Retrospective characterization of Newcastle Disease Virus Antrim '73 in relation to other epidemics, past and present.

In November 1973 Newcastle disease suddenly appeared in Northern Ireland, where the viscerotropic disease had not been seen in 3 1/2 years and the two Irelands had been regarded as largely disease free for 30 years. It was successfully controlled with only 36 confirmed affected layer flocks, plus 10 more slaughtered as 'dangerous contacts'. Contemporary investigations failed to reveal the source of the Irish epidemic. Using archival virus samples from most of the affected flocks, RT PCR was conducted with primers selected for all six NDV genes. Phylogenetic analyses of three genes, HN, M and F, confirmed vaccine as the cause of one of the outbreaks. The other six samples were identical and closely related to previous outbreaks in the United States and western Europe initiated by infected imported Latin American parrots. The probable cause of the epidemic followed from the importation from The Netherlands of bulk feed grains contaminated with infected pigeon faeces.

Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation.

The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-kappaB) in response to stimulation with tumor necrosis factor alpha (TNF-alpha) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-kappaB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-kappaB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-kappaB (IkappaB). In vitro kinase assays showed that RKIP antagonizes the activation of the IkappaB kinase (IKK) activity elicited by TNF-alpha. RKIP physically interacted with four kinases of the NF-kappaB activation pathway, NF-kappaB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKalpha, and IKKbeta. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-kappaB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.

Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein.

We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.

Neuronal Ca(2+) sensor 1. Characterization of the myristoylated protein, its cellular effects in permeabilized adrenal chromaffin cells, Ca(2+)-independent membrane association, and interaction with binding proteins, suggesting a role in rapid Ca(2+) signal transduction.

Overexpression of frequenin and its orthologue neuronal Ca(2+) sensor 1 (NCS-1) has been shown to increase evoked exocytosis in neurons and neuroendocrine cells. The site of action of NCS-1 and its biochemical targets that affect exocytosis are unknown. To allow further investigation of NCS-1 function, we have demonstrated that NCS-1 is a substrate for N-myristoyltransferase and generated recombinant myristoylated NCS-1. The bacterially expressed NCS-1 shows Ca(2+)-induced conformational changes. The possibility that NCS-1 directly interacts with the exocytotic machinery to enhance exocytosis was tested using digitonin-permeabilized chromaffin cells. Exogenous NCS-1 was retained in permeabilized cells but had no effect on Ca(2+)-dependent release of catecholamine. In addition, exogenous NCS-1 did not regulate cyclic nucleotide levels in this system. These data suggest that the effects of NCS-1 seen in intact cells are likely to be due to an action on the early steps of stimulus-secretion coupling or on Ca(2+) homeostasis. Myristoylated NCS-1 bound to membranes in the absence of Ca(2+) and endogenous NCS-1 was tightly membrane-associated. Using biotinylated NCS-1, a series of specific binding proteins were detected in cytosol, chromaffin granule membrane, and microsome fractions of adrenal medulla. These included proteins distinct from those detected by biotinylated calmodulin, demonstrating the presence of multiple specific Ca(2+)-independent and Ca(2+)-dependent binding proteins as putative targets for NCS-1 action. A model for NCS-1 function, from these data, indicates a constitutive membrane association independent of Ca(2+). This differs from the Ca(2+) myristoyl switch model for the closely related recoverin and suggests a possible action in rapid Ca(2+) signal transduction in response to local Ca(2+) signals.

Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP.

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

Heterotrimeric G-protein candidates for Ge in the ACTH secretory pathway.

The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used to identify candidate heterotrimeric G-proteins for G-exocytosis (Ge) which mediates calcium ion-stimulated adrenocorticotrophin (ACTH) secretion in this cell line. AtT-20 cells express several heterotrimeric G-protein alpha subunits; Gs alpha, Gt alpha, Gq alpha, G11alpha, G12alpha, G13alpha, G14alpha, G15alpha, Gz alpha, Gi2alpha, Gi3alpha, and Go alpha and so heterotrimeric G-protein selective agents were used to differentiate between these candidates. Agents which stimulate ACTH secretion via Ge were not pertussis toxin (PTX)-sensitive nor was cholera toxin (CTX) able to stimulate ACTH secretion from permeabilised cells in the absence of calcium. G-protein antagonists which inhibit activation of Gs, Gi, and Gq subfamilies did not attenuate Ge-stimulated ACTH secretion from permeabilised AtT-20 cells. In AtT-20 cells the stimulatory G-protein involved in the late stages of the ACTH secretory pathway does not belong to the Gs, Gi (with the exception of Gz) or Gq subfamilies of heterotrimeric G-proteins leaving Gz, G12 or G13 as the strongest candidates for Ge.

