PubMed Health⌕ Search

Biomedical subjects

G Aguilera

Publications and source records attributed to G Aguilera.

At least 127 records · Page 7Linked to original sources
PubMed1987

Involvement of vasopressin in the down-regulation of pituitary corticotropin-releasing factor receptors after adrenalectomy.

The role of vasopressin (VP) in the regulation of pituitary corticotropin-releasing factor (CRF) receptors was studied by examining the effects of adrenalectomy and VP infusion on pituitary CRF receptors in genetically VP-deficient rats (di/di) and Long-Evans control rats. Binding studies with [125I]Tyr-ovine CRF in 30,000 X g anterior pituitary membrane-rich fractions revealed similar characteristics for the CRF receptors in Long-Evans and di/di rats, with Kd values of 2.4 +/- 0.6 and 1.9 +/- 0.2 nM, respectively, and receptor concentrations of 278 +/- 31 and 286 +/- 43 fmol/mg, respectively. Two days after adrenalectomy, the pituitary CRF receptor concentration decreased by 72 +/- 4.2% in Long-Evans rats, but by only 20.3 +/- 5.6% in di/di rats. CRF receptor affinity was unchanged after adrenalectomy (Kd = 1.7 +/- 0.5 nM; n = 8). To determine whether VP deficiency is responsible for the smaller decrease in CRF receptor in di/di rats, the effect of exogenous VP infusion (100 ng/min) by sc osmotic minipumps was studied in adrenalectomized di/di rats. Two days after adrenalectomy, pituitary CRF receptors were reduced by 21 +/- 8% in control di/di rats, whereas a 77.7 +/- 1.8% decrease was observed in VP-infused di/di rats, comparable to the effect of adrenalectomy in Long-Evans rats. VP infusion also caused a significant 35 +/- 2% decrease in CRF receptors in the pituitaries of sham-operated di/di rats, with no change in CRF receptor affinity. In Sprague-Dawley rats, VP or CRF infusion (100 ng/min) decreased pituitary CRF receptors by 14 +/- 1.9% and 46 +/- 3%, respectively. However, the combined infusion of both peptides caused a 65% +/- 4.2 decrease, similar to that observed after adrenalectomy. In vitro incubation of quartered pituitaries with VP or CRF for 4 h reduced CRF receptors by 23.1 +/- 8.2% and 38.2 +/- 3.8%, respectively, while simultaneous preincubation with both peptides was followed by a decrease of 55.3 +/- 5.3%. These findings indicate that increased hypothalamic release of VP contributes to the down-regulation of pituitary CRF receptors after adrenalectomy.

Adrenalectomy↗
PubMed1987

Receptors and actions of corticotropin-releasing hormone in the primate pituitary gland.

Receptors for CRH were identified in the pituitary gland of several primate species, and their binding characteristics were compared to the ability of CRH to elicit ACTH and cAMP responses in vitro. Autoradiographic analysis of the binding of [125I]Tyr-ovine CRH to frozen pituitary sections revealed CRH receptors in the intermediate and anterior lobes of human, marmoset, and cynomolgus monkey pituitaries. In the cynomolgus monkey, a high density of CRH receptors was present throughout the anterior and intermediate lobes. In the human pituitary, binding was concentrated in the anteromedial portion of the gland, whereas in the marmoset, binding was dense in the intermediate lobe and scattered as clusters throughout the anterior lobe. In membrane-rich fractions from the cynomolgus pituitary binding of [125I]Tyr-ovine CRH was time and temperature dependent, and was specific for CRH-related peptides; specific binding was increased by divalent cations and inhibited by guanyl nucleotides. Scatchard analyses of the binding data revealed a single class of high affinity sites [Kd, 1.93 +/- 0.23 (+/- SEM) nM], with a receptor concentration of 605 +/- 121 fmol/mg. In marmoset pituitary membranes, there were fewer receptors (200 +/- 15 fmol/mg), in agreement with the lower autoradiographic density of CRH binding. In anterior pituitary cell cultures from cynomolgus monkeys, CRH caused a dose-dependent stimulation of cAMP production and ACTH release, with half-maximum effective concentrations in the range of the CRH receptor affinity. Vasopressin and norepinephrine stimulated ACTH release to a much lesser extent, but both potentiated the stimulatory effect of CRH. Angiotensin II had no effect alone, but it also potentiated the effect of CRH. These data demonstrate the presence of CRH receptors in the primate pituitary, with characteristics similar to those in other species in their binding properties, coupling to adenylate cyclase, and functional interactions with other regulators of ACTH secretion that mediate the stimulatory effect of the peptide in the corticotroph.

