpha 2AAR provides a unique tool with which to assess the relative contribution of K+ current activation and Ca2+ current suppression in mediating the cellular responses of alpha 2AAR. The wild-type alpha 2AAR suppresses basal and secretagogue-evoked adrenocorticotropic hormone (ACTH) release in a manner indistinguishable from response to the endogenous somatostatin receptor. In contrast, the D79N alpha 2AAR does not attenuate basal ACTH release and is only partially effective in suppressing ACTH secretion evoked by the secretagogue isoproterenol. Regulation of ACTH release evoked by 8-bromo-cAMP, which bypasses receptor regulation of cAMP synthesis, suggests that attenuation of cAMP production, although not sufficient for inhibition of ACTH secretion, nevertheless participates in a functionally relevant manner. Taken together, the present findings indicate that alpha 2AAR-mediated suppression of neuropeptide secretion requires concomitant regulation of K+ and Ca2+ currents in parallel with attenuation of cAMP production.

CRH induces corticotrope membrane depolarization and facilitates action potential firing. The increase in electrical excitability causes large oscillatory increases in cytosolic Ca2+ levels. In this study on highly enriched populations of cultured rat corticotropes, inhibitors were used to determine the contribution of the Na+ channel and Ca2+ channel subtypes to membrane excitability and cytosolic Ca2+ levels. Tetrodotoxin, an inhibitor of the voltage-dependent Na+ channel, inhibited a rapid initial component of the action potential, but generally did not influence spontaneous or CRH-induced firing frequency. Tetrodotoxin also had no effect on spontaneous or CRH-induced cytosolic Ca2+ levels. The L-type Ca2+ channel inhibitor nifedipine abolished spontaneous and CRH-induced action potentials and cytosolic Ca2+ transients, but did not eliminate the CRH-induced membrane depolarization or completely restore cytosolic Ca2+ to basal levels. Inhibition of P-type Ca2+ channels with omega-agatoxin-IVA decreased action potential firing frequency and reduced the CRH-induced increase in cytosolic Ca2+. The combination of nifedipine and omega-agatoxin-IVA abolished the CRH-induced rise in Ca2+, but did not abolish the membrane depolarization. Thus, cytosolic Ca2+ is mainly increased by CRH-induced action potentials that are completely dependent on L-type Ca2+ channels and partially regulated by P-type Ca2+ channels. CRH-induced Ca2+ entry also occurs independently of action potentials and is due to P-type, and possibly L-type, Ca2+ channels activated by the CRH-induced membrane depolarization.

The effect of chronic treatment with recombinant methionyl bovine somatotropin (USAN, sometribove) on anterior pituitary secretions and its target organs was investigated in six control and six sometribove-treated British Friesian cows. Cows averaged 112 and 119 d postpartum in their fourth lactation of treatment and, except for one control, had active corpora lutea. During each lactation, treated cows received sometribove injections (500 mg) every 2 wk (injection cycle) starting 60 +/- 3 d postpartum. On Day 9 of one injection cycle, blood was sampled for 390 min, starting 30 min before an intravenous injection of thyrotropin (TRH, 0.33 microgram/kg), corticotropin (100 microgram), and gonadotropin (GnRH, 200 micrograms)-releasing hormones. Baseline somatotropin (bST) and adrenocorticotropin (ACTH) were higher in sometribove-treated cows vs. controls (3.27 vs. 1.03 ng/ml and 35.24 vs. 19.28 pg/ml, respectively). Baseline total thyroxine, free thyroxine, triiodothyronine, prolactin, follicle stimulating and luteinizing hormones, estradiol, and progesterone (P4) were similar across treatments. Circulating cortisol levels did not differ between control and sometribove cows, indicating a reduced adrenal ACTH responsiveness in the latter. Releasing factors induced similar changes across treatments in hormones studied with the following exceptions: a bST spike was seen in control cows only, cortisol response to ACTH was reduced in treated cows, and a significantly higher P4 concentration was detected in the plasma of sometribove-treated cows, suggesting increased ovarian responsiveness to GnRH-stimulated P4 output. The study demonstrated reduced bST response to TRH, consistent with physiologic feedback mechanisms, whereas the release profiles of the other pituitary hormones were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous work showed the existence of receptors for arginine vasopressin (AVP) in the anterior pituitary; these receptors were classified as belonging to a distinct AVP receptor subtype, referred to as AVP-V1b receptors, and are thought to mediate the well documented ACTH-releasing activity of AVP. In the present work, high affinity receptors for another neurohypophyseal hormone, oxytocin (OT), were also shown to be present within the rat anterior pituitary; to this end, [125I]d(CH2)5[Tyr(Me)2Thr4Tyr-NH2(9)]OVT was used as a ligand in receptor binding studies. Experiments on dispersed rat anterior pituitary cells in a superfusion system confirmed earlier reports that OT acts as an additional secretagogue of ACTH, with significant effects first seen at concentrations as low as 1 nM. Further studies addressed the question of whether stimulation of ACTH release is mediated by OT receptors or whether receptors for AVP (V1b receptors) might serve this role. For this, highly selective agonist and antagonist ligands of the OT receptor and nonselective agonist and antagonist ligands of the V1b receptor were employed. Neither the OT receptor agonist Thr4Gly7OT nor the OT receptor antagonist des-Gly(NH2)9d(CH2)5-[Tyr(Me)2 Thr4]OVT displayed any influence on basal ACTH release, and des-Gly(NH2)9d(CH2)5-[Tyr(Me)2Thr4]OVT did not interfere with OT-induced ACTH release; these results indicated that OT promotes ACTH release through a receptor(s) other than the OT receptor itself. Evidence for the involvement of AVP V1b receptors was provided by the observation that the AVP receptor antagonist dP[Tyr(Me2)]AVP completely abolished OT-elicited increases in ACTH release. Thus, AVP V1b receptors mediate the actions of two structurally related peptides on ACTH secretion; the role of OT receptors in adenohypophyseal function remains to be determined.

