BBA - Molecular Cell Research (v.1497, #1)

Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions by Carine Michiels; Thierry Arnould; José Remacle (1-10).
The origin of several vascular pathologies involves sudden or recurrent oxygen deficiency. In this review, we examine what the biochemical and molecular responses of the endothelial cells to the lack of oxygen are and how these responses may account for the features observed in pathological situations, mainly by modifications of cell–cell interactions. Two major responses of the endothelial cells have been observed depending on the degree and duration of the oxygen deficiency. Firstly, acute hypoxia rapidly activates the endothelial cells to release inflammatory mediators and growth factors. These inflammatory mediators are able to recruit and promote the adherence of neutrophils to the endothelium where they become activated. The synthesis of platelet-activating factor plays a key role in this adherence process. Secondly, longer periods of hypoxia increase the expression of specific genes such as those encoding some cytokines as well as for the growth factors platelet-derived growth factor and vascular endothelial growth factor. The transcriptional induction of these genes is mediated through the activation of several transcription factors, the most important one being hypoxia inducible factor-1. The link between our knowledge of the signalling cascade of the cellular and molecular events initiated by hypoxia and their involvement in several vascular pathological situations, varicose veins, tumor angiogenesis and pulmonary hypertension is discussed briefly.
Keywords: Endothelial cell; Hypoxia; Leukocyte adherence; Hypoxia inducible factor-1;

The treatment of H4-IIE cells (an immortalised liver cell line derived from the Reuber rat hepatoma) with thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or pretreatment with EGTA, stimulated Ca2+ inflow (assayed using intracellular fluo-3 and a Ca2+ add-back protocol). No stimulation of Mn2+ inflow by thapsigargin was detected. Thapsigargin-stimulated Ca2+ inflow was inhibited by Gd3+ (maximal inhibition at 2 μM Gd3+), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca2+ channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca2+ inflow and release of Ca2+ from intracellular stores. No inhibition of either Ca2+ inflow or Ca2+ release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca2+ inflow by calmidazolium was only observed when the inhibitor was added before thapsigargin. Pretreatment of H4-IIE cells with pertussis toxin, or treatment with brefeldin A, did not inhibit thapsigargin-stimulated Ca2+ inflow. Compared with freshly isolated rat hepatocytes, H4-IIE cells exhibited a more diffuse actin cytoskeleton, and a more granular arrangement of the endoplasmic reticulum (ER). In contrast to freshly isolated hepatocytes, the arrangement of the ER in H4-IIE cells was not affected by pertussis toxin treatment. Western blot analysis of lysates of freshly isolated rat hepatocytes revealed two forms of Gi2α with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of Gi2α and substantially less total Gi2α than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca2+ channels which do not require calmodulin for activation and exhibit properties similar to those in freshly isolated rat hepatocytes, including susceptibility to inhibition by relatively high concentrations of voltage-operated Ca2+ channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require Gi2α for activation. Possible explanations for differences in the requirement for Gi2α in the activation of Ca2+ inflow are briefly discussed.
Keywords: Liver cell; H4-IIE cell; Store-operated Ca2+ channel; Thapsigargin; Calmodulin; GTP-binding regulatory protein;

Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn by Yi Wu; Yukio Ozaki; Katsue Inoue; Kaneo Satoh; Tsukasa Ohmori; Yutaka Yatomi; Koji Owada (27-36).
Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin αIIbβ3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 μM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (radioimmunoprecipitation assay) buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.
Keywords: Platelet; Tyrosine kinase; c-Src; Fyn; Tyr-530 dephosphorylation; Thrombin;

