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B J Canny

Publications and source records attributed to B J Canny.

51 records · Page 3Linked to original sources
PubMed1996

Prostaglandin E2 acts via the hypothalamus to stimulate ACTH secretion in the fetal sheep.

Prostaglandin (PG) E2 is secreted into the fetal circulation during late gestation in the sheep. Exogenous infusion of PGE2 is associated with robust increases in the circulating concentrations of ACTH and cortisol and it has therefore been proposed that endogenous PGE2 modulates the activity of the fetal HPA axis, which is the primary determinant of parturition and essential for the maturation of vital organ systems. The sites of action of PGE2 within the HPA axis have not been clearly established. We have compared the effects of PGE2 infusion on ACTH and cortisol concentrations in intact fetuses and in those whose pituitaries were surgically disconnected from hypothalamic control (hypothalamo-pituitary disconnected; HPD fetuses operated at 111-113 days gestational age). The effect of advancing gestational age on these responses was investigated by infusing PGE2 at 121, 131, 141 and 148 days of gestation. The functional integrity of the pituitary corticotrophs was tested by injecting CRH (1 microgram) into intact and HPD fetuses at day 125. This test was repeated for the HPD fetuses at 160 days. The responsiveness of the adrenal glands was also tested by injecting 2.5 micrograms/kg synthetic ACTH1-24 (Synacthen) into both groups of fetuses at day 135, with the HPD group retested at day 155. PGE2 infusion was associated with a robust increase (P < 0.001) in plasma immunoreactive (ir) ACTH in intact fetuses at all gestational ages while HPD fetuses did not respond, except at day 148 when the response was small. Similarly, cortisol concentrations were increased (P < 0.001) during PGE2 infusion in intact fetuses but not in HPD fetuses, except for a minor increase at 148 days. The response of irACTH to exogenous CRH was similar in intact and HPD fetuses at 125 days and this response was maintained in HPD fetuses at day 160. The cortisol response of intact fetuses to ACTH1-24 exceeded that of HPD fetuses at day 135 and, in HPD fetuses, this response was unchanged when they were retested at 155 days, indicating that their adrenal responsiveness did not wane with time. We conclude that the effects of PGE2 on the fetal HPA axis are exerted predominantly or exclusively at a level above the pituitary gland.

Adrenocorticotropic Hormone↗
PubMed1993

Pituitary adenylate cyclase-activating polypeptide regulates cytosolic Ca2+ in rat gonadotropes and somatotropes through different intracellular mechanisms.

The hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates an increase in the cytoplasmic free Ca2+ ion concentration ([Ca2+]i) in GH-secreting somatotropes and LH-secreting gonadotropes of the rat anterior pituitary gland. The dynamics of the PACAP-induced Ca2+ responses and their dependence on extracellular Ca2+ are markedly different in the two cell types, suggesting separate mechanisms of action of PACAP in somatotropes and gonadotropes. The present study reports a full characterization of the Ca2+ responses seen in the two cell types over a wide range of PACAP concentrations. In addition, the involvement of the cAMP-dependent protein kinase (PKA) system in the mediation of PACAP-stimulated Ca2+ was tested using the R-isomer of cAMP (RpcAMPs) as a specific inhibitor of PKA. In identified somatotropes, PACAP (10(-11)-10(-6) M) stimulated Ca2+ responses in 49% of the cells tested, with two types of Ca2+ response profile observed. The first was a slow rise in [Ca2+]i to a new level (Ca2+ step; 28% of somatotropes); the second was characterized by repetitive transient rises in [Ca2+]i (Ca2+ transients; 21% of somatotropes). The range of PACAP concentrations tested (10(-11)-10(-6) M) did not markedly alter the number of cells responding or the type of response observed. In some gonadotropes, PACAP stimulated Ca2+ step responses similar to those seen in somatotropes; however, the most common response observed was a rapid, high amplitude, but transient spike of [Ca2+]i, which was often accompanied by rapid oscillations in [Ca2+]i. This response profile was termed a Ca2+ spike-oscillations response, and the proportion of cells exhibiting this response increased from 25% at a PACAP concentration of 10(-11) M to 73% at 10(-6) M PACAP. PACAP-induced Ca2+ responses in somatotropes were blocked by pretreatment with the cAMP antagonist RpcAMPs (10(-3) M). In addition, other factors known to increase cAMP in somatotropes (GH-releasing factor and 8-bromo-cAMP) stimulated Ca2+ responses in these cells that were qualitatively similar to those induced by PACAP. In contrast, the Ca2+ responses in gonadotropes were insensitive to the cAMP antagonist RpcAMPs, and the membrane-permeable cAMP analog 8-bromo-cAMP failed to stimulate responses in this cell type. These results suggest that the intracellular mechanisms of PACAP action in the two cell types are markedly different. In somatotropes, the rise in [Ca2+]i stimulated by PACAP is probably through the production of cAMP and the activation of protein kinase-A.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Corticotropin-releasing factor, but not arginine vasopressin, stimulates concentration-dependent increases in ACTH secretion from a single corticotrope. Implications for intracellular signals in stimulus-secretion coupling.

