Protein kinase C (PKC) and neuropeptidergic stimulation of alveolar macrophages (AMs) (original) (raw)
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Protein Kinase C Mediates the Effect of Vasopressin in Pituitary Corticotrophs
Molecular Endocrinology, 1989
The role of protein kinase C (PKC) on vasopressin (VP) action was investigated by inhibition of endogenous PKC using prolonged incubation of the cells with phorbol ester, and by direct measurement of PKC activity in pituitary cells. Preincubation of the cells for 6 h with 100 ni wi TPA at 37 C resulted in a 90% decrease in total PKC activity. In the PKCdepleted cells, cAMP responses to stimulation with 100 nwi CRF for 30 min were normal, but the potentiating effects of VP and PMA on CRF-stimulated cAMP production were abolished. The stimulation of ACTH secretion by VP and PMA alone was also abolished in PKC-depleted cells. PKC activity in cytosolic and detergent-solubilized membrane fractions from enriched pituitary corticotrophs obtained by centrifugal elutriation, was directly measured by enzymatic assays and by immunoblotting techniques. Basal PKC activity was higher in the cytosol than in the membranes (8.43 ± 0.47 and 1.93 ± 0.11 pmol 32 P incorporated/10 min, respectively). After incubation of the cells with VP for 15 min or [ 3 H] phorbol-12-myristate-13-acetate (PMA) for 30 min, PKC activity in cytosol was decreased by 40% and 89%, respectively, while the activity in the membrane was increased by 138% and 405%, respectively. Such VP-and PMA-induced translocation of PKC was also observed when the enzyme content in the cytosol and the membranes was measured by immunoblotting using a specific anti-PKC antibody and [ 12S l]protein A. Autoradiographic analysis of immunoblots revealed an 80 kilodalton band characteristic of PKC, with OD higher in the cytosolic than in the membrane fractions. Consistent with the enzymatic assay, 15-min treatment with VP or PMA caused a marked decrease in OD of cytosolic PKC, accompanied by a corresponding increase in the membrane-associated enzyme. These data demonstrate that PKC is essential for the effect of VP in the pituitary corticotroph. This observation, in conjunction with the ability of VP to cause redistribution
Cancer Research, 1989
The protein designated 7B2 is a recently discovered pituitary polypeptide which is selectively expressed in cells containing secretory granules, such as neurons and endocrine cells. Northern blot analysis of 7B2 gene expression in small cell lung carcinoma (SCLC) cell lines revealed that 7B2 was expressed in all nine cell lines of the classic type tested, but in six of seven SCLC cell lines of the variant type, 7B2 expression could not be detected. In only one of four non-SCLC cell lines tested, 7B2 was expressed. Furthermore, in 16 primary human non-SCLCs, no or only very low expression of 7B2 was found. In the eight primary human SCLCs tested, expression of 7B2 appeared variable: three exhibited a high level of expression; three a low level; while in two cases, expression was very low or not detectable at all. Finally, the three carcinoid tumors tested expressed very high levels of 7B2 mRNA. These data indicate that the 7B2 gene is a useful marker not only to discriminate between classic and variant types of SCLC cell lines, but also in human lung cancer diagnosis.
Molecular Endocrinology, 1987
pearing as a doublet of 78/80 kilodaltons. Long-term treatment (24 h) of cells with 0.6 HM TPA caused the specific loss of immunologically reactive PKC. Consistently, TPA pretreatment decreased the amount of phosphatidylserine-dependent protein kinase activity measured in vitro by 90%. In control cells, vasopressin (AVP) stimulated ACTH secretion and potentiated ACTH secretion stimulated by CRF. After a 24-h treatment with 0.6 MM TPA, secretory responses to AVP and the potentiating effect of AVP on CRF action were completely abolished. In contrast, CRF action on ACTH secretion, thought to be mediated by cAMP, was unaffected. Similarly, forskolin-and 8 bromo-cAMP-induced ACTH secretion remained unchanged after TPA pretreatment. These results indicate a crucial role for PKC in mediating the effects of AVP on ACTH secretion and on the potentiating action of AVP on CRF-induced secretion from corticotropic cells of the anterior pituitary. (Molecular Endocrinology 1: 555-560, 1987)
CNS distribution of a novel pituitary protein ‘7B2’: localization in secretory and synaptic vesicles
Brain Research, 1986
Key words: immunoreactivity of a pituitary protein '7B2' --central nervous system --subcellular organization --molecular form A detailed study of the immunoreactivity distribution of a novel pituitary protein, '7B2', within specific brain regions and in spinal cord revealed a complex network of neurons and fibers. Immunoreactive cell bodies were found exclusively in the hypothalamus. Stained fibers were more widely distributed and encompassed nearly 100 structures, including several limbic and autonomic nuclei. Biogenic amine centers such as substantia nigra, raphe nuclei and locus coeruleus have also contained immunoreactive 7B2. Subcellular fractionation using differential centrifugation and Percoll gradient revealed the preferential localization of this protein within granule-enriched fractions of rat neurointermediate lobe of the hypophysis. In agreement, ultrastructural studies revealed the presence of 7B2 within secretory-like granules in the median eminence and within synaptic vesicles of selected extrahypothalamic nuclei. Based upon migration on SDS-PAGE, the apparent molecular weight of the major form of 7B2 extracted from different brain regions was found to be 22,000, identical to that of the pituitary form.