Release from apoptosis correlates with tumor progression in the AKR lymphoma by Sigalit Kay; Natalie Donin; Moshe Michowitz; Dina Katzenelson; Jehuda Hiss; Ginnette Schibi; Asher Pinchassov; Judith Leibovici (37-50).
Disturbance of apoptosis is an established factor in tumorigenesis. The role of apoptosis in tumor progression is not yet clear. In the present study we compared the tendency to spontaneous apoptosis (and the proliferative capacity) of tumor cells derived from primary (PT) and metastatic tumor (MT) cells of several AKR lymphoma variants. Apoptosis-related gene expression was also compared. Our results indicate that release from apoptosis has a role in the tumor progression of this T cell lymphoma. At the cellular level, a markedly lower apoptotic tendency was observed in MT than in PT cells. The existence of macrophages only in PT also supports the presence of apoptotic cells in local but not in MTs. By contrast, proliferative capacity does not determine tumor aggressiveness in this system. At the molecular level, we found a higher staining intensity for bcl-2 in MT than in PT cells, suggesting that bcl-2 might be responsible for the reduced apoptosis in MT compared to PT cells. Evidence for p53 overexpression was found in the MT cells of one of the variants but in none of the PT. Comparison of Fas receptor, unexpectedly showed an increased expression in MT versus PT cells, possibly indicating resistance to Fas-induced apoptosis in the MT cells.
Keywords: Apoptosis resistance; Tumor progression; Primary versus metastatic tumor; Apoptosis-related gene expression; Metastatic phenotype; AKR lymphoma;

The nitroxide, Tempol, can protect tissue from oxidative damage by removing superoxide and by oxidizing Fe(II) to Fe(III), thus decreasing formation of the hydroxyl radical. However, long-term exposure to Tempol can damage cells. The oxidation of Fe could have profound effects on Fe metabolism in cells, yet this has not been previously studied. In the present investigation, the effects of Tempol on the synthesis of the Fe storage protein, ferritin, and its ability to store Fe were studied in cultured lens epithelial cells (LEC). In addition, the effects of short- and long-term Tempol treatment on the resistance of LEC to oxidative stress were determined. Tempol had a clear dose-dependent inhibitory effect on ferritin synthesis noted at 6 h. By 20 h, ferritin synthesis returned toward normal levels. However, Fe incorporation into ferritin was decreased by almost 90% by the highest dose of Tempol, even at the 20-h time point. The decrease in Fe incorporation into ferritin was accompanied by a significant increase in the LMW pool of Fe. After short-term (4 h) treatment with Tempol, LEC were protected against the toxic effects of tertiary butyl hydroperoxide. However, after longer term treatment (20 h), Tempol itself had a toxic effect and did not afford protection. Indeed, at the higher doses, Tempol significantly reduced the ability of the cells to withstand oxidative stress. The redistribution of Fe within the cell after 20 h of Tempol treatment appears to render the cells more vulnerable to oxidative stress. The deleterious effects of Tempol on LEC are likely due to its effects on Fe metabolism, perhaps by reducing the availability of Fe for incorporation into ferritin and Fe-dependent enzymes as well as enlarging a low molecular weight pool of Fe which may be capable of catalyzing damaging free radical reactions.
Keywords: Ferritin; Fe metabolism; Lens; Cataract; Lens epithelial cell; Tempol; Oxidative damage; Peroxide; Free radical;

Pro-inflammatory prostaglandins are known to be first catabolized by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to inactive metabolites. This enzyme is under regulatory control by various inflammation-related agents. Regulation of this enzyme was investigated in human promonocytic U937 cells. 15-PGDH activity was found to be optimally induced by phorbol 12-myristate 13-acetate (PMA) at 10 nM after 24 h of treatment. The induction was blocked by staurosporine or GF 109203X indicating that the induction was mediated by protein kinase C. The induction by PMA was inhibited by the concurrent addition of dexamethasone. Nearly complete inhibition was observed at 50 nM. Other glucocorticoids, such as hydrocortisone and corticosterone, but not sex hormones, were also inhibitory. Inhibition by dexamethasone could be reversed by the concurrent addition of antagonist mifepristone (RU-486) indicating that the inhibition was a receptor-mediated event. Either induction by PMA or inhibition by dexamethasone the 15-PGDH activity correlated well with the enzyme protein expression as shown by the Western blot analysis. These results provide the first evidence that prostaglandin catabolism is regulated by glucocorticoids at the therapeutic level.
Keywords: Dexamethasone; Prostaglandin; Dehydrogenase; Inflammation; Monocyte;