The two fundamental parameters of corticotropin (ACTH) secretion are the number of secreting corticotropes and the amount of ACTH secreted by each cell. We have measured these parameters in rat corticotropes in response to increasing concentrations of corticotropin-releasing factor (CRF) or arginine vasopressin (AVP). Increasing concentrations of AVP stimulated more corticotropes to secrete, while the amount of ACTH each cell secreted remained relatively fixed (nongraded secretory response). Conversely, increasing concentrations of CRF stimulated more ACTH secretion per cell (graded secretory response), while the number of secretory cells remained relatively constant. When viewed from the perspective of a single corticotrope, it was clear that CRF and AVP induced completely distinct specific responses. We have previously shown, and provide further evidence here, that secretory responses to CRF or AVP occur in the same cell. It is therefore apparent that a single corticotrope is able to generate either a graded, or a nongraded secretory response. We have also considered the potential intracellular changes that must direct graded or nongraded secretion. It is generally accepted that CRF stimulates activation of adenylate cyclase, whereas AVP activates phosphoinositidase in pituitary corticotropes. Our findings, and others surveyed here, suggest that the activation of adenylate cyclase results in graded secretion, while the activation of phosphoinositidase induces the nongraded secretion. Graded or nongraded secretion may therefore be linked to specific second messengers. It is hypothesized that the inositol 1,4,5-trisphosphate-mediated release of an intracellular Ca2+ store constitutes a mechanism whereby phosphoinositidase-coupled hormones set in motion the nongraded secretory response. These findings suggest novel functions for individual second messengers.

Adrenalectomy↗
PubMed1992

Paracrine communication regulates adrenocorticotropin secretion.

Local communication among cells of the anterior pituitary appears to play an important role in the regulation of ACTH secretion. Dissociated pituitary cells were plated as a monolayer at decreasing concentrations of cells (increasing the distance between cells and, thus, decreasing their potential interactions), and ACTH secretion was measured from individual corticotropes using a specific reverse hemolytic plaque assay. There was a critical intercell distance above which significant changes in the number of CRF-responsive corticotropes were observed. Provided that this critical distance was not exceeded the number of secretory corticotropes in response to CRF (10 nM) was relatively constant, thereby defining a fraction of corticotropes that was robustly CRF responsive. In contrast, when this critical distance between cells was exceeded, the number of CRF-responsive corticotropes progressively increased to almost double their original number, thereby defining a second fraction of CRF-responsive corticotropes that was previously repressed. These observations suggest the presence of a paracrine factor that profoundly inhibits CRF-stimulated ACTH secretion from a repressed fraction of corticotropes. Further independent studies confirmed and extended these observations. We identified the cellular source of the inhibitory factor as the robustly CRF-responsive fraction of corticotropes. Pituitary cells were identified by reverse hemolytic plaque assay and then destroyed using a laser photoablation procedure that did not compromise the remaining cells. The pituitary cells were separated by a distance at which the inhibitory factor was fully effective. Destruction of the cellular source of the paracrine inhibition would, therefore, allow secretion from the previously repressed fraction of corticotropes. Accordingly, when robustly CRF-responsive corticotropes were destroyed, a significant number of previously repressed corticotropes appeared in a second assay. Destruction of somatotropes or a cell adjacent to a robustly CRF-responsive corticotrope did not alter the number of CRF-stimulated corticotropes among the remaining cells. We conclude that a paracrine factor liberated by the robustly CRF-responsive corticotropes inhibits ACTH secretion from the repressed fraction of corticotropes. The robustly CRF-responsive corticotropes appear unresponsive to the effects of the factor, and the repressed corticotropes are unlikely to secrete it. A role of this paracrine communication is to hold corticotropes in reserve and, therefore, prevent the severe depletion of hormone. This form of paracrine communication may be a specialized adaption among cells where the physiological setting demands robust secretory responses to multiple stimuli. The experimental paradigms developed here may be extremely useful for 1) screening potential paracrine factors and 2) determining whether the secretion of the paracrine factor is regulated by adrenal or hypothalamic hormones.