Adrenocorticotropic Hormone↗
PubMed1987

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors.

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.

Adrenal Cortex↗
PubMed1987

Brain receptors for hypothalamic hormones.

Angiotensin II and CRF are but two of the several regulatory peptides which exert specific actions in the brain that are complementary with their peripheral effects upon end organs such as the anterior pituitary and adrenal glands. In the pituitary, the two peptides act in a coordinate manner on the corticotroph to regulate ACTH release. In the adrenal gland, angiotensin II receptors are abundant in the zona glomerulosa but are also present in the medulla, where the occurrence of CRF receptors and actions on catecholamine release reveals an additional site at which the two peptides exert related actions, in this case in the peripheral neuroendocrine system. Within the brain, the mapping of AII and CRF binding sites by topical autoradiography has provided new information about the distribution and potential functions of receptors for the two peptides. The central receptors for AII are distributed in a characteristic pattern in brain regions concerned with drinking, regulation of adrenergic function and arterial blood pressure, and control of pituitary hormone secretion. Thus, in addition to its recognized modulatory effects in the peripheral adrenergic system, angiotensin II may be involved in the central control of catecholamine release and action. A central action of AII on the release of regulatory peptides such as vasopressin and CRF, both of which are present in neurones of the paraventricular nucleus, is indicated by the high concentration of AII receptors in this region. Also, the high density of AII receptors in the median eminence suggests that AII modulates the hypothalamic secretion of neuropeptides such as CRF by actions at their site of release, as well as on the cell bodies of neurones responsible for peptide synthesis. The highly localized pattern of AII receptors at numerous specific sites in the brain differs from the more general distribution of many other CNS receptors, and reflects the selective actions of AII on discrete neural systems that subserve precisely integrated functions within the central nervous system. The widespread distribution of CRF receptors, with prominent localization in the cortical and limbic regions, is consistent with the more general neuroregulatory actions of CRF in the brain, and with the presence of immunoreactive CRF in several regions of the brain including the cortex, limbic system, and centers involved in the control of autonomic function. The cortical and limbic receptors are clearly relevant to the effects of centrally administered CRF on both behavioral and visceral responses, with prominent autonomic changes including increased catecholamine release and hypertension.

Angiotensin II↗
PubMed1986

Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells.

The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs.

Animals↗
PubMed1986

Distribution of corticotropin-releasing factor receptors in primate brain.

