Zymogen granules of mouse parotid acinar cells are acidified in situ in an ATP-dependent manner

We used molecular biological and patch-clamp techniques to identify the Ca2+-activated K+ channel genes in mouse parotid acinar cells. Two types of K+ channels were activated by intracellular Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the intermediate and maxi-K classes of Ca2+-activated K+ channels, typified by the mIK1 ( Kcnn4) and mSlo ( Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo β-subunits ( Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each β-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from β1 knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the β4-subunit.

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Phosphorylation of myristoylated alanine-rich C kinase substrate is involved in the cAMP-dependent amylase release in parotid acinar cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90536.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. G1382-G1390 ◽

Cited By ~ 19

Author(s):

Keitaro Satoh ◽

Miwako Matsuki-Fukushima ◽

Bing Qi ◽

Ming-Yu Guo ◽

Takanori Narita ◽

...

Keyword(s):

Protein Kinase ◽

Acinar Cells ◽

Cellular Migration ◽

Intracellular Camp ◽

Dependent Manner ◽

Amylase Release ◽

Kinase Substrate ◽

Parotid Acinar Cells ◽

Dependent Protein ◽

Rat Parotid

Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major cellular substrate for protein kinase C (PKC). MARCKS has been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as phagocytosis, endocytosis, and exocytosis. The involvement of MARCKS phosphorylation in secretory function has been reported in Ca2+-mediated exocytosis. In rat parotid acinar cells, the activation of β-adrenergic receptors provokes exocytotic amylase release via accumulation of intracellular cAMP levels. Here, we studied the involvement of MARCKS phosphorylation in the cAMP-dependent amylase release in rat parotid acinar cells. MARCKS protein was detected in rat parotid acinar cells by Western blotting. The β-adrenergic agonist isoproterenol (IPR) induced MARCKS phosphorylation in a time-dependent manner. Translocation of a part of phosphorylated MARCKS from the membrane to the cytosol and enhancement of MARCKS phosphorylation at the apical membrane site induced by IPR were observed by immunohistochemistry. H89, a cAMP-dependent protein kinase (PKA) inhibitor, inhibited the IPR-induced MARCKS phosphorylation. The PKCδ inhibitor rottlerin inhibited the IPR-induced MARCKS phosphorylation and amylase release. IPR activated PKCδ, and the effects of IPR were inhibited by the PKA inhibitors. A MARCKS-related peptide partially inhibited the IPR-induced amylase release. These findings suggest that MARCKS phosphorylation via the activation of PKCδ, which is downstream of PKA activation, is involved in the cAMP-dependent amylase release in parotid acinar cells.

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Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

Biochemical Journal ◽

10.1042/bj3630059 ◽

2002 ◽

Vol 363 (1) ◽

pp. 59-66 ◽

Cited By ~ 7

Author(s):

Akihiro NEZU ◽

Akihiko TANIMURA ◽

Takao MORITA ◽

Kazuharu IRIE ◽

Toshihiko YAJIMA ◽

...

Keyword(s):

Ryanodine Receptors ◽

Critical Role ◽

Acinar Cells ◽

Ion Concentration ◽

Pcr Analysis ◽

Zymogen Granule ◽

Zymogen Granules ◽

Weak Band ◽

Parotid Acinar Cells ◽

Rat Parotid

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the β-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca2+ signalling as intracellular Ca2+ stores, changes in cytosolic free Ca2+ ion concentration ([Ca2+]i) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca2+]i induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca2+ wave towards the basolateral region. The magnitude of the [Ca2+]i response and the speed of the Ca2+ wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP3R) was performed on zymogen granule membranes and microsomes using anti-IP3R antibodies. The immunoreactivity of all three IP3Rs was clearly observed in the microsomal preparations. Although a weak band of IP3R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca2+ signalling in rat parotid acinar cells.