Arachidonic acid metabolites have been implicated in the regulation of ACTH secretion. To define further which eicosanoid(s) is primarily involved, we examined the effects of both inhibitors of the three arachidonate metabolic pathways (cyclooxygenase, lipoxygenase, and epoxygenase) and specific eicosanoid products on ACTH secretion by rat pituitary corticotrophs in a microperifusion system. CRF stimulates sustained ACTH release that is mediated by protein kinase-A-induced extracellular Ca2+ (Cae2+) influx via L-type voltage-sensitive calcium channels (VSCC). Arginine vasopressin (AVP) stimulates an initial spike phase of ACTH release that presumably is mediated by inositol 1,4,5-trisphosphate-induced intracellular Ca2+ (Cai2+) release, followed by a sustained plateau phase of ACTH release that is mediated by protein kinase-C-induced Cae2+ influx via L-type VSCC. Pretreatment for 15 min with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 microM), but not the cyclooxygenase inhibitor indomethacin (10 microM) or the epoxygenase inhibitor SKF525A (100 microM) inhibited the sustained response to CRF by 48% and the initial spike response to AVP by 38%. NDGA-induced inhibition was not reversed by indomethacin or SKF525A, alone or in combination, precluding arachidonate shunting into other pathways. However, the results suggested that epoxygenase metabolites may have a minor stimulatory and cyclooxygenase metabolites may have a minor inhibitory effect on ACTH secretion. Preexposure to NDGA suppressed by 43% the sustained response to 8-bromo-cAMP, which directly activates protein kinase-A; by 57% the sustained response to dioctanolglycerol, which directly activates protein kinase-C; and by 59% the spike-type response to ionomycin, which releases Cai2+ by an inositol 1,4,5-trisphosphate-independent mechanism. These results suggest that NDGA either inhibits the production of a lipoxygenase metabolite involved in Cae2+ influx and/or Cai2+ release or acts other than by inhibiting lipoxygenase, such as by directly blocking membrane transport of Cae2+. The three major lipoxygenase metabolites tested, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acid (HETE), all stimulated sustained ACTH release in a dose-dependent manner. At a concentration of 2 microM, 12(S)-HETE was 4.7 and 2.5 times more potent than 5(S)- and 15(S)-HETE, respectively, and completely reversed NDGA inhibition of both CRF- and AVP-stimulated ACTH secretion. The ACTH-releasing activity of 12(S)-HETE was inhibited 26% by removing Cae2+ and 54% by both removing Cae2+ and depleting Cai2+, indicating either that 12(S)-HETE facilitates transmembrane Ca2+ transfer or that increased cytosolic Ca2+ is necessary for 12(S)-HETE's action.(ABSTRACT TRUNCATED AT 400 WORDS)

Joining Peptide (JP) is a 30 amino acid fragment separating the N-terminal peptide and ACTH within their common polypeptide precursor POMC. Using antibody Jamie directed against the C-terminal amidated residue Glu-NH2 we studied the molecular weight forms and the variations of plasma immunoreactive (IR)-JP in man under various physiological, pharmacological, and pathological conditions. In 21 plasma samples from patients with ACTH hypersecretory syndromes from pituitary and nonpituitary tumors, IR-JP had the same elution pattern on Sephadex G-75 showing a predominant, if not single, peak corresponding to a mol wt of 7000 as expected for a JP-homodimer. Normal male volunteers at 0800 h had plasma IR-JP values ranging from undetectable (< 6 pmol/L) to 28 pmol/L; all values were suppressed by the overnight 1 mg dexamethasone test. Plasma IR-JP had circadian variations and responded to the metyrapone test in a manner strictly similar to that of ACTH and lipotropins (LPHs). One hundred and fifteen plasma samples covering a large range of pathological ACTH values (from 10(0) to 10(4) pmol/L) were also assayed by the JP and LPH RIAs. All three immunoreactivities strongly correlated with each other with a molar ratio close to 1:1. Discrepancies were observed in two situations where both IR-JP and IR-LPH were much higher than ACTH: they occurred in some patients with the ectopic ACTH syndrome and in all patients with chronic renal failure; they are explained by the further degradation of ACTH into corticotropin-like intermediary lobe peptide in the first case, by the prolonged plasma half-life of JP and LPH, compared to that of ACTH, in the second case. These data show that JP is a normal end-product of POMC processing in man which circulates in blood mainly as a homodimer. It provides yet another marker of the overall corticotroph function and may be used to unravel abnormal POMC processing in some nonpituitary tumors.