The distribution and properties of receptors for corticotropin-releasing factor (CRF) were analyzed in the brain of cynomolgus monkeys. Binding of [125I]tyrosine-labeled ovine CRF to frontal cortex and amygdala membrane-rich fractions was saturable, specific, and time- and temperature-dependent, reaching equilibrium in 30 min at 23 degrees C. Scatchard analysis of the binding data indicated one class of high-affinity sites with a Kd of 1 nM and a concentration of 125 fmol/mg (approximately equal to 30% of the receptor number in monkey anterior pituitary membranes). As in the rat pituitary and brain, CRF receptors in monkey cerebral cortex and amygdala were coupled to adenylate cyclase. Autoradiographic analysis of specific CRF binding in brain sections revealed that the receptors were widely distributed in the cerebral cortex and limbic system. Receptor density was highest in the pars tuberalis of the pituitary and throughout the cerebral cortex, specifically in the prefrontal, frontal, orbital, cingulate, insular, and temporal areas, and in the cerebellar cortex. A very high binding density was also present in the hippocampus, mainly in the dentate gyrus, and in the arcuate nucleus and nucleus tuberis lateralis. A high binding density was present in the amygdaloid complex and mamillary bodies, olfactory tubercle, and medial portion of the dorsomedial nucleus of the thalamus. A moderate binding density was found in the nucleus accumbens, claustrum, caudate-putamen, paraventricular and posterior lateral nuclei of the thalamus, inferior colliculus, and dorsal parabrachial nucleus. A low binding density was present in the superior colliculus, locus coeruleus, substantia gelatinosa, preoptic area, septal area, and bed nucleus of the stria terminalis. These data demonstrate that receptors for CRF are present within the primate brain at areas related to the central control of visceral function and behavior, suggesting that brain CRF may serve as a neurotransmitter in the coordination of endocrine and neural mechanisms involved in the response to stress.

Adenylyl Cyclases↗
PubMed1986

Receptor-mediated actions of corticotropin-releasing factor in pituitary gland and nervous system.

High-affinity corticotropin-releasing factor (CRF) receptors which mediate the actions of the hypothalamic peptide on adrenocorticotropic hormone (ACTH) release have been identified in the rat anterior pituitary gland. Occupancy of the pituitary receptor by CRF agonists stimulates ACTH release via activation of adenylate cyclase and cyclic adenosine monophosphate dependent protein kinase. In the regulation of ACTH secretion, the effects of CRF on the corticotroph are integrated with the stimulatory actions of cyclic adenosine monophosphate-independent stimuli such as angiotensin II, vasopressin and norepinephrine, and the inhibitory effects of glucocorticoids and somatostatin. In contrast to the major importance of the inhibitory effect of glucocorticoid feedback on ACTH secretion, somatostatin has relatively little effect on CRF-stimulated ACTH release in the normal rat corticotroph. Following adrenalectomy, the progressive elevation of plasma ACTH levels is accompanied by a concomitant decrease in pituitary CRF receptors. The postadrenalectomy loss of CRF receptors, which is prevented by dexamethasone treatment, is caused by a combination of occupancy and processing of the pituitary sites during increased secretion of the hypothalamic peptide. Recently, specific receptors for CRF have been localized in the rat and monkey brain and adrenal medulla, where they are also coupled to adenylate cyclase. Brain CRF receptors are most abundant in the cerebral and cerebellar cortices and in structures related to the limbic system and control of the autonomic nervous system. The actions of CRF on the central and peripheral nervous systems, as well as on the pituitary gland, emphasize the role of CRF as a key hormone in the integrated response to stress.

Adrenocorticotropic Hormone↗
PubMed1986

Predominant release of vasopressin vs. corticotropin-releasing factor from the isolated median eminence after adrenalectomy.

In an in vitro system, we have demonstrated concomitant release of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) from the median eminence (ME) of normal and adrenalectomized rats. The ME were incubated in a Krebs-Ringer bicarbonate medium, CRF and AVP released into the medium were measured by radioimmunoassay. The release of both neuropeptides was stimulated by increasing concentrations of potassium (28-56 mM) in the incubation medium, or by addition of veratridine (5-20 microM). In both cases the release process was dependent on the presence of calcium in the incubation medium. Interestingly, potassium-induced release was found to be relatively insensitive to calcium channel antagonists that are potent inhibitors in smooth and cardiac muscle. The sodium channel antagonist, tetrodotoxin (1 microM), completely blocked the effect of veratridine, while no change was seen in the response to potassium. Adrenalectomy increased the ratio of AVP:CRF release from 2:1 in ME removed from sham-operated rats to 8:1 in ME from the adrenalectomized group. We suggest that this ratio of AVP:CRF release may also pertain in vivo, and that AVP could be the predominant corticotropic stimulus in adrenalectomized rats.