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Two modes of regulation of the phospholipase C-linked substance-P receptor in rat parotid acinar cells

Biochemical Journal ◽

10.1042/bj2530459 ◽

1988 ◽

Vol 253 (2) ◽

pp. 459-466 ◽

Cited By ~ 28

Author(s):

H Sugiya ◽

J F Obie ◽

J W Putney

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Substance P ◽

Acinar Cells ◽

Control Mechanisms ◽

Dependent Manner ◽

Parotid Acinar Cells ◽

Kinase C ◽

Substance P Receptor ◽

Rat Parotid

In rat parotid acinar cells prelabelled with [3H]inositol, substance P (100 nM) induced the formation of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 reached a maximum 7 s after substance P stimulation, and thereafter decreased and reached a stable value at 60 s. When the cells were exposed to substance P for 10, 30, 60, or 300 s, washed, and re-exposed to this peptide, the formation of [3H]inositol trisphosphate (InsP3) was attenuated in a time-dependent manner. In the cells pretreated as described above, the number of [3H]substance-P-binding sites (Bmax) was also decreased. Possible role(s) of Ca2+ and protein kinase (protein kinase C) control mechanisms in regulating substance P responses were investigated. Desensitization of substance P-induced InsP3 was not affected by the Ca2+ ionophore ionomycin, nor was it dependent on Ca2+ mobilization. On the other hand, in the presence of 4 beta-phorbol 12,13-dibutyrate (PDBu) and 12-O-tetradecanoyl-4 beta-phorbol 13-acetate, known activators of protein kinase C, substance P-induced InsP3 formation was inhibited. However, PDBu had no effect on [3H]substance P binding, whether present during the assay or when cells were pretreated. The persistent desensitization of InsP3 formation induced by substance P was not affected by PDBu. These results suggest that the persistent desensitization of InsP3 formation induced by substance P is a homologous process involving down-regulation of the substance P receptor; the mechanism does not appear to involve, or to be affected by, the Ca2+ or protein kinase C signalling systems. Protein kinase C activation can, however, inhibit substance P-induced InsP3 formation, which may indicate the presence of a negative-feedback control on the substance P pathway.

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5-Hydroxytryptamine synthesis, storage, and secretion from rat pancreatic acinar cells

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y84-124 ◽

1984 ◽

Vol 62 (7) ◽

pp. 755-761 ◽

Cited By ~ 4

Author(s):

Pierre Falardeau ◽

Seymour Heisler

Keyword(s):

Biological Function ◽

De Novo ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Dependent Manner ◽

Zymogen Granules ◽

De Novo Synthesis ◽

Concentration Dependent Manner ◽

Granule Fraction ◽

Stimulation Of

Pancreatic acinar cells are known to synthesize serotonin and dopamine from extracellular precursors. In this study, we found that small amounts of serotonin, alone, were preferentially stored in zymogen granules of acinar cells. Serotonin was apparently incorporated into mature rather than newly formed granules. This was based on the fact that the amine, rather than the newly synthesized zymogen protein, appeared first in the purified granule fraction; additionally, concentrations of cycloheximide, which markedly inhibited de novo synthesis of zymogen protein, did not affect the incorporation of serotonin into the granule fraction. Serotonin, synthesized by the acinar cells, can be secreted along with amylase in a time- and concentration-dependent manner following stimulation of acinar cells by a variety of pancreatic secretagogues. Whether serotonin secreted into pancreatic juice has a biological function remains unknown.

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Evidence for colocalization and interaction between 37 and 39 kDa isoforms of secretory carrier membrane proteins (SCAMPs)

Journal of Cell Science ◽

10.1242/jcs.110.13.1533 ◽

1997 ◽

Vol 110 (13) ◽

pp. 1533-1541 ◽

Cited By ~ 2

Author(s):

T.T. Wu ◽

J.D. Castle

Keyword(s):