1. The present studies were undertaken to determine the effects of arginine vasopressin (AVP), corticotrophin-releasing factor (CRF) and AVP in combination with CRF on proopiomelanocortin (POMC) gene expression, and in the release and biosynthesis of POMC products (i.e. ACTH). 2. After a 3 hr treatment, AVP (10(-7) M), CRF (10(-7) M) and AVP (10(-7) M) in combination with CRF (10(-7) M) stimulated ACTH release by 291 +/- 19.2%, 377.4 +/- 25.6% and 462.1 +/- 38.4% (P < 0.01), respectively, with respect to basal secretion; while ACTH content diminished by 84.0 +/- 4.6%, 81.3 +/- 2.1% and 71.0 +/- 1.5% (P < 0.01), respectively, with respect to basal. Total POMC mRNA levels were not affected after a 3 hr treatment. 3. When cells were treated with a wide range of AVP concentrations (10(-11)-10(-7) M) to which we added different concentrations of CRF, modulation of AVP-induced ACTH release was most effective at CRF concentrations of < 10(-10) M. 4. Prolonged (3-6 hr) exposure to as low as 10(-10) M CRF or 10(-9) M AVP resulted in homologous desensitization of ACTH secretion. However, these pretreated cells were able to respond to a further challenge of CRF and AVP. This paper provides new information on the dose ranges over which desensitization and cross-sensitization between CRF and AVP secretory effects take place in mouse anterior pituitary cells.

1. During spring, decreased sensitivity of the adrenal cortex to adrenocorticotrophic hormone (ACTH) in the sand rat inhabiting the Béni-Abbès area (Algeria), results in a reduction in the production of cortisol. Thus the secretion of ACTH is enhanced, becoming maximal in June and presumably also during the following weeks. 2. Increase in ACTH secretion, together with a slightly increased adrenal sensitivity, is likely to stimulate corticosteroidogenesis throughout the summer. 3. In autumn, as levels of cortisol are high, the negative feedback increases leading to a reduction in ACTH production. 4. An increase in the adrenocortical sensitivity to ACTH allows a high production of cortisol until February.

The purpose of this study was to determine whether normal morphological development occurs in pituitary corticotrophs deprived of products of the hypothalamic paraventricular nucleus (PVN), e.g. corticotropin releasing hormone and arginine vasopressin (AVP), after PVN lesions. In addition, we have attempted to ascertain if the neurophysin/AVP-positive fibers innervating the fetal sheep anterior pituitary are affected by PVN lesions. The experimental groups consisted of fetal sheep in which 1) hypothalamic PVN lesions were placed at 118-122 days gestation (dGA) and the fetuses subsequently harvested while still in utero at 157 dGA or more (PVNX; n = 5); 2) sham PVN lesions were placed at 118-122 dGA and subsequently harvested as newborn lambs immediately after birth at 146.5 +/- 0.9 (mean +/- SEM) dGA combined with two uninstrumented fetuses harvested at 144 dGA or more but not in labor (perinatal; n = 6); and 3) no instrumentation was placed, and the fetuses were harvested at 120 dGA (control; n = 4). Two ACTH-immunoreactive cell types were seen in the anterior pituitary: 1) fetal cells: large and variably stained, often columnar, occurring in clusters and arranged in palisades; and 2) adult cells: smaller, darkly staining, and angular, occurring singly or in small groups. Quantification of the distribution of the two ACTH cell types was performed by scanning sections from a one in six series from each pituitary and estimating the percent area of each section in the well that showed adult type staining only. The observer was blind to the treatment group assignment of the sections. The estimated percentages of the portion of the pituitaries of each group that contained adult-type cells only were as follows: PVNX, 42.8 +/- 10.0%; perinatal, 90.9 +/- 2.1%; and control, 3.7 +/- 1.1% (mean +/- SEM; P less than 0.05 for all comparisons). There were no qualitative differences between all groups in the appearance of neurophysin-positive fibers innervating the anterior pituitary. AVP staining was strong in the internal zone of the median eminence in all groups, but was absent in the external zone of PVNX fetuses only. The intermediate pituitary lobes stained darkly in all groups. We conclude that lesions of the PVN at 120 dGA delay development of fetal pituitary corticotrophs, but have no effect on the presence of neurophysin-positive nerve fibers in the anterior pituitary.

Interferon-gamma (IFN-gamma) is known to inhibit the release of ACTH, PRL, and GH by rat anterior pituitary (AP) cells, stimulated by appropriate hypothalamic releasing factors. In the present study we examined the mechanisms underlying this inhibition. Dose-response studies, revealing a maximal inhibitory effect with an IFN-gamma dose as small as 10 U/ml, suggested the existence of a limiting intermediate step. In addition, in perifusion experiments with aggregates of established hormone-secreting tumor cell lines (AtT-20 and GH3), IFN-gamma had no inhibitory effect, suggesting that an accessory cell type was involved. Studies on differentially enriched cell populations of normal rat AP, obtained by velocity and buoyant density sedimentation, indicated that the inhibitory effect of IFN-gamma on stimulated ACTH and GH release was absent in those populations that contained only few folliculo-stellate (FS) cells. The presence of a minimal proportion of FS cells was found to be necessary for the inhibition to be manifest. This was seen in monolayers, but also in cultures of AP cell aggregates, which are well known to closely mimic the behavior of the AP gland in vivo. Definitive evidence for the role of FS cells was obtained by reconstitutive coculture experiments; FS cell-poor populations, which by themselves resisted the inhibitory effect of IFN-gamma, became sensitive when cocultured with an FS cell-rich population. Basal ACTH and GH release were not influenced by preincubation with IFN-gamma in either original or fractionated AP cell populations. In contrast, basal PRL release was inhibited in both systems. In cultures of AP cell populations, separated by velocity sedimentation, this inhibition showed a pattern similar to that observed for stimulated release of ACTH and GH, i.e. more inhibition in fractions with a higher proportion of FS cells. However, in FS cell-poor cultures, inhibition of basal PRL release did occur, although to a lesser degree than in FS cell-rich cultures. Our results indicate that IFN-gamma affects AP hormone secretion through the FS cell. In addition, they suggest that IFN-gamma and the FS cell constitute a system through which the pituitary gland perceives changes in the activation state of the immune system.