Neuronal Ca2+ sensor 1, the mammalian homologue of frequenin, is expressed in chromaffin and PC12 cells and regulates neurosecretion from dense-core granules.

Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission in Drosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J. , Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.

Lack of bioeffects of ultrasound energy after intravenous administration of FS069 (Optison) in the anesthetized rabbit.

The current study was designed to provide a sensitive in vivo model to maximize the potential bioeffects (measured by hemolysis) of B-mode ultrasound energy in combination with FS069 (Optison). B-mode ultrasound energy was delivered to anesthetized male New Zealand white rabbits with a phased array 5 MHz transducer on a Hewlett-Packard Sonos 1500 ultrasonograph, with transmit level set to maximum (40 dB, approx 135 W/cm2). FS069 (Optison), latex particles in human albumin, or human albumin alone (vehicle) was infused via an ear vein at 0.6 mL/kg. No statistically significant changes were noted in serum free hemoglobin or lactate dehydrogenase either over time or between groups.

2',3'-Cyclic nucleotide 3'-phosphodiesterase is associated with mitochondria in diverse adrenal cell types.

In this study, we have examined the expression and intracellular localisation of the myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in bovine adrenal medullary chromaffin cell cultures. By immunoblotting, using two distinct anti-CNP monoclonal antibodies, CNP was detected in medullary cell cultures and expression of CNP was confirmed by reverse transcription and PCR amplification. CNP did not leak from digitonin-permeabilised chromaffin cells, suggesting that there is no cytosolic pool of this protein. Immunofluorescence studies with both antibodies showed that all cells in the medullary chromaffin cell culture were stained with a punctate appearance consistent with an intracellular localisation for CNP. More specifically it was demonstrated that CNP is co-localised with mitochondria. Various cell types in chromaffin cell cultures were stained with a mitochondrial pattern and CNP staining was co-localised with mitochondrial staining. These results show that CNP is a widely expressed protein that is associated with mitochondria and provides new clues as to its cellular function outside of myelin structures.

Similar effects of alpha- and beta-SNAP on Ca(2+)-regulated exocytosis.

Soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAP) proteins function in Ca(2+)-regulated exocytosis. Recent work (Schiavo et al. (1996) Nature 378, 733-736) based on in vitro protein interactions has raised the possibility that alpha- and beta-SNAPs have distinct roles in exocytosis. We have examined this possibility by comparing the activities of recombinant alpha- and beta-SNAPs. Both of these proteins were able to similarly bind NSF and activate its ATPase activity but to a lesser extent than gamma-SNAP. When introduced into digitoninpermeabilised chromaffin cells, both alpha- and beta-SNAP stimulated Ca(2+)-regulated exocytosis in a MgATP-dependent manner. The dose-response relationships for these proteins were essentially the same and addition of both proteins did not lead to any further increase in exocytosis above that due to each protein alone. We conclude that alpha- and beta-SNAPs are interchangeable isoforms with similar functions in regulated exocytosis.

The roles of adenosine 3',5'-cyclic monophosphate-dependent protein kinase A and protein kinase C in stimulus-secretion coupling in AtT-20 cells.