Adrenocorticotropic Hormone↗
PubMed1992

Pituitary adenylate cyclase-activating polypeptide specifically increases cytosolic calcium ion concentration in rat gonadotropes and somatotropes.

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is a potent stimulator of cAMP accumulation in the anterior pituitary gland, though its physiological function has yet to be defined. To establish the target cells of PACAP action we have measured PACAP-induced changes in cytosolic free calcium ion concentration ([Ca2+]i) in single identified anterior pituitary cells. This was achieved by combining fura-2 videomicroscopy, to measure [Ca2+]i, and reverse hemolytic plaque assays, to identify the secreted hormone. PACAP (100 nM) increased [Ca2+]i in 32% of all pituitary cells. These responses were predominantly seen in identified gonadotropes and somatotropes, but rarely in corticotropes or lactotropes. PACAP induced two forms of Ca2+ response in gonadotropes; a "Ca2+ spike" (independent of extracellular Ca2+) in 72% of responding gonadotropes, and an extracellular Ca(2+)-dependent "Ca2+ plateau" (28% of cells). In somatotropes, PACAP stimulated either Ca2+ plateau responses (58% of responding somatotropes) or repetitive "Ca2+ transients" (42% of cells), both of which were dependent upon extracellular Ca2+. PACAP, therefore, produces distinct changes in [Ca2+]i in gonadotropes and somatotropes, which may be related to distinct intracellular messenger pathways. The identification of these cell types as targets of PACAP action suggests a role in the regulation of reproduction and growth.

Animals↗
PubMed1992

Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors.

GH secretion has been thought traditionally to be regulated by the two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF). Recent evidence has suggested that other factors may be involved. These factors include the natural ligand for the synthetic hexapeptide GH-releasing peptide (GHRP) and the putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PA-CAP). Accordingly, we examined the effects of GHRP and PACAP on GH secretion at the single cell level using the reverse hemolytic plaque assay which allows distinction of effects on the number of secreting cells and the amount of hormone each cell secretes. Both factors stimulated GH secretion in a dose-dependent fashion, with PACAP being more effective. PACAP increased both the number of cells secreting and the mean amount of hormone secreted per cell. In contrast, GHRP increased the number of secreting cells, although it had no effect on the amount of secretion per cell. GH secretion induced by GHRH, GHRP, and PACAP was inhibited by SRIF, but the effect was predominantly on the number of cells secreting rather than the amount secreted per cell. Specific antagonists to GHRP and GHRH inhibited GH secretion induced by the respective agonist but not that induced by the other factor nor by PACAP. These findings confirm the complex nature of the regulation of GH secretion at the level of the somatotrope. At least three factors, operating via distinct receptors, are able to increase GH secretion. In addition, they ascribe a potential physiological role for the hitherto putative hypophysiotropic factor PACAP.

Animals↗
PubMed1991

Distinct classes of corticotropes mediate corticotropin-releasing hormone- and arginine vasopressin-stimulated adrenocorticotropin release.