Cholinergic mechanisms have been implicated in the regulation of anterior pituitary hormone secretion. The present study was designed to determine the effect of a single injection of an organophosphate acetylcholinesterase inhibitor, diisopropylfluorophosphate (DFP), on anterior pituitary function in male rats. DFP increased serum ACTH (2.7-fold) and corticosterone (9.1-fold), while suppressing TSH, PRL, LH, and GH by up to 95%. The earliest response was at 1 hr, with a duration of at least 18 hr for TSH and LH. Responses were similar in adrenalectomized animals. After DFP, responses to hypothalamic releasing factors were normal for TSH, GH, and ACTH, but significantly blunted for PRL and LH. TSH suppression was partially prevented by combined therapy with a nicotinic (mecamylamine) and a muscarinic (atropine) antagonist. TSH suppression was partially reversed by immunoneutralization with somatostatin antibody, and PRL suppression was completely prevented by a dopamine antagonist (haloperidol). Atropine alone prevented the effects on corticosterone. TSH pituitary content and TSH-beta mRNA were reduced by 37 and 22%, respectively, by DFP. In contrast, PRL mRNA was unchanged but PRL content was increased 3-fold. We conclude that cholinesterase inhibition evokes a multiplicity of effects on anterior pituitary function. There is a hierarchy of responses, with corticosterone being the most and TSH the least sensitive. There is evidence for inhibition at both the hypothalamic and pituitary levels, involving both nicotinic and muscarinic receptors. Although cholinesterase inhibition is the proximate event, other neurotransmitter pathways involved in TSH and PRL suppression are somatostatin and dopamine, respectively.

Although it has been demonstrated that glucocorticoids can inhibit basal and stimulus-induced increase in fetal plasma ACTH concentrations, the site(s) of action of the glucocorticoids in the fetal hypothalamo-pituitary axis are unclear. We have investigated the ontogeny of the negative feedback effect of cortisol on basal and CRF-stimulated ACTH secretion in intact fetal sheep and in fetal sheep after surgical disconnection of the hypothalamus and pituitary (HPD). In saline-pretreated fetal sheep there was a significant increase in plasma ACTH concentrations in response to an ovine CRF (oCRF) bolus (1 microgram) in HPD and intact fetuses at 120-128 days and at 138-149 days gestation. In the intact group the ACTH response at 120-240 min after oCRF was significantly lower (P less than 0.01) after 138 days compared with before 128 days. In this group the cortisol response during the first hour after oCRF was significantly greater (P less than 0.005) after 138 days than in the younger fetuses. In the HPD group there was no significant difference between the two gestational age ranges in either the ACTH or cortisol responses to oCRF. Between 120 and 128 days gestation, increasing plasma cortisol concentrations (110-170 nmol/liter above baseline values) by exogenous infusion had no significant effect on the basal fetal plasma ACTH concentrations in HPD or in intact fetuses. In contrast, after 138 days gestation, the basal plasma ACTH concentrations were significantly decreased by 50% during the cortisol infusion period in the intact but not in the HPD group. It would appear therefore, that the development of the negative feedback action of cortisol to suppress basal fetal plasma concentrations of ACTH is dependent on the presence of a functional fetal hypothalamus. In HPD and intact fetuses cortisol infusion abolished the fetal ACTH response to the oCRF bolus from as early as 120 days gestation. Thus the negative feedback action of cortisol on the CRF-induced increases in fetal plasma ACTH concentrations can occur directly at the fetal pituitary.

The present studies were designed to evaluate the binding characteristics of arginine vasopressin (AVP), using rat anterior pituitary dispersed cells, in correlation with the biological activity of the peptide. Synthetic AVP released ACTH from dispersed anterior pituitary cells in a concentration-dependent fashion, with a minimal effective dose of 10(-10) M. [3H] AVP, the ligand for binding studies, showed full biological activity at different concentrations. Saturable and high affinity binding sites for [3H]AVP were obtained using dispersed anterior pituitary cells. Specific binding reached equilibrium by 180 min at 22 C, and rapid and complete dissociation was obtained after the addition of excess AVP. Scatchard analysis of the data indicated a single class of binding sites, with an apparent Kd of 1.63 nM and a binding capacity (Bmax) of 10.7 fmol/10(6) cells, at 37 C. When cells were incubated at 22 C, Kd (7.63 nM) and Bmax (39 fmol/10(6) cells) values were within the same order of magnitude. AVP effectively inhibited [3H]AVP binding with an IC50 of 1.5 X 10(-7) M, while oxytocin showed a somewhat higher IC50 (0.9 X 10(-6) M) and did not achieve complete inhibition of binding. An AVP analog with a ring substitution ( Asu1 ,6 AVP) showed decreased displacement capacity. Both oxytocin and the AVP analog showed weak ACTH-releasing activity compared to synthetic AVP. This indicates that modifications in the tail and/or ring portion of the AVP molecule reduce both the binding affinity and ACTH-releasing activities of these peptides. Other structurally unrelated peptides with intrinsic ACTH-releasing activity, such as rat corticotropin-releasing factor and angiotensin II, had no affinity for AVP-binding sites. The results indicate that specific AVP-binding sites in anterior pituitary cells are closely correlated with the intrinsic ability of the peptide to elicit ACTH release.