The ACTH-secreting mouse AtT-20/D16-16 anterior pituitary tumour cell line was used to study adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) and protein kinase C (PKC) involvement in stimulus-secretion coupling pathways. In permeabilised AtT-20 cells under calcium ion-free conditions, forskolin (1O mu M), CRH-41 (1OOnM), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 mu M) but not mastoparan (10 mu M) stimulated cAMP accumulation. Measurement of ACTH secretion under identical incubation conditions revealed that GTP-gamma-S and mastoparan significantly stimulated ACTH secretion but forskolin and CRH-41 did not. This dissociates cAMP accumulation from ACTH secretion under calcium ion-free conditions and indicated that the effects of mastoparan and GTP-gamma-S on ACTH secretion are not mediated by cAMP production. Calcium ions (1 nM to I mM) stimulated ACTH secretion from electrically permeabilised cells in a concentration-dependent manner. cAMP (100 mu M) and phorbol 12-myristate 13-acetate (PMA; 100 nM) synergistically enhanced the response to calcium ions. cAMP did not stimulate ACTH secretion in the absence of calcium ions nor did it alter the concentrations at which calcium stimulated ACTH secretion. This suggests that stimulation of ACTH secretion via the calcium-dependent pathway is necessary before any cAMP-mediated enhancement of secretion is manifest. PMA, however, did stimulate ACTH secretion in the absence of calcium ions, indicating distinct mechanisms for PKC-evoked secretion. Co-incubation with cAMP and PMA did not exceed the secretory response obtained with the combination of PMA and calcium ions. CRH-41 (1 pM to 100 nM) and forskolin (1 nM to 100 mu M) stimulated ACTH secretion from intact cells in a concentration-dependent manner. Co-incubation with PMA (100 nM) further enhanced the ACTH response to CRH-41 and forskolin; the effects were simply additive. The present study indicates that there are distinct roles for PKA and PKC in stimulus-secretion coupling in AtT-20 cells. The PKA-dependent pathway, acting in concert with the calcium messenger system, serves as part of the stimulus-secretion coupling pathway by which activation of CRH-41 receptors control ACTH secretion. The PKC-dependent pathway, in contrast, seems to be independent of the calcium messenger system and may represent a separate control mechanism of ACTH secretion.

Myocardial contrast echocardiography in experimental coronary artery occlusion with a new intravenously administered contrast agent.

A new intravenously administered ultrasound contrast agent was studied in eight dogs during intermittent coronary artery occlusion. The area of the myocardial contrast defect was compared with that of the acute wall motion abnormality induced by coronary occlusion. A close correlation was found between these two independent measures of acute myocardial ischemia. The peak change in myocardial intensity during coronary occlusion was significantly less than for the same segment before ischemia and for a remote nonischemic segment. This new, intravenously administered ultrasound contrast agent can be used to evaluate the spatial distribution of hypoperfused myocardium and should therefore prove valuable in the clinical evaluation of ischemic syndromes.

Effects of mastoparan upon the late stages of the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of the effects of mastoparan upon the late stages of the adrenocorticotrophin (ACTH) secretory pathway. 2. Mastoparan (10(-8)-10(-5) M), an activator of heterotrimeric guanosine 5'-triphosphate binding proteins (G-proteins), stimulated ACTH secretion from electrically-permeabilized AtT-20 cells in a concentration-dependent manner in the effective absence of calcium ions with a threshold of 10(-6) M. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (10(-8)-10(-4) M) also stimulated ACTH secretion from electrically-permeabilized AtT-20 cells in a concentration-dependent manner in the effective absence of calcium ions with a threshold of 10(-6) M. This GTP-gamma-S-evoked secretion is consistent with previous studies which demonstrated that a G-protein, termed GE, mediates calcium evoked ACTH secretion from AtT-20 cells. GTP-gamma-S-evoked secretion however was not as great as that obtained in response to mastoparan. 3. Both mastoparan (10(-5) M) and GTP-gamma-S (10(-4) M) stimulated ACTH secretion from electrically-permeabilized AtT20 cells in a time-dependent manner. A time of 30 min was adopted as the standard incubation period for the study of both mastoparan and GTP-gamma-S-stimulated ACTH secretion from permeabilized AtT-20 cells. 4. Mastoparan (10(-8)-10(-5) M) stimulated ACTH secretion from permeabilized AtT-20 cells to the same extent in the presence and absence of the protein kinase C (PKC) inhibitor, chelerythrine chloride (10(-5) M). 5. Mastoparan (10-8 10-5 M)-stimulated ACTH secretion from permeabilized AtT-20 cells was significantly reduced in the presence of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S, 10-4 M).6. The mastoparan analogue, Mas-7 (10-8-10-5 M) stimulated ACTH secretion from permeabilized AtT-20 cells to a greater extent than mastoparan (10-8 10-5 M) however, the mastoparan analogue Mas-17 (10-8- 10-5 M) had no effect upon ACTH secretion from permeabilized AtT-20 cells.7. Mastoparan (10-8-10-5 M) stimulated ACTH secretion from permeabilized AtT-20 cells in the presence and absence of ATP, normally present in the standard permeabilization medium at a concentration of 5 mM. Mastoparan (10-8- 10-5 M)-stimulated ACTH secretion as well as control secretion was reduced when ATP was omitted.8. The results of the present study demonstrate that mastoparan stimulated ACTH secretion from permeabilized AtT-20 cells and displayed characteristics consistent with calcium ion- and GTP-y-gamma-S-stimulated ACTH secretion from permeabilized AtT-20 cells. This suggests that in permeabilized AtT-20 cells, mastoparan directly activates GE and that this G-protein may be a heterotrimeric G-protein. This study also suggests mastoparan may be a useful alternative to GTP-gamma-S as a means of directly activating GE.