The Journal of Physiology, 1999
In many neuroendocrine cells of the anterior pituitary gland, activation of the protein kinase C (PKC) intracellular signalling pathway leads to sustained cellular excitability and neurosecretion although the cellular mechanisms and targets for PKC are poorly understood (Ozawa & Sand, 1986; Mason et al. 1988). In anterior pituitary corticotrophs PKC mediates the sustained phase of adrenocorticotrophin (ACTH) secretion stimulated by activation of the phospholipase C pathway by the hypothalamic secretagogue vasopressin (Carvallo & Aguilera, 1989; Oki et al. 1990). Vasopressin elicits a biphasic elevation of intracellular free calcium (Corcuff et al. 1993; Tse & Lee, 1998) and during the sustained phase of calcium influx stimulates PKC translocation and enhances PKC activity at the plasma membrane, an effect that is mimicked by the cell-permeant PKC-activating phorbol esters (Carvallo & Aguilera, 1989). In AtT20 mouse corticotroph cells phorbol-ester-mediated activation of PKC has been proposed to exert effects both distal and proximal to voltage-dependent calcium influx, which may result from activation of different PKC isoforms (McFerran et al. 1995) to elicit ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995). Intracellular free calcium measurements in AtT20 cells suggest that PKC-induced calcium influx results, at least in part, from inhibition of TEA-sensitive potassium conductances. This inhibition results in membrane depolarization and subsequent, indirect, enhancement of voltage-gated calcium influx (Reisine & Guild, 1987; Reisine, 1989). However, ionic conductances regulated by PKC activation in corticotrophs have not been identified. In rat GHÚC1 pituitary cells activation of PKC results in inhibition of the TEA-sensitive large conductance calciumand voltage-activated potassium (BK) channels (Shipston & Armstrong, 1996), which act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997). Furthermore, BK channels are an important target for cellular regulation by two distinct, physiologically relevant, intracellular signalling pathways in AtT20 corticotrophs. Activation of the cAMP-dependent protein kinase pathway results in inhibition of BK channels leading to a robust
Chemosensory Functions for Pulmonary Neuroendocrine Cells
American Journal of Respiratory Cell and Molecular Biology, 2014
The mammalian airways are sensitive to inhaled stimuli, and airway diseases are characterized by hypersensitivity to volatile stimuli, such as perfumes, industrial solvents, and others. However, the identity and function of the cells in the airway that can sense volatile chemicals remain uncertain, particularly in humans. Here, we show that solitary pulmonary neuroendocrine cells (PNECs), which are morphologically distinct and physiologically undefined, might serve as chemosensory cells in human airways. This conclusion is based on our finding that some human PNECs expressed members of the olfactory receptor (OR) family in vivo and in primary cell culture, and are anatomically positioned in the airway epithelium to respond to inhaled volatile chemicals. Furthermore, apical exposure of primaryculture human airway epithelial cells to volatile chemicals decreased levels of serotonin in PNECs, and the led to the release of the neuropeptide calcitonin gene-related peptide (CGRP) to the basal medium. These data suggest that volatile stimulation of PNECs can lead to the secretion of factors that are capable of stimulating the corresponding receptors in the lung epithelium. We also found that the distribution of serotonin and neuropeptide receptors may change in chronic obstructive pulmonary disease, suggesting that increased PNEC-dependent chemoresponsiveness might contribute to the altered sensitivity to volatile stimuli in this disease. Together, these data indicate that human airway epithelia harbor specialized cells that respond to volatile chemical stimuli, and may help to explain clinical observations of odorant-induced airway reactions.
Developmental expression of protein kinase C subspecies in rat brain-pituitary axis
Molecular and Cellular Endocrinology, 1994
We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKCa, PKCfl and PKCe when brains from 6-day-old and 3-month-old female rats were compared, whereas PKCr and PKC6 increased markedly during this period. On the other hand, brain PKC~ decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKCa, PKCfl and PKC~ when 6-day-old and 3-month-old female rats were compared, while PKCr was not detected. The major band of pituitary PKCd (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change. On the other hand PKCe which appeared as one band (92 kDa) in the 6-day-old pituitaries, increased markedly in the 3-month-old rat and was followed by a second major band (68 kDa) which was not observed in the juvenile rat. The increase in the PKC-positive cells and the decrease in histone phosphorylation in the developing pituitary is probably due to the marked dominance of PKCe in the adult rat pituitary.