Plasma corticosterone level and corticotropin (ACTH) content of the anterior pituitary (AP) were measured after various cuts around the medial basal hypothalamus (MBH). Seven to eight days after placing a complete cut around MBH there was no change in plasma corticosterone or ACTH content of AP; the slight rise in ACTH concentration was offset by the decreased weight of AP. Twenty eight days after a frontal cut there was no change in plasma corticosterone and in bioactive or immunoreactive ACTH concentration of AP.

Tissue levels of bioactive and immunoactive ACTH were measured in both the anterior and neuro-intermediate lobes of the rat pituitary. Similar concentrations of bioactive (65 ng/mg) and immunoactive (83 ng/mg) ACTH were found in the anterior lobes control rats. A 2-min ether stress had no effect on either bioactive or immunoactive ACTH levels in the anterior lobe. Twenty-four h after adrenalectomy the anterior lobe content of both bioactive and immunoactive ACTH decreased only to return to supranormal levels 21 days after the operation. A 30-min neurogenic stress had no effect on anterior lobe bioactive ACTH content but reduced the immunoactive ACTH level to 50 ng/mg. Synthetic alphah-17-39 ACTH was used in our radio-immunoassay in order to measure the C-terminal ACTH activity of the neuro-intermediate lobe. The concentration of such C-terminal activity in control rats (890 ng alphah-17-39 ACTH/mg) considerably exceeded the amount of bioactive ACTH (15 ng/mg). This is presumably due primarily to the presence of the so-called corticotropin-like intermediate lobe-peptide (CLIP). The amounts of bioactive or C-terminal immunoactive ACTH in the neuro-intermediate lobe were not affected by ether stress nor short term (24-h) or long term (21-day) adrenalectomy. Neuro-intermediate lobe bioactive ACTH decreased (to 8 ng/mg) only with the introduction of a 30-min neurogenic stress. Neurogenic stress had no effect on the concentration of CLIP, but when the stress was imposed 24 h after adrenalectomy, a significant reduction was observed. The data support the presence of bioactive ACTH in the intermediate lobe of the rat pituitary and suggest that such ACTH is preferentially released by neurogenic stress and not appreciably regulated by circulating levels of glucocorticoids. Until the biological function and/or target organ of CLIP is identified, the significance of the changes in tissue levels of C-terminal immunoactive ACTH will remain unknown.

To investigate the possible effect of GABA on the corticotropic system, the potent GABA antagonist picrotoxin was injected into two groups of 14 female rats at 07.00 h and 19.00 h, respectively. A single subconvulsive I.p. injection dramatically raised plasma ACTH and corticosterone, and thereafter suppressed the circadian rhythm of ACTH, but not of corticosterone, for 24 h in the group injected at 07.00 h and for 48 h in the one injected at 19.00 h and increased mean hormonal levels. Results are discussed in the light of the possibility that inhibition by the GABAergic system and stimulation by the serotoninergic system might be components of the mechanism controlling the circadian rhythm of ACTH.

CRF-41 and vasopressin are the major part of the hypothalamic complex responsible for the release of ACTH. The potentiated response of the corticotroph to mixtures of the peptides and their co-localization in the same neurosecretory vesicles would indicate their obligatory co-secretion in many stress situations. The occurrence of CRF at peripheral sites especially in the placenta and in the plasma of women in their third trimester of pregnancy is suggestive of further roles for this peptide although care should be exercised in interpreting simple immunohistochemical staining in certain tissues. When ACTH is released from the pituitary several other peptides which form part of its precursor are co-released. The effect of these peptides on the adrenal gland and the way their activity is expressed has led to a post-secretional proteolytic control mechanism being proposed.

After adrenalectomy, the plasma levels of adrenocorticotropic hormone (corticotropin, ACTH)/endorphin peptides in rats rise dramatically in the first 4 hr while pituitary peptide levels fall sharply. Eight hours after adrenalectomy, plasma levels are near control values again but they then increase continuously over the next 8 days. Proopiomelanocortin (POMC) mRNA levels in the anterior pituitary (quantitated by hybridization with cloned POMC cDNA) increase 2-fold in the first 24 hours, reaching 15- to 20-fold the control level 18 days after adrenalectomy. When dexamethasone is administered to rats 8 days after adrenalectomy, the above events are reversed. Plasma ACTH falls to control levels within 2 hr whereas anterior pituitary POMC mRNA requires 5 days of treatment for return to control levels. The levels of POMC mRNA in the neurointermediate lobe and the hypothalamus are not altered by either treatment. Adrenalectomy increases transcription of the POMC gene in the anterior pituitary approximately 20-fold and halves transcription of the growth hormone gene within 1 hr of operation. Administration of dexamethasone immediately after adrenalectomy suppresses the increase in transcription of the POMC gene and increases the transcription of the growth hormone gene. Transcription of the POMC gene(s) in the neurointermediate lobe is not altered by either of these treatments.