Adrenalectomy↗
PubMed1986

Regulation of primate angiotensin II receptors during altered sodium intake.

In the rat, angiotensin II receptors of the adrenal glomerulosa and smooth muscle undergo reciprocal regulatory changes that parallel the changes in target cell sensitivity to angiotensin II during altered sodium intake. In primates, the relative importance of angiotensin II receptor regulation during sodium-induced changes in angiotensin II sensitivity is not clear. To evaluate the role of angiotensin II receptor regulation in the primate, we analyzed the changes in angiotensin II receptors of adrenal and bladder membrane-rich particles after 4 to 6 days of high or low sodium intake in the monkey (Macaca fascicularis). Consistent with the decreased pressor response to angiotensin II, smooth muscle angiotensin II receptors were fewer in sodium-restricted monkeys (93 +/- 17 fmol/mg) than in sodium-loaded monkeys (171 +/- 6 fmol/mg). However, in contrast to the rat, changes in zona glomerulosa angiotensin II receptors in monkey adrenal were similar to those in smooth muscle, decreasing with sodium restriction and increasing with sodium loading (344 +/- 64 and 660 +/- 68 fmol/mg, respectively). There was no change in angiotensin II receptor affinity in either smooth muscle or adrenal particles during altered sodium intake. Concomitant with the decrease in adrenal angiotensin II receptors, 18-hydroxylase activity was increased twofold in adrenal mitochondria from sodium-restricted monkeys (74 +/- 8 fmol/mg/min) compared with sodium-loaded animals (28 +/- 11 fmol/mg/min). The increased sensitivity of the primate adrenal to angiotensin II despite a fall in angiotensin II receptors indicates that full activation of steroidogenesis by angiotensin II can be maintained with partial receptor occupancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1986

Participation of voltage-dependent calcium channels in the regulation of adrenal glomerulosa function by angiotensin II and potassium.

The stimulation of aldosterone secretion from adrenal glomerulosa cells by angiotensin II (AII), potassium, and ACTH is highly dependent on the extracellular calcium concentration. To evaluate the role of voltage-dependent calcium channels in aldosterone production, we analyzed the actions and binding of calcium channel antagonists in collagenase-dispersed adrenal glomerulosa cells and membrane-rich particles. In rat glomerulosa cells, nifedipine caused dose-dependent inhibition of the aldosterone responses to AII and potassium, with half-maximum inhibitory concentration (IC50) of 100 nM, but had no effect on ACTH or 8-bromo-cAMP stimulated steroidogenesis in adrenal glomerulosa and fasciculata cells. Binding studies with [3H]nitrendipine in adrenal glomerulosa cells revealed a high affinity site with dissociation constant (Kd) of 0.4 +/- 0.1 nM, similar to that described in other tissues but about 100-fold lower than the IC50 for blockade of aldosterone production. However, Scatchard analysis of binding data from three of seven experiments in isolated adrenal glomerulosa cells revealed a low affinity site with Kd of 130 nM, in agreement with the IC50 for the effect of nifedipine on aldosterone production. In rat adrenal particles, nitrendipine-binding sites were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Furthermore, there was a close correlation (r = 0.92) between the concentrations of nitrendipine-binding sites and AII receptors in the different zones of the adrenal in rat, dog, and cow, suggesting a functional relationship between AII receptors and calcium channels. These studies have shown a major and selective role of voltage-dependent calcium channels in the control of aldosterone secretion by the major physiological regulators, AII and potassium.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Involvement of protein kinase C in the regulation of adrenocorticotropin release from rat anterior pituitary cells.

