Trypsinization of bovine parathyroid cells abolishes Ca2+-regulated parathyroid hormone secretion

1989 ◽  
Vol 122 (1) ◽  
pp. 213-218 ◽  
Author(s):  
R. Muff ◽  
J. A. Fischer
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Adrenergic Receptors ◽  
Hormone Secretion ◽  
Dependent Manner ◽  
Kinase C ◽  
And Control ◽  
Β Adrenergic Receptors ◽  
Dose Dependent

ABSTRACT The secretion of parathyroid hormone (PTH) is inversely related to the extracellular Ca2+ concentration (Cae2+). To test the hypothesis that a Ca2+ sensor on the surface of parathyroid cells is involved in Ca2+-regulated PTH secretion, limited trypsinization of bovine parathyroid cells was carried out. Treatment with trypsin (1·1–10 mg/ml) inhibited, in a dose-dependent manner, PTH secretion stimulated by lowering Cae2+ from 2·0 to 0·5 mmol/l. In control cells, activation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced PTH secretion at 2·0 mmol Cae2+/1 but not at 0·5 mmol Cae2+/1. In trypsinized cells, however, TPA enhanced PTH secretion at both 0·5 and 2·0 mmol Cae2+/1. Isoproterenol-stimulated PTH secretion was maintained in trypsinized cells, but reduced cyclic AMP production revealed that some β-adrenergic receptors were destroyed. The cytosolic free Ca2+ concentration (Cai2+), as measured with fura-2, was raised within seconds in response to increasing Cae2+ from 0·5 to 2·0 mmol/l and was then lowered within 1 min to a sustained plateau; the changes were the same in trypsinized and control cells. In conclusion, trypsinization of parathyroid cells abolished Ca2+-regulated PTH secretion without affecting Cai2+. Journal of Endocrinology (1989) 122, 213–218

Effect of phorbol myristate acetate on secretion of parathyroid hormone

1988 ◽  
Vol 254 (1) ◽  
pp. E63-E70 ◽  
Author(s):  
J. J. Morrissey
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Membrane Fraction ◽  
Hormone Secretion ◽  
Myristate Acetate ◽  
Kinase C ◽  
Low Calcium ◽  
Protein Kinase C Activity ◽  
High Calcium

The influence of phorbol myristate acetate (PMA), an activator of protein kinase c, on the secretion of parathyroid hormone from collagenase-dispersed bovine parathyroid cells was tested. The cells were incubated at low (0.5 mM) or high (2.0 mM) concentrations of calcium in the medium, and the hormone secreted into the medium was measured by a radioimmunoassay that recognizes both intact and C-terminal fragments of hormone. At low calcium, the secretory rate averaged 32 +/- 3.8 ng.h-1.(10(5) cells)-1. The addition of 1.6 microM PMA did not affect secretion. At high calcium there was a significant suppression of secretion by 38% to 19.8 +/- 3 ng.h-1.(10(5) cells)-1. The addition of 1.6 microM PMA significantly stimulated hormone secretion to 35.8 +/- 8 ng.h-1.(10(5) cells)-1, a rate indistinguishable from low calcium. This stimulatory effect of PMA at high calcium was seen at PMA concentrations as low as 1.6 nM, did not occur with a biologically inactive 4 alpha-isomer of phorbol ester, and was independent of changes in cellular adenosine 3',5'-cyclic monophosphate levels. Examination of 32P-labeled phosphoproteins by two-dimensional gel electrophoresis revealed acidic proteins of approximately 20,000 and 100,000 Da that were phosphorylated at low and high calcium + 1.6 microM PMA but not at high calcium alone. The protein kinase c activity associated with the membrane fraction of parathyroid cells significantly decreased 40% when the cells were incubated at high vs. low calcium. The data suggest that calcium may regulate parathyroid hormone secretion through changes in protein kinase c activity of the membrane fraction of the cell and protein phosphorylation.

Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2

1994 ◽  
Vol 131 (5) ◽  
pp. 510-515 ◽  
Author(s):  
Osamu Kozawa ◽  
Haruhiko Tokuda ◽  
Atsushi Suzuki ◽  
Jun Kotoyori ◽  
Yoshiaki Ito ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Phosphoinositide Hydrolysis ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent

Kozawa O, Tokuda H, Suzuki A, Kotoyori J, Ito Y, Oiso Y. Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2. Eur J Endocrinol 1994;131:510–15. ISSN 0804–4643 It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2α stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2α-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2α in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2α- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2α-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2α-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells. Osamu Kozawa, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan

Inhibition of PAH transport by parathyroid hormone in OK cells: involvement of protein kinase C pathway

1997 ◽  
Vol 273 (5) ◽  
pp. F674-F679 ◽  
Author(s):  
Junya Nagai ◽  
Ikuko Yano ◽  
Yukiya Hashimoto ◽  
Mikihisa Takano ◽  
Ken-Ichi Inui
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Regulatory Control ◽  
Dependent Manner ◽  
Transcellular Transport ◽  
Ok Cells ◽  
Kinase C ◽  
Kinase A

We have previously shown that the p-aminohippurate (PAH) transport system in OK kidney epithelial cell line is under the regulatory control of protein kinase C. Parathyroid hormone (PTH) could activate protein kinase C, as well as protein kinase A, in OK cells. In the present study, the effect of PTH on PAH transport was studied in OK cells. PTH inhibited the transcellular transport of PAH from the basal to the apical side, as well as the accumulation of PAH in OK cells. Basolateral PAH uptake was inhibited by PTH in a dose- and time-dependent manner. Protein kinase A activators did not affect the transcellular transport or the accumulation of PAH. The PTH-induced inhibition of the accumulation of PAH was blocked by a protein kinase C inhibitor staurosporine. These results suggest that PTH inhibits the PAH transport in OK cells and that the messenger system mediated by protein kinase C, not protein kinase A, plays an important role in the regulation of PAH transport by PTH.

scholarly journals Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

1998 ◽  
Vol 9 (5) ◽  
pp. 1209-1220 ◽  
Author(s):  
Alexander V. Chibalin ◽  
Juleen R. Zierath ◽  
Adrian I. Katz ◽  
Per-Olof Berggren ◽  
Alejandro M. Bertorello
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Α Subunit ◽  
Kinase C ◽  
Dose Dependent ◽  
Α Subunits ◽  
K Activity ◽  
Phosphatidylinositol 3

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

scholarly journals Regulation of prostaglandin synthesis by protein kinase C in mouse peritoneal macrophages

1989 ◽  
Vol 260 (2) ◽  
pp. 471-478 ◽  
Author(s):  
H J Pfannkuche ◽  
V Kaever ◽  
D Gemsa ◽  
K Resch
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Membrane Phospholipids ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Mouse Peritoneal Macrophages

Resident mouse peritoneal macrophages synthesized and released prostaglandins (PGs) when challenged with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (DiC8). Both stimuli were found to activate Ca2+/phospholipid-dependent protein kinase C (PKC). 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (‘H-7’) and D-sphingosine, known to inhibit PKC by different mechanisms, were able to decrease the PKC activity of macrophages in a dose-dependent manner. Addition of either PKC inhibitor decreased PG synthesis and also the release of arachidonic acid (AA) from phospholipids induced by TPA or DiC8. Simultaneously TPA or DiC8 also decreased incorporation of free AA into membrane phospholipids of macrophages. AA incorporation could be restored, however, by pretreatment with the PKC inhibitors. Our results demonstrate an involvement of PKC in the regulation of PG synthesis in mouse peritoneal macrophages and provide further evidence that reacylation of released fatty acids may be an important regulatory step.

scholarly journals Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

2015 ◽  
Vol 36 (2) ◽  
pp. 555-568 ◽  
Author(s):  
Jiaoqian Ying ◽  
Yuan Zhang ◽  
Shan Gong ◽  
Zhigang Chang ◽  
Xiaofeng Zhou ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Dependent Manner ◽  
The Novel ◽  
Inotropic Effects ◽  
Kinase C ◽  
Melanocortin 4 Receptor ◽  
Dose Dependent ◽  
Ca2 Channels

Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca2+ currents (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased ICa,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-β-S or anti-Gβ antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca2+-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKCθ), and PKCθ inhibition abolished the decrease in ICa,L induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca2+ channels via the MC4-R that couples sequentially to the βγ subunits of Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes.

scholarly journals Entry of influenza viruses into cells is inhibited by a highly specific protein kinase C inhibitor