The fine structural characteristics of normal rat corticotrophs stained with anti-porcine ACTH1-39 serum were studied. At the ultrastructure level immunoreactive corticotrophs appear to comprise four distinct cell types: (1) large stellate cells (Siperstein cells) containing granules (170-250 nm in diameter) arranged in a peripheral row and usually embracing an acidophil; (2) elongate spindle-shaped cells (Moriarty cells) in which the secretory granules (170-250 nm in diameter) are distributed in a row or in small clusters in the peripheral cytoplasm: (3) oval or polygonal cells filled only with small secretory granules (130-170 nm in diameter), resembling the "acidophil of small granules type" (Yoshimura et al. 1974); and (4) polygonal or stellate cells filled with secretory granules of varying diameters (180-300 nm in diameter) and occasionally embracing an acidophil. The first type is the most common, but the others are infrequent. It is concluded that the criteria of Siperstein and Miller (1970) do not necessarily include all categories of rat corticotrophs.

High molecular weight ACTH fractions, obtained through gel filtration of boiled rat anterior pituitary extract, induced a marked increase in corticosterone production from isolated rat adrenal cells in the presence of low concentrations of ACTH-(1-24). This indicates the presence of heat-stable factors augmenting the steroidogenic action of ACTH in the rat anterior pituitary. We also noted that these factors potentiated the activity not only of ACTH-1(1-24) but also of ACTH-(1-8). The ACTH-potentiating factors in rat anterior pituitary extract are possibly present in heterogeneous forms according to their molecular weights (8,000, 10,000 and 15,000), their mobility in ion-exchange chromatography and their content in RIA-ACTH activity. Of these three forms, the former comigrated with biological ACTH activity. The remaining two forms were free of it. Since the effect of potentiating factors on modified ACTH-(1-9), shown to be less susceptible to proteolytic degradation from ACTH-(1-24), was similar to the effect on ACTH-(1-24), it is suggested that potentiation was not due to an inhibition of ACTH proteolysis.

The effects of adrenocortical and gonadal steroids on the secretion in vitro of ACTH by adenohypophysial segments and corticotrophin releasing factor (CRF) by isolated hypothalami were studied in the rat. Corticosterone (1.25 X 10(-6) mol/l), betamethasone (2.5 X 10(-8) mol/l) and progesterone (2.5 X 10(-7) mol/l) reduced the hypothalamic extract-induced secretion of ACTH by pituitary tissue in vitro but aldosterone (2 X 10(-7) mol/l), testosterone, androsterone, androstenedione (2 x 10(-7) mol/l), oestradiol, oestriol, and oestrone (10(-6) mol/l) did not. Corticosterone (2.5 +/- 10(-9) mol/l), aldosterone (2 X 10(-8) mol/l) and betamethasone (2 X 10(-10) mol/l) inhibited and oestradiol, oestriol and oestrone (10(-8) - 10(-6) mol/l) potentiated the production of CRF by isolated hypothalami which occurred when acetylcholine or 5-hydroxytryptamine were added to the incubation medium but progesterone (2.5 X 10(-7) mol/l), testosterone, androsterone and androstenedione (2 X 10(-7) mol/l) had no effects. The results indicate that hypothalamo-pituitary-adrenocorticotrophic activity may be modified not only by glucocorticoids but also by other steroids.

A biotin-conjugated synthetic corticotropin releasing factor (B-CRF) was prepared and characterized. Its biological activity and binding affinity were compared with that of unlabeled synthetic CRF. Both forms of the releasing factor were equipotent in in vitro studies measuring the release of corticotropin (ACTH) (ED50 = 1 nM). The IC50 in the binding assays was 1.5 nM for CRF and 4 nM for B-CRF. Dual avidin-biotin peroxidase complex stains were then used in pituitary monolayer cultures to visualize receptivity to the releasing factor and to confirm opiocortin storage in the target cells. All corticotropes showed stain for B-CRF. The percentage of cells that were double-labeled for ACTH and CRF increased with the dose of B-CRF during a four hour incubation period. The CRF stain was abolished, however, when an excess of unlabeled CRF was added to compete with B-CRF. The distribution of the B-CRF and ACTH stains varied in the cells with the time of exposure to the analog. These studies show that biotin-conjugate CRF is a potent analog that can be demonstrated cytochemically on cells identified immunocytochemically as corticotropes. It can be used to follow important events associated with CRF stimulation including the rapid internalization of CRF coupled with the mobilization of corticotropin stores and the formation of cellular processes.