The involvement of protein kinase C in normal corticotroph function was studied by analysis of the effects of the phorbol ester derivative phorbol 12-myristate-13-acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (DOG) on basal and stimulated ACTH release in cultured rat anterior pituitary cells. Incubation of rat pituitary cells with increasing concentrations of PMA or DOG caused dose-related increases in ACTH release up to 13.4 +/- 2.1- and 10.1 +/- 0.9-fold, respectively, similar to that caused by CRF (9.8 +/- 1.6-fold). Also, stimulation of endogenous diglyceride formation by phospholipase C (100 mU/ml) stimulated ACTH release by 2.5 +/- 0.1-fold. In cells incubated with maximum stimulatory concentrations of CRF (10 nM) or 8-bromo-cAMP (8-Br-cAMP; 5 mM), addition of either 100 microM DOG or 100 nM PMA caused significantly higher ACTH responses than those obtained with CRF, 8-Br-cAMP, DOG, or PMA alone. 8-Br-cAMP (5 mM) and 10 nM CRF significantly increased the effect of 100 nM PMA by 1.4 +/- 0.2- and 1.5 +/- 0.1-fold, respectively. Combinations of 10 nM CRF with either vasopressin (VP) or angiotensin II (AII) increased ACTH secretion to values higher than those produced by CRF, VP, or AII alone. However, addition of maximal stimulatory concentrations of VP or AII (10 nM) did not further increase the effects of either PMA alone or PMA/CRF combinations, indicating that their mechanisms of action may be similar to that of PMA. These results indicate that in addition to the established cAMP-dependent mechanism, stimulation of ACTH release in normal pituitary cells may be elicited by activation of protein kinase C. The evidence also suggests that protein kinase C is involved during stimulation of ACTH release by the cAMP-independent regulators VP and AII and in the synergistic effects of VP and AII with CRF on the corticotroph.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Selective enhancement of angiotensin II- and potassium-stimulated aldosterone secretion by the calcium channel agonist BAY K 8644.

Recent studies with dihydropyridine calcium channel antagonists have indicated that voltage-sensitive calcium channels (VSCC) play a major role in the control of aldosterone secretion. The modulation of VSCC by physiological regulators of zona glomerulosa function was further evaluated by analysis of the actions of the dihydropyridine calcium channel agonist BAY K 8644 (BK 8644) on basal and stimulated aldosterone production in isolated rat glomerulosa cells. In the presence of normal K+ concentrations (3.5-4.5 mM), only high concentrations of BK 8644 (greater than or equal to 100 nM) stimulated aldosterone secretion. However, addition of 10 nM BK 8644 markedly enhanced steroid production (70% over control) in cells stimulated by incubation in 7.5 mM K+ or 0.1 nM angiotensin II (AII). Greater enhancement was achieved with 1 microM BK 8644, with aldosterone secretion 150% and 300% above control levels for K+ and AII, respectively. In AII-stimulated cells, 30 nM BK 8644 enhanced aldosterone secretion at all peptide concentrations studied, including a 70% increase in the maximum steroid response, with no change in sensitivity to AII. In K+-stimulated cells, the effects of BK 8644 were dependent on the medium concentration of K+. At submaximally stimulating K+ concentrations (less than 9 mM), 30 nM BK 8644 increased the sensitivity of glomerulosa cells to K+ with no change in the maximal aldosterone response. However, at supramaximally stimulating concentrations of K+ (greater than 10 mM), BK 8644 reduced aldosterone production by 50%. In contrast to the effects of BK 8644 on cells stimulated with K+ or AII, the channel agonist had no effect on the action of ACTH. The ability of BK 8644 to enhance the maximum aldosterone response to AII suggests that AII, unlike K+, does not fully activate the Ca2+ influx pathway that leads to aldosterone secretion. Since BK 8644 is believed to facilitate Ca2+ influx primarily through previously activated channels, these results suggest that VSCC in the rat glomerulosa cell are partially operative under basal conditions, and that the same types of channels are further activated by AII and K+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Biphasic inhibition of adrenocorticotropin release by corticosterone in cultured anterior pituitary cells.