Total cortisol, free cortisol, and growth hormone associated with brief social separation experiences in young macaques.

Many behavioral, immunological, and physiological consequences or brief maternal separation in bonnet (Macaca radiata) and pigtail monkeys (Macaca nemistrina) have been documented. However, the impact of social separation on plasma cortisol and growth hormone is unknown for these particular species. In the present study, the behavioral and endocrinological consequences of a 2-week maternal separation in socially housed infant bonnet and pigtail monkeys were followed. In seven pairs (separated and matched control) of bonnet and six pairs of pigtail infants, plasma was obtained under baseline, separated, and reunion conditions twice weekly for the duration of the study. Blood samples were obtained from both infants of the pair in approximately 10 min. Plasma total cortisol, free cortisol, and growth hormone were measured in these samples. Focal animal behavioral observations were made on all subjects twice daily throughout the study period. In both species, total cortisol and free cortisol rose immediately following maternal separation in comparison to the matched nonseparated controls and returned to basal levels (e.g., that of matched nonseparated controls) following reunion with the mother. In contrast, plasma growth hormone rose only in the pigtail infants over a time course that peaked around the time of reunion. Multiple regression techniques indicated for the first week of separation, in the separated but not control subjects, that mean plasma free and total cortisol was positively related to distress behaviors (vocalization and postural slouch) observed during this week and negatively related to social behaviors (play and proximity to others) noted during the same period. In contrast, plasma growth hormone was related to both species and sex of the subjects but unrelated to behavioral variables.

Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein kinase C (PKC)-mediated enhancement of calcium- and guanine nucleotide-evoked adrenocorticotrophin (ACTH) secretion. 2. A profile of the PKC isozymes present in AtT-20 cells was obtained by Western blotting analysis and it was found that AtT-20 cells express the alpha, beta, epsilon and zeta isoforms of PKC. 3. PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically-permeabilized cells. Phorbol 12-myristate 13-acetate (PMA, EC50 = 1 +/- 0.05 nM, which activates all isozymes of PKC, except the zeta isozyme), thymeleatoxin (TMX, EC50 = 10 +/- 0.5 nM, which activates the alpha, beta and gamma isozymes) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA, EC50 = 3 +/- 0.5 nM, a beta 1-selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration-dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA-stimulated ACTH secretion was only 60-70% of that obtained in response to PMA or TMX. 4. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 100 nM to 10 microM. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated. PMA (100 nM) and TMX (100 nM)significantly enhanced ACTH secretion in the effective absence of calcium (i.e. where the free calcium concentration is nM).5. GTP-gamma-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner with a threshold of 1 micro M. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) increased the amount of ACTH secretion evoked by every concentration of GTP-gamma-S investigated.6. The PKC inhibitor, chelerythrine chloride (10 micro M), blocked the PMA (100 nM)-evoked enhancement of calcium- and GTP-micro-S-stimulated ACTH secretion but did not significantly alter calcium- or GTP-micro-S-evoked secretion itself.7. The present paper indicates that AtT-20 cells express multiple isoforms of PKC and that these act at different sites in the secretory pathway for ACTH secretion. The alpha and epsilon isozymes of PKC can act distal to calcium entry to modulate the ability of increased cytosolic calcium concentrations to stimulate ACTH secretion. This site of action is either at the level of, or at some stage distal to, a GTP-binding protein which mediates the effects of calcium upon ACTH secretion. The beta isozyme of PKC may act ata stage early in the secretory pathway to regulate the cytosolic calcium concentration.