Transforming growth factor-β1 (TGF-β1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-β1 is not clearly understood. In this study, we found that the addition of TGF-β1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-β1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-β1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-β1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF.
Keywords: Transforming growth factor-β1; Vascular endothelial growth factor; MC3T3-E1 cell; Protein kinase C;

Mice genetically deficient in growth and differentiation factor 8 (GDF8/myostatin) had markedly increased muscle fiber numbers and fiber hypertrophy. In the regenerating muscle of mice possessing FGF6 mutation, fiber remodeling was delayed. Although myostatin and FGF6 may be important for the maintenance, regeneration and/or hypertrophy of muscle, little work has been done on the possible role of these proteins in adult muscle in vivo. Using Western blot and immunohistochemical analysis, we investigated, in rats, the distribution of myostatin, FGF6 and LIF proteins between slow- and fast-type muscles, and the adaptive response of these proteins in mechanically overloaded muscles, in regenerating muscles following bupivacaine injection and in denervated muscles after section of the sciatic nerve. The amounts of myostatin and LIF protein were markedly greater in normal slow-type muscles. In the soleus muscle, myostatin and LIF proteins were detected at the site of the myonucleus in both slow-twitch and fast-twitch fibers. In contrast, FGF6 protein was selectively expressed in normal fast-type muscles. Mechanical overloading rapidly enhanced the myostatin and LIF but not FGF6 protein level. In the regenerating muscles, marked diminution of myostatin and FGF6 was observed besides enhancement of LIF. Denervation of fast-type muscles rapidly increased the LIF, but decreased the FGF6 expression. Therefore, the increased expressions of myostatin and LIF play an important role in muscle hypertrophy following mechanical overloading. The marked reduction of FGF6 in the hypertrophied and regenerating muscle would imply that FGF6 regulates muscle differentiation but not proliferation of satellite cells and/or myoblasts.
Keywords: Growth and differentiation factor 8/myostatin; Fibroblast growth factor 6; Leukemia inhibitory factor; Muscle regeneration; Muscle hypertrophy;

Prolactin (PRL) stimulates lactogenesis in mammary cells and mitogenesis in a variety of cell types including Nb2 cells. Studies indicate that a different composite of signaling pathways is involved in the PRL stimulation of mitogenesis as compared to lactogenesis. In the present studies, PRL is shown to stimulate the tyrosyl phosphorylation of all three isoforms of Shc proteins in Nb2 cells (mitogenesis), but not in the mammary gland. Maximal phosphorylation of the Shc proteins is expressed between 10 and 15 min after a 50-ng/ml PRL treatment. In addition, there is an increased association between the Grb2 protein and Shc proteins upon PRL stimulation. However, no increased association between JAK2 and Shc proteins was observed in either the Nb2 cells or mammary tissues.
Keywords: Prolactin; Nb2 cell; Mouse mammary gland; Shc protein; Tyrosyl phosphorylation;

Molecular cloning of a ferret angiotensin II AT1 receptor reveals the importance of position 163 for Losartan binding by Mylène J. Gosselin; Patrice C. Leclerc; Mannix Auger-Messier; Gaétan Guillemette; Emanuel Escher; Richard Leduc (94-102).
A complementary DNA for the angiotensin II (AngII) type 1 (AT1) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT1) of 359 amino acids having high homologies (93–99%) to other mammalian AT1 receptor counterparts. When fAT1 was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue 125I-[Sar1, Bpa8]AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT1 non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT1 receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT1 receptor had a high affinity for the peptide antagonist [Sar1, Ile8]AngII (K d of 5.8±1.4 nM) but a low affinity for the AT1 selective non-peptidic antagonist DuP 753 (K d of 91±15.6 nM). Interestingly, when we substituted Thr163 with an Ala residue, which occupies this position in many mammalian AT1 receptors, we restored the high affinity of this receptor for Dup 753 (11.7±5.13 nM). These results suggest that position 163 of the AT1 receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.
Keywords: Angiotensin II type 1 receptor; Gene cloning; Receptor binding; Losartan; Angiotensin II type 1 receptor antagonist; Mustela putorius furo;