The inhibition of ACTH secretion by glucocorticoids in vivo is biphasic, with rapid early suppression followed by transient recovery and a late inhibitory phase. To evaluate whether this biphasic effect of glucocorticoids occurs at the pituitary level, the effects of corticosterone (B) on stimulated ACTH release were analyzed in rat anterior pituitary cell cultures. Preincubation with 1 microM B inhibited the ACTH response to 10 nM CRF in a biphasic manner, with rapid inhibition after 10-40 min of preincubation, followed by partial recovery between 40-80 min, and a later phase of inhibition after 80-140 min. Preincubation with B for 40 or 120 min caused a dose-dependent suppression of CRF-stimulated ACTH release, with ED50 values of 416 +/- 21 and 45 +/- 12 nM B, respectively. Pretreatment with B also caused a biphasic inhibitory effect on the stimulatory action of vasopressin, angiotensin II, and norepinephrine on ACTH release. However, addition of these stimuli in combination with CRF surmounted B inhibition of CRF-stimulated ACTH release. B also inhibited the ACTH-releasing effects of postreceptor stimuli, including 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and 1,2-dioctanoylglycerol. In the presence of cycloheximide (10 microM), the early inhibitory effect of B was unchanged, but the delayed effect was decreased. Whereas preincubation with B for 40 min inhibited ACTH release, but not total intracellular plus released ACTH, preincubation for 120 min decreased both released and total ACTH. These findings demonstrate that the two inhibitory effects of B on ACTH release differ in their kinetics, steroid sensitivity, and dependence on protein synthesis. The inhibitory effect of B on ACTH responses to stimuli with different mechanisms of action suggests that the suppressive effects of B are mainly exerted at a site distal to the formation of the second messengers involved in hormonal activation of ACTH release.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Role of arachidonic acid in the regulation of adrenocorticotropin release from rat anterior pituitary cell cultures.

In addition to cAMP-dependent mechanisms, stimulation of pituitary ACTH secretion by various stimuli, including CRF, may involve phospholipid and arachidonic acid turnover. To determine the role of phospholipase A2 activation in corticotroph function, we studied the effect of exogenous arachidonic acid, phospholipase A2, and the phospholipase A2 activator melittin on ACTH release in cultured rat anterior pituitary cells. Incubation with 1-100 micron arachidonic acid, 0.01-1 micron melittin, 0.1-10 U/ml phospholipase A2, and 0.01-10 nM CRF caused dose-dependent increases in ACTH release to 8.1 +/- 1.1- (+/- SE), 16.2 +/- 0.9-, 13.6 +/- 1.2-, and 2.9 +/- 0.3-fold; respectively. The participation of the major pathways of arachidonic acid metabolism in the control of ACTH release was analyzed in cells treated with nordihydroguaiaretic acid, a lipoxygenase inhibitor; indomethacin, a cycloxygenase inhibitor; and 5,8,11,14-eicosatetraynoic acid, an inhibitor of both pathways. The effects of arachidonic acid, melittin, and CRF were partially blocked by 10 micron nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid, but were significantly enhanced by 10 micron indomethacin. These results suggest that arachidonic acid is mainly metabolized through the lipoxygenase pathway to a stimulatory metabolite and, to a lesser extent, through the cycloxygenase pathway to an inhibitory metabolite. Arachidonic acid release from anterior pituitary cells labeled with [3H]arachidonic was analyzed during cell column perifusion and stimulation by CRF and other secretagogues. Two-minute pulses of CRF (10 nM), vasopressin (10 nM) and phorbol 12-myristate 13-acetate (100 nM) caused immediate 1.5- to 2-fold increases in [3H]arachidonic acid release, and melittin (100 nM) caused a 5-fold increase in [3H]arachidonic acid release. The ability of both exogenously added and endogenously generated arachidonic acid to stimulate ACTH secretion, together with the stimulation of arachidonic acid release by ACTH secretagogues and the attenuation of stimulated ACTH release by lipoxygenase blockers, indicate that lipoxygenase products of arachidonic acid metabolism participate in the control of ACTH secretion.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1986

Cytochemical studies of corticotropin-releasing factor (CRF) receptors in anterior lobe corticotropes: binding, glucocorticoid regulation, and endocytosis of [biotinyl-Ser1]CRF.