ACTH release from the anterior pituitary gland is principally driven by the two hypothalamic hormones, corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). Using the reverse hemolytic plaque assay, we have compared the effects of CRH and AVP on ACTH release from individual, dispersed pituitary cells. A small percent (0.36 +/- 0.06%) of pituitary cells formed plaques when exposed to medium alone. AVP caused 3.44 +/- 0.10% of cells to form plaques (P less than 0.01 compared with medium alone), CRH produced 4.85 +/- 0.20% plaque-forming cells (P less than 0.01 compared with AVP), and the combination of CRH and AVP produced a still greater percent of plaque-forming cells (5.80 +/- 0.20%, P less than 0.01 compared with CRH alone). A double reverse hemolytic plaque assay was then employed to examine whether some cells formed plaques only in the presence of one or other secretagogue. Using this technique we found clear evidence of cells that formed plaques in response to CRH but not AVP (P less than 0.005); CRH or AVP (P less than 0.0001), and CRH and AVP (P less than 0.05). There was no evidence of a corticotrope forming a plaque with AVP but not CRH (P = 0.52). Thus there appears to be functionally distinct classes of corticotropes. These findings have important implications for our understanding of the relative responsiveness of the pituitary to hypothalamic secretagogues and provide a new physiological perspective on recent reports of stress-specific hypothalamic responses regulating ACTH release.

Adrenocorticotropic Hormone↗
PubMed1990

Adrenocorticotropin responses to endogenous and exogenous secretagogues in the sheep: specificity of glucocorticoid action.

Glucocorticoids act upon the hypothalamus and pituitary to regulate both corticotropin-releasing factors and adrenocorticotropin (ACTH) in a classical negative feedback loop. There are two distinct receptor systems in the central nervous system for glucocorticoids: type I corticosterone/cortisol-preferring, predominantly hippocampal receptors, and type II dexamethasone-binding receptors with a much broader distribution. To determine the relative roles played by these two receptors in determining basal and stress-induced ACTH/cortisol levels in the sheep, we have conducted two series of experiments. In the first, we infused 4 sheep with cortisol (50 micrograms/h), or dexamethasone (20 micrograms/h) for 17 h, collected 10-min blood samples for 3 h for basal hormone levels, and then subjected the sheep to an audiovisual stress (barking dog), 10 micrograms of ovine corticotropin-releasing factor (oCRF) 1 h later, and 100 U of insulin another 2 h later. Dexamethasone reduced ACTH and cortisol levels, both basal and in response to stress; cortisol infusion had no effect on any parameters; oCRF did not raise ACTH levels. In the second experiment, cortisol (100 micrograms/h) alone was infused into 4 sheep for 17 h, and blood samples were collected for 3 h for basal hormone levels; this was followed by the injection of 10 micrograms oCRF, and 2 h later a barking dog was introduced into the sheep shed. Again, cortisol infusion did not affect basal ACTH or cortisol levels, though both cortisol-infused and control animals had ACTH responses to oCRF when it was not preceded by the audiovisual stress. ACTH and cortisol levels were higher in the 2 h following oCRF injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1990

Arginine vasopressin and corticotropin releasing factor: binding to ovine anterior pituitary membranes.

In the sheep, in contrast to the rat, arginine vasopressin (AVP) is a more potent stimulus to ACTH secretion from the anterior pituitary (AP) than CRF. To further explore this difference, we have compared [3H]AVP and [125I]-[Nle21 Tyr32]ovine CRF binding in membranes prepared from rat and sheep AP. Between species, no difference in affinity of binding was found for either ligand. In contrast, the concentration of AVP receptors in sheep AP was twice that in rat, whereas that of CRF receptors was only one tenth. AVP receptor concentration in sheep AP was not altered by chronic (10 day) dexamethasone administration, but fell to 60% of control after chronic (60 day) hypothalamo-pituitary-disconnection. The increased level of AVP receptors and the much lower level of CRF receptors in sheep compared with rat may thus provide an explanation for our previous findings of increased sensitivity to AVP and a very poor response to CRF in stimulating ACTH release from the sheep AP. In addition the finding that AVP receptor numbers are reduced in the hypothalamo-pituitary-disconnected sheep suggests that hypothalamic factors may play a role in regulating AVP receptor concentration in the ovine AP gland.