The aim of the study was to investigate, by coupled immunocytochemical and stereologic methods, the effects of long-term intraventricular administration of ovine CRF (oCRF) on ACTH-immunoreactive cells of the rat anterior pituitary and to compare this effect with that of adrenalectomy. CRF-treated rats received 100 ng of oCRF daily for 8 days while control rats were administered with bovine serum albumin (BSA). After BSA administration number of ACTH-immunoreactive cells/mm2 was greater than in intact rats and CRF lowered their number, however, they were still more numerous than in intact rats. Neither average area nor average volume of studied cells were changed in control and CRF-treated animals if compared with intact ones. The total number of ACTH-immunoreactive cells was higher in the glands of BSA and CRF-treated rats than in intact animals. On the other hand, 60 h after bilateral adrenalectomy a marked increase in both, average area and average volume of ACTH-immunoreactive cells was found. The different response of rat corticotropes to intraventricular CRF administration and adrenalectomy may depend on the presence of operating negative corticosterone feedback in animals with adrenal glands intact. Moreover, the present study revealed the lack of correlation between the average area and average volume of ACTH-immunoreactive cells and the weight of the adrenal gland.

Previous work in our laboratory has shown that stimulation of beta 2-adrenergic receptors on mouse anterior pituitary tumor cells causes the secretion of immunoreactive adrenocorticotropin (ACTH). The present study was designed to test the hypothesis that catecholamines can cause the release of ACTH in vivo by the direct stimulation of beta 2-adrenergic receptors in the rat anterior pituitary. Systemic administration of a beta-adrenergic receptor agonist (-)-isoproterenol resulted in an increase in plasma ACTH levels in intact animals and in rats with transected pituitary stalks. This effect could be blocked by the beta-adrenergic receptor antagonist, propranolol, but not by the specific beta 1-adrenergic receptor antagonist, practolol. Salmefamol, a beta 2-adrenergic receptor agonist also elevated plasma ACTH levels in stalk-sectioned animals. Dexamethasone, a glucocorticoid that inhibits the synthesis and release of ACTH from the anterior pituitary but not the intermediate lobe, prevented the elevation of ACTH secretion by (-)-isoproterenol in stalk-transected rats. These data indicate that beta 2-adrenergic receptors are present on anterior pituitary cells and suggest that catecholamines can directly stimulate ACTH secretion.

Recent electrophysiological studies from this laboratory demonstrated that anterior lobe corticotropes exhibited a tetrodotoxin-sensitive sodium current and two types of voltage-dependent calcium currents, consisting of low threshold (transient) and high threshold (long lasting) components. The present report describes cytophysiological and cytochemical studies that used specific blockers of each of these currents to assess their role in the regulation of CRF binding and ACTH secretion and storage. Two dihydropyridines, nimodipine and the pure antagonist enantiomer (-)R202-791, which block high threshold Ca2+ channels, decreased 1 h basal release by 54-74% and CRF-mediated (5 min or 3 h) release completely. Percentages of CRF-bound cells were reduced as much as 74%; however, the inhibitory effect on percentages of CRF-bound cells could be reversed by adding 10 nM Bay K 8644, (a pure dihydropyridine agonist) with the antagonists. CdCl2, which blocks both high and low threshold calcium currents, inhibited basal and CRF-stimulated ACTH release, but only the highest concentration (0.1 mM) reduced percentages of CRF-bound cells. Involvement of the low threshold Ca2+ channels could not be proved by adding dihydropyridine antagonists with 0.1 mM CdCl2. Basal and CRF-mediated ACTH release were blocked by the potent sodium channel blocker tetrodotoxin, and the highest concentration (3 microM) reduced percentages of CRF-bound cells. Basal (1 h) and CRF-stimulated (5 min) ACTH release were also inhibited in medium containing 1 mM EGTA and no Ca2+; however, percentages of CRF-bound cells were within the normal range. Densitometric analysis of stains for ACTH showed an increase in the concentration of stain per cell after a 1-h exposure to the highest concentrations of the inhibitors or to no Ca2+ and 1 mM EGTA coupled with a significant (10%) decrease in corticotrope cell area. Finally, in the last series of tests, the Bay K 8644 agonist or arginine vasopressin were used to study mechanisms of augmentation of basal or CRF-mediated ACTH release. Bay K 8644 augmented basal release in a concentration of 1 microM and CRF-mediated release in a concentration of 100 nM or 1 microM. After pretreatment with either Bay K 8644 or arginine vasopressin (10 nM) there was a significant (30%) increase in the percentage of CRF-bound cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The forms of immunoreactive beta-endorphin-sized material in extracts of anterior and intermediate-posterior pituitary from the rat examined by the ion exchange chromatography method of Zakarian & Smyth. The anterior pituitary primarily contained material that co-migrated with synthetic camel beta-endorphin(1-31), whereas the intermediate-posterior pituitary contained relatively little such material. The majority of immunoactive beta-endorphin-sized peptides in the intermediate pituitary eluted at lower concentrations of NaCl than did camel beta-endorphin. Conditions were developed for the stable, long-term tissue culture of dissociated intermediate-posterior pituitary cells. Extracts of cells maintained in tissue culture for 18 h or nine days had the same content of immunoreactive beta-endorphin, 16k fragment, ACTH(18-39) (or CLIP) and ACTH(17-24). Throughout the nine days in culture, characteristic cells that could be immunostained with antibodies to various regions of pro-ACTH/endorphin were present; during the time in culture, non-reactive background cells multiplied rapidly. The major proteolytic processing of pro-ACTH/endorphin remained characteristic of intermediate pituitary tissue throughout the nine days in tissue culture, and did not become similar to the simpler pattern of proteolytic processing found in the anterior pituitary.