Regulation of human PLD1 and PLD2 by calcium and protein kinase C by Abdur R Siddiqi; Geraldine E Srajer; Christina C Leslie (103-114).
Numerous studies show that PLD is activated in cells by calcium and by protein kinase C (PKC). We found that human PLD1 and PLD2 expressed in Sf9 cells can be activated by calcium-mobilizing agonists and by co-expression with PKCα. The calcium-mobilizing agonists A23187 and CryIC toxin triggered large increases in phosphatidylethanol (PtdEth) production in Sf9 cells over-expressing PLD1 and PLD2, but not in vector controls. PLD activation by these agonists was largely dependent on extracellular calcium. Membrane assays demonstrated significant PLD1 and PLD2 activity in the absence of divalent cations, which could be enhanced by low levels of calcium either in the presence or absence of magnesium. PLD1 but not PLD2 activity was slightly enhanced by magnesium. Treatment of Sf9 cells expressing PLD1 and PLD2 with PMA resulted in little PtdEth production. However, a significant and comparable formation of PtdEth occurred when PLD1 or PLD2 were co-expressed with PKCα, but not PKCδ, and was further augmented by PMA. In contrast to PLD1, co-expressing PLD2 with PKCα or PKCδ further enhanced A23187-induced PtdEth production. Immunoprecipitation experiments demonstrated that PLD1 and PLD2 associated with the PKC isoforms in Sf9 cells. Furthermore, in membrane reconstitution assays, both PLD1 and PLD2 could be stimulated by calmodulin and PKCα-enriched cytosol. The results indicate that PLD2 as well as PLD1 is subject to agonist-induced activation in intact cells and can be regulated by calcium and PKC.
Keywords: PLD1; PLD2; PKC; Calcium;

Previous works of our group demonstrated that xenobiotic metabolism by brain microsomes or cultured cerebral cells may promote the formation of reactive oxygen species. In order to characterise the risk of oxidative stress to both the central nervous system and the blood-brain barrier, we measured in the present work the release of superoxide in the culture medium of rat cerebrovascular endothelial cells during the metabolism of menadione, anthraquinone, diquat or nitrofurazone. Assays were run in the same experimental conditions on primary cultures of rat neurones and astrocytes. Quinone metabolism efficiently produced superoxide, but the production of radicals during the metabolism of diquat or nitrofurazone was very low, as a probable result of their reduced transport inside the cells. In all cell types assayed, superoxide production was time- and concentration-dependent, and cultured astrocytes always produced the highest amounts of radicals. Superoxide formation by microsomes prepared from the cultured cells was decreased by immunoinhibition of NADPH-cytochrome P450 reductase or by its irreversible inhibition by diphenyliodonium chloride, suggesting the involvement of this flavoprotein in radical production. Cerebrovascular endothelial cells cultured on collagen-coated filters produced equivalent amounts of superoxide both at their luminal side and through the artificial basement membrane, suggesting that in vivo, endothelial superoxide production may endanger adjacent astrocytes and neurones.
Keywords: Superoxide; Drug metabolism; Blood-brain barrier; Neurone; Astrocyte; NADPH-cytochrome P450 reductase;