Animals↗
PubMed1989

The posterior pituitary regulates prolactin, but not adrenocorticotropin or gonadotropin, secretion in the sheep.

The aim of this study was to determine the role of the posterior pituitary gland in the control of PRL, LH, FSH, and ACTH secretion in sheep. Posterior pituitary function was removed in ovariectomized ewes by electrical lesioning of the hypothalamo-neurohypophysial tract immediately posterior to the stalk-median eminence (LESION); controls were subjected to sham surgery (SHAM). LESION caused a 2-fold increase in plasma PRL concentrations on days 1-3 after surgery. Thereafter, concentrations gradually declined until they were similar to those in SHAM ewes. There was no change in plasma concentrations of LH, FSH, or ACTH after LESION. Plasma PRL responses to insulin in SHAM ewes were completely abolished, and the plasma PRL response to chlorpromazine was reduced to almost half by LESION. In contrast, audiovisual stress (barking dog) and serotonin challenge caused an immediate release of PRL in both LESION and SHAM ewes, with the amplitude of the responses indistinguishable between groups. LESION had no effect on the plasma ACTH responses to audiovisual stress, insulin, or serotonin. We conclude that the posterior pituitary gland is involved in the regulation of PRL under some circumstances, but not of LH, FSH, or ACTH secretion in the sheep. Accordingly, changes in PRL release after hypothalamopituitary disconnection in this species may reflect a loss of posterior lobe function rather than the removal of hypothalamic inputs. In addition, the PRL response to insulin is dependent on a functional posterior pituitary gland, whereas responses to audiovisual stress and serotonin appear to rely on inputs to the pituitary gland via the median eminence and the long hypothalamo-hypophysial portal blood vessels.

Adrenocorticotropic Hormone↗
PubMed1989

Glucocorticoids regulate ovine hypophysial portal levels of corticotropin-releasing factor and arginine vasopressin in a stress-specific manner.

Hypophysial portal circulation levels of immunoreactive (ir) CRF, immunoreactive arginine vasopressin (ir-AVP), and systemic ir-ACTH and cortisol were determined in dexamethasone-infused (20 micrograms/h) ewes, both under basal conditions and in response to audiovisual and hypoglycemic stress. Glucocorticoid infusion lowered mean basal levels of ir-CRF (P less than 0.05), ir-ACTH (P less than 0.05), and cortisol (P less than 0.05), but not of ir-AVP. Audiovisual stress led to coordinate release of ir-AVP and ir-CRF, and although the hypothalamic response to this stress was not altered in dexamethasone (DEX)-infused sheep, the pituitary-adrenal response was blocked, suggesting that glucocorticoids act on the pituitary rather than on higher centers to inhibit this response. In contrast, hypoglycemia led to preferential release of ir-AVP over ir-CRF, and DEX infusion delayed and inhibited both hypothalamic and pituitary responses. Ketamine injection also led to preferential release of ir-AVP, but neither the hypothalamic nor pituitary-adrenal responses were affected by DEX infusion. These results suggest that different stressors evoke different patterns of hypothalamic secretagogue release, and that glucocorticoids act in a site-specific fashion to regulate the hypothalamo-pituitary-adrenal responses to stress.

Animals↗
PubMed1988

Innervation of the sheep adrenal cortex: an immunohistochemical study with rat corticotrophin-releasing factor antiserum.