Attempts have been made to establish whether the immunoreactive corticotrophin (IR-ACTH) reserve was impaired in old age during surgical stress and whether an exhaustion of the IR-ACTH reserve could be traced postoperatively. Recognition of the degenerative changes of the senescent adenohypophysis has made this investigation essential in an effort to analyze factors responsible for the high risk involved in surgery on the aged. In the study 18 young and 14 elderly patients were subjected to elective abdominal or thoracic surgery, and the IR-ACTH response was determined. No significant age-related difference in response to surgery was found. On the 5th day after operation an intravenous metyrapone test was carried out, and the IR-ACTH response in plasma determined. In the elderly, the IR-ACTH response to metyrapone was not found to be inferior to that in the young patients. It was concluded that the IR-ACTH reserve was unimpaired during surgery in old age and that exhaustion was not in evidence based on unimpaired IR-ACTH response during repeated stress in the postoperative period. Therefore, decreased ACTH reserve seems not to be a factor involved in the higher surgical risk in the elderly.

AtT-20 cells comprise a mouse anterior pituitary tumor cell line that synthesizes and secretes adrenocorticotropin hormone (ACTH). beta-Adrenergic receptors were characterized on AtT-20 cells using receptor binding methodology and the ability of beta-receptor agonists to stimulate intracellular cyclic adenosine 3':5'-monophosphate (cAMP) formation and the release of ACTH immunoreactivity. The density of beta-receptors on membrane preparations of these cells is 64 fmol/mg of protein and their affinity constant (KD value) for tritiated dihydroalprenolol is 11 nM. The binding of [3H] dihydroalprenolol to AtT-20 cells is stereoselectively inhibited by propranolol and isoproterenol but is not affected by phentolamine. The beta-receptors on these cells appear to be of the beta 2-receptor subtype since a selective beta 2-receptor agonist, salmefamol, can inhibit [3H]dihydroalprenolol binding, whereas practolol, a beta 1-receptor blocker, is ineffective. (-)-Isoproterenol stimulates cAMP formation in AtT-20 cells and this effect is blocked by dl-propranolol. Both l-epinephrine and l-norepinephrine induce dose-dependent increases in cAMP formation with the former agonist being more potent. Salmefamol also stimulates cAMP formation in these cells. The secretion of ACTH from AtT-20 cells is induced by (-)-isoproterenol as well as by other adrenergic agonists. The isoproterenol effect on ACTH release is stereoselective, calcium dependent, and blocked by dl-propranolol but not by phentolamine or practolol.

The effects of selective agonists and antagonists of type 1 (V1) and type 2 (V2) vasopressin receptors on the secretion of ACTH in vitro by segments of adenohypophysial tissue and in vivo in rats pretreated with pentobarbitone and chlorpromazine were studied in the presence and absence of the 41 amino acid-containing peptide, corticotrophin-releasing factor-41 (CRF-41). The non-selective vasopressin receptor agonist, arginine vasopressin (AVP) and the V1-receptor agonist, felypressin caused dose-related increases in ACTH release in vivo and in vitro but the V2-receptor agonist, desmopressin was only weakly active in this respect. Their actions in vitro were antagonized competitively by the V1-receptor antagonist, d(C2H5)2-AVP, but were unaffected by the V2-receptor antagonist, d(CH2)5-D-Iso2-Thr4-AVP. Arginine vasopressin, felypressin and desmopressins in concentrations considerably lower than those necessary to elicit directly the release of ACTH, potentiated, in a dose-related manner, the activity of CRF-41 in vitro. The potentiating effects were not antagonized by the V2-receptor antagonist or by low concentrations of the V1-receptor antagonist. At a higher concentration, the V1-receptor antagonist reduced, but did not abolish, the potentiating effects of AVP and its analogues. However, at this concentration, it also exhibited weak intrinsic activity and, like the agonists, potentiated the response to CRF-41. The results suggest that the direct effect of AVP on ACTH release is mediated by V1-like receptors. The vasopressin receptors involved in the potentiation of CRF-41 activity appear to be different.

A multi-column perifusion system was used to investigate the dynamics of the dose-response relationships of ACTH release by ovine pituitary cells when stimulated by both corticotrophin-releasing hormone (CRF) and arginine vasopressin (AVP) given alone and in combination. A dose-response relationship was obtained when 10-min pulses were given at 60-min intervals over the range of 0.002-2000 nmol CRF/1 and 1-2000 nmol AVP/1, with a minimum effective concentration of 0.02 nmol CRF/1 or 1 nmol AVP/1. When AVP was given together with CRF, the expected potentiation of the ACTH response occurred when compared with the summed response of these secretagogues given separately. At the higher concentrations of CRF and AVP used, the ACTH responses to repeated pulses decreased with time during the experiment. The rate of this loss of responsiveness was significantly correlated to the size of the response to the first pulse (for CRF: r = 0.89, P less than 0.01; for AVP: r = 0.95, P less than 0.01), being greatest when the response was potentiated by adding the secretagogues together (for CRF plus AVP: r = 0.95, P less than 0.01). Reduced availability of receptors or changes in intracellular transduction processes may contribute to this desensitization. Reduced levels of secretable ACTH do not appear to be implicated because desensitization to pulses of one secretagogue did not cause equivalent desensitization to the other. In addition, cells stimulated continuously with submaximal levels of either secretagogue showed desensitization while more ACTH was still available for release to higher levels of stimulant.(ABSTRACT TRUNCATED AT 250 WORDS)