A new synthetic biotinylated N-terminal analog of CRF was used to study its binding, endocytosis, and route of processing. The new analog was fully bioactive compared to rat(r) CRF in an ACTH bioassay and pituitary membrane receptor assay. In 1- to 4-day cultured pituitary cell monolayers, CRF receptors were labeled for 1-biotinylated rCRF (1-bio-CRF) and demonstrated with avidin-fluorescein (Ar-Fl) in living cells or with avidin-biotin-peroxidase complex (ABC) in fixed cells. The percentages of fluorescein-labeled cells were comparable to those stained with the ABC technique. The specificity of binding by 1-bio-CRF was shown by the ability of unlabeled CRF to inhibit staining, whereas 100 nM arginine vasopressin, angiotensin II, or somatostatin was without effect. Treatment of the cells with 100 nM glucocorticoids for 1 or 24 h before the 3-min stimulation with 1-bio-CRF caused a 50-60% reduction in the percentage of cells stained with either ABC or Av-Fl. Pretreatment with vehicle, a lower dose of corticosterone (10 nM), or other steroids (dihydrotestosterone or epitestosterone) did not decrease the percentage of cells stained with the ABC technique. The lower dose of glucocorticoids decreased the stain with Av-Fl by 50% after 1 h of pretreatment. Electron microscopic analysis of binding and endocytosis of 1-bio-CRF stained with the ABC technique showed patches of stain on coated pits and microvilli during the first 3 min of exposure. Internalization in vesicles and receptosomes was also seen during the first 3 min. Some stain was found on secretion granules as early as 1 min after exposure. Five minutes after exposure, the stain was in receptosomes, Golgi cisternae, condensing vesicles in the transreticular Golgi region, and 23% of the granules in the cytoplasm. During the later exposure periods (15-30 min), stain was also seen in multivesicular bodies. The correlative light and electron microscopic studies showed that 1-bio-CRF is rapidly internalized after patching on the surface and is ultimately found in multivesicular bodies or granules. We postulate that these structures are involved in processing or degradation of the ligand.

Adrenocorticotropic Hormone↗
PubMed1986

Effects of 17 beta-estradiol on angiotensin II receptors and prolactin release in cultured pituitary cells.

Angiotensin II (AII) binds to specific receptors in the lactotroph and stimulates PRL secretion from isolated rat pituitary cells. Since estrogens exert major regulatory actions on PRL secretion, the effects of estradiol (E2) on pituitary AII receptors and PRL responses were studied in vivo and in cultured rat anterior pituitary cells. In female rats, treatment with E2-containing Silastic capsules for 4 days caused a significant increase in PRA from 1.3 to 3 ng/ml X min and a 38% decrease in the binding of [125I]AII to anterior pituitary membrane-rich fractions (P less than 0.01). In vitro studies showed that treatment of cultured anterior pituitary cells with 1 nM E2 for 4 days caused a 57 +/- 6% decrease in AII receptor concentration with no change in binding affinity. Reduction of AII receptors by E2 in 4-day cultures was dose dependent and was demonstrable with E2 concentrations that occur in plasma during the estrous cycle (0.01-1 nM). The decrease in AII receptors in cells incubated with 1 nM E2 was near maximum after 24 h of culture, and results were similar when receptor concentrations were calculated per unit protein or per cell. Despite the substantial decrease in AII receptors, E2 treatment did not specifically decrease the responsiveness of the pituitary cells to AII stimulation. Thus, PRL responses to AII (10 nM) or TRH (100 nM) were unchanged after 1 day of E2 treatment and were increased after 4 days of treatment. These findings demonstrate that E2 has a direct inhibitory action on expression of pituitary AII receptors that is not accompanied by a decrease in AII-stimulated PRL secretion. In the rat pituitary, estrogen modulation of postreceptor events is the predominant determinant of lactotroph responsiveness during stimulation of PRL release by AII.

Animals↗