Using indirect immunohistochemistry and an antiserum raised against rat corticotrophin-releasing factor (CRF) we have outlined an asymmetric network of cells and varicose fibers in sheep adrenal cortex. This network was not associated with the larger splanchnic nerves, but was occasionally found in small bundles or with blood vessels; in most instances fibers were found weaving independently through cortical parenchyma. A plexus of fibers was found in the zona reticularis, with a few fibers ramifying into adjacent medulla. Uni or bipolar cells were found throughout the cortex, with the greatest frequency at the corticomedullary junction; a multipolar-type cell was also found in this area. Staining of varicose structures and most cells was abolished by incubation with excess rat CRF 1-41, but not by ovine CRF or a range of other peptides. Though the immunoreactive species has not as yet been identified, it may thus share homology with sequences present in rat but not ovine CRF.

Adrenal Cortex↗
PubMed1988

Post-translational processing of pro-opiomelanocortin in the Brattleboro (di/di) rat pituitary.

In homozygous (di/di) Brattleboro rats, pro-opiomelanocortin (POMC) synthesis and processing, and the release of its products, is by definition not under the control of hypothalamic arginine vasopressin (AVP) in contrast with normal rats or heterozygote (di/+) littermates. To explore the role of AVP on these parameters, we have compared homozygotes and heterozygotes in terms of POMC mRNA levels, pituitary content of immunoreactive (ir)-ACTH, ir-beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH) and ir-N-acetyl-EP (NacEP), plasma levels of ir-ACTH and ir-beta-EP, and RP-HPLC profiles of the various forms of ir-alpha-MSH and ir-NacEP. Homozygous rats had immeasurably low levels of hypothalamic ir-AVP, in contrast with their heterozygote littermates; hypothalamic ir-corticotropin releasing factor did not differ between strains. No difference between-strains was seen in levels of POMC mRNA; elevated levels of all pituitary peptides, except ir-ACTH, were found in di/di rats; plasma levels of both ir-ACTH and ir-beta-EP were lower in di/di rats; pituitary ir-alpha-MSH RP-HPLC profiles were similar in both strains, but those for ir-NacEP showed a striking increase in Nac alpha-EP in di/di rats. We interpret these data as evidence for decreased degradation/retarded release of POMC products from the di/di anterior pituitary, for increased processing of POMC products to shorter forms in both anterior pituitary and neuro-intermediate lobe, and thus for a role of AVP in the processing of POMC, as well as POMC synthesis and ACTH/beta-EP release.

Adrenocorticotropic Hormone↗
PubMed1987

Concomitant dopaminergic and glucocorticoid control of pituitary proopiomelanocortin messenger ribonucleic acid and beta-endorphin levels.

Synthesis and secretion of POMC-derived peptides appear to be differentially regulated in the anterior pituitary (AP) and neurointermediate lobe (NIL). In the AP, glucocorticoids inhibit, and CRF and arginine vasopressin stimulate, synthesis of POMC and release of immunoreactive (ir)-beta-endorphin (beta EP); in the NIL, synthesis and release of POMC and its derivatives are under tonic inhibitory dopaminergic control. There is, however, evidence for some overlap of these control mechanisms under certain circumstances. In the present study we have used specific RIA and Northern blot analysis to examine the effects of chronic treatment with dopaminergic agents and dexamethasone (DM) (both alone and in combination) on AP and NIL content of ir-beta EP and POMC messenger RNA (mRNA), and/or hypothalamic ir-arginine vasopressin and ir-CRF content. In the NIL, the dopamine agonist bromocriptine reduced and the antagonist haloperidol raised both POMC mRNA and ir-beta EP content. Long term DM treatment did not alter NIL ir-beta EP content in the intact rat, but increased levels of POMC mRNA. DM abolished the haloperidol-induced increase in NIL ir-beta EP content but further increased the haloperidol-induced rise in POMC mRNA. DM treatment lowered both ir-beta EP and POMC mRNA in the AP as well as lowering levels of hypothalamic ir-CRF. In DM-treated rats, haloperidol partially restored AP ir-beta EP and POMC mRNA to control untreated levels. These findings further support the proposition that both dopaminergic agents and glucocorticoids can modulate POMC mRNA levels and/or tissue content of ir-beta EP in both the NIL and AP of the rat. The effects of DM on the NIL, both alone or with haloperidol, suggest that glucocorticoids may have both direct and indirect effects on POMC gene expression in this tissue.

Animals↗