The effects of calyculin A upon calcium-, guanine nucleotides- and phorbol 12-myristate 13-acetate-stimulated ACTH secretion from AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein phosphatase involvement in the late stages of the secretory pathway for adrenocorticotrophin (ACTH) secretion. The effects of the type 1 and 2 phosphatase inhibitor calyculin A upon calcium-, guanine nucleotide- and phorbol 12-myristate 13-acetate (PMA)-stimulated ACTH secretion from electrically-permeabilized AtT-20 cells were studied. 2. Calyculin A (1 nM-1 microM) inhibited both calcium (10 microM)- and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (100 microM)-evoked ACTH secretion from permeabilized cells in a concentration-dependent manner. These effects were maximal with 100 nM calyculin A. 3. ACTH secretion was stimulated from electrically-permeabilized cells when the cytosolic free calcium ion concentration, controlled by calcium-EGTA buffers, was raised over the concentration range of 100 nM to 10 microM. This calcium-stimulated ACTH secretion was inhibited by co-incubation with calyculin A (100 nM). 4. GTP-gamma-S (10 nM-100 microM) stimulated ACTH secretion from permeabilized cells at concentrations greater than 1 microM GTP-gamma-S. Co-incubation with calyculin A (100 nM) inhibited this stimulation of ACTH secretion observed at these concentrations of GTP-gamma-S. 5. PMA (100 nM) significantly stimulated ACTH secretion from permeabilized cells in the absence of either calcium and guanine nucleotides and this action was enhanced by calyculin A (100 nM). Furthermore, an inhibition of GTP-gamma-S (100 microM)-stimulated ACTH secretion observed in the presence of calyculin A (100 nM) was not observed in the presence of PMA (100 nM). 6. The results of the present study indicate that dephosphorylation by phosphatases plays an important role in stimulus-secretion coupling in AtT-20 cells and is involved in mediating the effects of GE upon the secretory apparatus in these cells. Furthermore, the point of regulation of the secretory response by PKC which underlies the ability of PKC to amplify the calcium/GE system may lie distal to both GE and these phosphatases.

Effects of protein kinase C activators upon the late stages of the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of phorbol 12-myristate 13-acetate (PMA)-mediated enhancement of calcium-evoked adrenocorticotrophin (ACTH) secretion. 2. PMA stimulated ACTH secretion from intact cells in a concentration-dependent manner. Other phorbol esters; phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD) and diacylglycerol analogues; 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG) also stimulated ACTH release from intact AtT-20 cells. This would suggest that activation of protein kinase C (PKC) stimulates ACTH secretion from AtT-20 cells. 3. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 10(-7) M to 10(-5) M. PMA (10(-7) M) enhanced the amount of ACTH secreted at every concentration of calcium investigated. The PKC inhibitor, chelerythrine (10(-5) M) blocked the PMA (10(-7) M)-evoked enhancement of calcium (10(-5) M)-stimulated ACTH secretion but did not alter significantly the calcium (10(-5) M)-evoked secretion itself. This suggests that PKC modulates the secretory response to increases in intracellular calcium but does not mediate the effects of calcium. 4. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S, 10(-5) M) stimulated ACTH secretion from permeabilized cells in the absence of calcium and was additive with calcium-evoked ACTH secretion up to a maximum value which could be achieved by calcium acting alone. This suggests that a GTP-binding protein mediates the secretory response to increases in the intracellular calcium. PMA (10-7 M) enhanced ACTH secretion stimulated by the combination of calcium and GTP-gamma-S (10-5 M).5. GTP-gamma-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner with a threshold of 10-6 M. PMA (10-7 M) increased the amount of ACTH secretion evoked by every concentration of GTP-gamma-S investigated. Chelerythrine (10-s M) blocked the PMA (10-7 M)-evoked enhancement of GTP-gamma-S (10-4 M)-stimulated ACTH secretion but did not significantly alter GTP-gamma-S(10-4 M)-evoked secretion itself. This suggests that PKC modulates the secretory response to GTP-gamma-S but does not mediate the effects of GTP-gamma-S.6. GTP-gamma-S (10-8-10-4-M) stimulated ACTH secretion from permeabilized cells either in the presence or absence of ATP (5 mM) indicating that its effects on secretion are ATP-independent.7. The results of the present study support the hypothesis that, in AtT-20 cells, PMA is acting at some site distal to calcium entry which modulates the ability of an increase in cytosolic calcium concentration to stimulate ACTH secretion. This site of action is either at the level of or at some stage distal to a GTP-binding protein which mediates the effects of calcium upon secretion.8. PMA, unlike adenosine 3':5'-cyclic monophosphate (cyclic AMP) (Guild, 1991), can stimulate ACTH secretion from permeabilized cells in the absence of added calcium and guanine nucleotides which suggests that PMA and cyclic AMP are acting through distinct mechanisms at this post calcium site of action.