We have previously demonstrated that chemically oversulfated fucoidan (OSF) but not native fucoidan (NF) effectively suppresses the tube structure formation by human umbilical vein endothelial cells (HUVEC) on the basement membrane preparation, Matrigel. In this study, using more defined systems where basic fibroblast growth factor (bFGF) induces the tube formation by HUVEC on collagen gel, we investigated the mechanism responsible for the inhibition of angiogenesis by OSF in vitro. Unlike NF and desulfated fucoidan (desF), OSF potently inhibited the bFGF-induced HUVEC migration and tube formation. ELISA for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the culture media indicated that OSF increased the bFGF-induced release of PAI-1 antigen, but not of t-PA antigen. Analyses of the binding of bFGF to HUVEC surfaces and the following protein tyrosine phosphorylation revealed that OSF could promote the cell binding and autophosphorylation of 140 and 160 kDa receptors. In heparitinase-treated HUVEC, contrarily, the bFGF binding and PAI-1 release were decreased by OSF. These results suggest that OSF is a highly sulfated unique polysaccharide that can promote the binding of bFGF to the heparan sulfate molecules required for binding to the high affinity receptors with tyrosine kinase activity. The resultant increase in PAI-1 release may play a key role for the prevention of cell migration accompanied by matrix proteolysis.
Keywords: Fucoidan; Basic fibroblast growth factor; Angiogenesis; Plasminogen activator inhibitor-1; Endothelial cell (human umbilical vein);

During G1 to S phase transition, D-type cyclins form complexes with cyclin-dependent kinases (Cdk), which in turn phosphorylate retinoblastoma gene product (Rb) and inhibit its growth-inhibitory function, leading ultimately to cell proliferation. We report here a novel finding that D1 and D2 cyclins are induced in macrophages by antiproliferative factor gamma interferon (IFNγ). The induction appears to be transcriptional activation of the D cyclin genes, since indirect events such as IFNγ-induced colony-stimulating factor-1 (CSF-1) autocrine stimulation, alteration of D1 and D2 mRNA stability and lipopolysaccharide contamination in commercial IFNγ preparations play no roles. In contrast to CSF-1, IFNγ neither induces D1–Cdk4 complex formation and Rb hyperphosphorylation nor interferes with CSF-1-stimulated D1–Cdk4 interaction and Rb phosphorylation, while it completely blocks CSF-1-stimulated cell proliferation. This study suggests that induction of D1 and D2 cyclins is not necessarily associated with cell cycle progression, and D cyclins may have cell cycle-independent functions in response to IFNγ.
Keywords: Gamma interferon; Colony-stimulating factor-1; D cyclin; Macrophage; Signal transduction;

Peroxisome proliferator-activated receptor γ1 (PPARγ1) expresses in rat mesangial cells and PPARγ agonists modulate its differentiation by Tamotsu Asano; Masanori Wakisaka; Mototaka Yoshinari; Kenzo Iino; Kazuo Sonoki; Masanori Iwase; Masatoshi Fujishima (148-154).
Thiazolidinediones, synthetic ligands of peroxisome proliferator-activated receptor γ (PPARγ), are reported to have direct beneficial effects on diabetic nephropathy without lowering blood glucose levels in human and rat. We hypothesized these effects of thiazolidinediones might be derived from PPARγ activation of kidney cells, and we examined the expression of PPARγ and the effect of PPARγ agonists, troglitazone and 15-deoxy-δ-prostaglandin J2 (15d-PGJ2), on the proliferation and differentiation in rat mesangial cells. A single band of mRNA of PPARγ with a predicted size was detected in reverse transcription-polymerase chain reaction products (RT-PCR) using established PCR probes of PPARγ. PPARγ protein in rat mesangial cells was identified as PPARγ1 by a Western blot. In a gel mobility shift assay to determine a binding activity of PPARγ, the nuclear protein from rat mesangial cells bound to a 32P-labeled oligonucleotide probe, including PPAR response elements. A synthetic and a natural ligand of PPARγ, troglitazone and 15d-PGJ2, decreased thymidine incorporation in a dose dependent manner. After 7 days incubation with troglitazone and 15d-PGJ2, α-smooth muscle actin expression, a marker of mesangial cell de-differentiation, was decreased significantly compared to that of control. These results indicate that PPARγ1 is expressing in rat mesangial cells, and PPARγ1 activation with its agonists modulates the proliferation and differentiation of cultured rat mesangial cells.
Keywords: Rat mesangial cell; Peroxisome proliferator-activated receptor γ; Cell differentiation; Troglitazone;