2000 ◽  
Vol 81 (11) ◽  
pp. 2697-2705 ◽  
Author(s):  
Christiana N. Root ◽  
Elizabeth G. Wills ◽  
LaShonn L. McNair ◽  
Gary R. Whittaker
Keyword(s):  
Influenza Virus ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent

Following binding to cell surface sialic acid, entry of influenza viruses into cells is mediated by endocytosis. Productive entry of influenza virus requires the low-pH environment of the late endosome for fusion and release of the virus into the cytoplasm and transport of the virus genome into the nucleus. We investigated novel mechanisms to inhibit influenza virus infection using highly specific inhibitors of protein kinase C. We found that one inhibitor, bisindolylmaleimide I, prevented replication of influenza A virus in a dose-dependent manner when added at the time of infection, but had little specific effect when added 2 h after infection had commenced. Virus yields dropped by more than 3 log units in the presence of micromolar levels of bisindolylmaleimide I. Influenza B virus replication was also inhibited by bisindolylmaleimide at micromolar concentrations. We carried out experiments to determine the point in infection that was blocked by bisindolylmaleimide I, and determined that entry of viral ribonucleoproteins (vRNPs) into the nucleus was prevented. Upon drug washout vRNP nuclear entry resumed, showing that bisindolylmaleimide I is reversible. Bisindolylmaleimide I did not affect virus binding and was apparently not acting as a weak base, because its effects were independent of the pH of the external growth medium. These experiments show that bisindolylmaleimide I blocks replication of different types of influenza virus in a dose-dependent and reversible manner, and that virus entry into the cell is inhibited.

scholarly journals Inhibitory action of polyamines on protein kinase C association to membranes

1987 ◽  
Vol 247 (1) ◽  
pp. 175-180 ◽  
Author(s):  
M Moruzzi ◽  
B Barbiroli ◽  
M G Monti ◽  
B Tadolini ◽  
G Hakim ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Activation Process ◽  
Dependent Manner ◽  
Kinase C ◽  
Association Reaction ◽  
Dose Dependent ◽  
Inside Out

Physiological activation of protein kinase C requires the interaction of this enzyme with cellular membranes [Nishizuka (1986) Science 233, 305-312]. In the present work a reconstituted system of protein kinase C and human inside-out erythrocyte vesicles was utilized to study the effect in vitro of naturally occurring polyamines on the activation process of protein kinase C. The active membrane-associated complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The association reaction of the enzyme to membrane was rapid, being complete within 1 min at 25 degrees C. The addition of polyamines, particularly spermine, greatly decreased in a dose-dependent manner the amount of protein kinase C bound to membranes (i.e. in the activated form). The effect observed was quite specific, since it was dependent on the chemical structure of the polyamine and it was manifest at micromolar concentrations of the polycation; the order of potency was spermine greater than spermidine greater than putrescine. A characterization of this effect is presented and possible physiological implications are discussed.

Thromboxane A2-stimulated phospholipase D in osteoblast-like cells: possible involvement of PKC

1995 ◽  
Vol 269 (3) ◽  
pp. E524-E529 ◽  
Author(s):  
J. Shinoda ◽  
A. Suzuki ◽  
Y. Oiso ◽  
O. Kozawa
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Inositol Phosphates ◽  
Specific Inhibitor ◽  
Thromboxane A2 ◽  
Dependent Manner ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Dose Dependent

We examined the effect of thromboxane A2 (TxA2) on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of TxA2, stimulated the formations of both choline and inositol phosphates in a dose-dependent manner in the range between 10 nM and 10 microM. The formation of choline stimulated by a combination of STA2 and 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C-activating phorbol ester, was not additive. 1-(5-Isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinases, suppressed the formation of choline induced by STA2 as well as that by TPA, but 20 microM N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a control for H-7 as a protein kinase C inhibitor, had little effect. Calphostin C, a potent and specific inhibitor of protein kinase C, also suppressed the formation of choline induced by STA2. The STA2-induced formation of choline was significantly reduced by chelating extracellular Ca2+ with ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid. STA2 dose dependently stimulated 45Ca2+ influx from extracellular space. STA2 stimulated DNA synthesis of MC3T3-E1 cells and increased the number of these cells. These results suggest that TxA2 stimulates phospholipase D in osteoblast-like cells, resulting in the direction of their proliferation, and that the activation of protein kinase C is involved in the stimulation of phospholipase D.

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