Membrane Proteins ◽

Acinar Cells ◽

Chemical Crosslinking ◽

Vesicle Membrane ◽

Parotid Acinar Cells ◽

Single Protein ◽

Transport Vesicle ◽

Potential Formation ◽

Secretory Carrier Membrane Proteins

Secretory carrier membrane proteins (SCAMPs) are proteins of post-Golgi recycling carriers, including regulated secretory organelles. The two major size variants, SCAMP1 (37 kDa) and SCAMP2 (39 kDa), extensively colocalize in membranes of fibroblasts and parotid acinar cells based on immunocytochemistry and velocity centrifugation, although the relative amounts of each variant may differ in selected organelles. SCAMP1, and to a lesser extent, SCAMP2, are substrates for chemical crosslinking in situ, and the recognizable crosslinking products of SCAMP1 suggest potential formation of homomultimers. SCAMP1 and SCAMP2 can be co-immunoprecipitated following detergent solubilization, using antibodies that specifically react with only one of the variants. Both the localization and interactions of SCAMPs are reiterated using transfected SCAMP1 that is epitope tagged (myc) at either the NH2 or COOH terminus and an anti-myc antibody. Like other transport vesicle membrane proteins, SCAMPs form complexes that apparently include homomultimers. Furthermore, these studies suggest that both SCAMP1 and SCAMP2 may function together in a single protein complex.

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Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

Biochemical Journal ◽

10.1042/0264-6021:3630059 ◽

2002 ◽

Vol 363 (1) ◽

pp. 59 ◽

Cited By ~ 11

Author(s):

Akihiro NEZU ◽

Akihiko TANIMURA ◽

Takao MORITA ◽

Kazuharu IRIE ◽

Toshihiko YAJIMA ◽

...

Keyword(s):

Acinar Cells ◽

Zymogen Granules ◽

Parotid Acinar Cells ◽

Rat Parotid

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The regulation of the phosphorylation of inositol 1,3,4-trisphosphate in cell-free preparations and its relevance to the formation of inositol 1,3,4,6-tetrakisphosphate in agonist-stimulated rat parotid acinar cells

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)47191-3 ◽

1989 ◽

Vol 264 (33) ◽

pp. 19871-19878

Author(s):

P J Hughes ◽

A R Hughes ◽

J W Putney ◽

S B Shears

Keyword(s):

Acinar Cells ◽

Parotid Acinar Cells ◽

Rat Parotid

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Exposure to Electromagnetic Fields (EMF) from Submarine Power Cables Can Trigger Strength-Dependent Behavioural and Physiological Responses in Edible Crab, Cancer pagurus (L.)

Journal of Marine Science and Engineering ◽

10.3390/jmse9070776 ◽

2021 ◽

Vol 9 (7) ◽

pp. 776

Author(s):

Kevin Scott ◽

Petra Harsanyi ◽

Blair A. A. Easton ◽

Althea J. R. Piper ◽

Corentine M. V. Rochas ◽

...

Keyword(s):

Electromagnetic Field ◽

Policy Making ◽

Physiological Responses ◽

Dependent Manner ◽

Power Cables ◽

Environmental Assessments ◽

Cancer Pagurus ◽

Commercially Important ◽

Total Haemocyte Count

The current study investigated the effects of different strength Electromagnetic Field (EMF) exposure (250 µT, 500 µT, 1000 µT) on the commercially important decapod, edible crab (Cancer pagurus, Linnaeus, 1758). Stress related parameters were measured (l-Lactate, d-Glucose, Total Haemocyte Count (THC)) in addition to behavioural and response parameters (shelter preference and time spent resting/roaming) over 24 h periods. EMF strengths of 250 µT were found to have limited physiological and behavioural impacts. Exposure to 500 µT and 1000 µT were found to disrupt the l-Lactate and d-Glucose circadian rhythm and alter THC. Crabs showed a clear attraction to EMF exposed (500 µT and 1000 µT) shelters with a significant reduction in time spent roaming. Consequently, EMF emitted from MREDs will likely affect crabs in a strength-dependent manner thus highlighting the need for reliable in-situ measurements. This information is essential for policy making, environmental assessments, and in understanding the impacts of increased anthropogenic EMF on marine organisms.

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