A possible role for Ca2+/calmodulin-dependent protein kinase IV during pancreatic acinar stimulus–secretion coupling by Hitoshi Yoshida; Fumihiko Nozu; Tim O. Lankisch; Keiji Mitamura; Chung Owyang; Yasuhiro Tsunoda (155-167).
Ca2+/calmodulin-dependent protein kinases (CaMKs) are important intracellular mediators in the mediation of stimulus–secretion coupling and excitation–contraction coupling in a wide variety of cell types. We attempted to identify and characterize the functional roles of CaMK in mediating pancreatic enzyme secretion. Immunoprecipitation and immunoblotting studies using a CaMKII or CaMKIV antibody showed that rat pancreatic acini expressed both CaMKII and CaMKIV. Phosphotransferase activities of CaMKs were measured by a radioenzyme assay (REA) using autocamtide II, peptide γ and myosin P-light chain as substrates. Although CaMKII and CaMKIV use autocamtide II as a substrate, peptide γ is more efficiently phosphorylated by CaMKIV than by CaMKII. Intact acini were stimulated with cholecystokinin (CCK)-8, carbachol (CCh) and the high-affinity CCK-A receptor agonist, CCK-OPE, and the cell lysates were used for REA. CCK-8, CCh and CCK-OPE caused a concentration-dependent increase in CaMKs activities. When autocamtide II was used, maximal increases were 1.5–1.8-fold over basal (20.2±2.0 pmol/min/mg protein), with peaks occurring at 20 min after cell stimulation. In separate studies that used peptide γ, CCK-8, CCh and CCK-OPE dose-dependently increased CaMKIV activities. Maximal increases were 1.5–2.4-fold over basal (30.7±3.2 pmol/min/mg protein) with peaks occurring at 20 min after cell stimulation. Peak increases after cell stimulation induced by peptide γ were 1.8–2.8-fold higher than those induced by autocamtide II. CCK-8, CCh and CCK-OPE also significantly increased phosphotransferase activities of myosin light chain kinase (MLCK) substrate (basal: 4.4±0.7 pmol/min/mg protein). However, maximal increases induced by MLCK substrate were less than 10% of those occurring in peptide γ. Characteristics of the phosphotransferase activity were also different between autocamtide II and peptide γ. When autocamtide II was used, elimination of medium Ca2+ in either cell lysates or intact cells resulted in a significant decrease in the activity, whereas it had no or little effect when peptide γ was used. This suggests that Ca2+ influx from the extracellular space is not fully required for CaMKIV activity and Ca2+ is not a prerequisite for phosphotransferase activity once CaMKIV is activated by either intracellular Ca2+ release or intracellular Ca2+ oscillations. The specific CaMKII inhibitor KN-62 (50 μM) had no effect on the CaMKIV activity and pancreatic enzyme secretion elicited by CCK-8, CCh and CCK-OPE. The specific MLCK inhibitor, ML-9 (10 μM), also did not inhibit CCK-8-stimulated pancreatic amylase secretion. In contrast, wide spectrum CaMK inhibitors, K-252a (1 μM) and KT5926 (3 μM), significantly inhibited CaMKIV activities and enzyme secretion evoked by secretagogues. Thus, CaMKIV appears to be an important intracellular mediator during stimulus–secretion coupling of rat pancreatic acinar cells.
Keywords: Calmodulin-dependent protein kinase; Cholecystokinin; Carbachol; Stimulus–secretion coupling; Pancreatic acinus (rat);

A septin gene homologue designated Pyrenopeziza brassicae septin 1 (Pbs1) has been identified and cloned from the plant pathogenic fungus Pyrenopeziza brassicae and its expression analysed. Pbs1 is present in both mating types and in a single copy within each genome and is transcribed in proportionate levels during both vegetative and sexual growth.
Keywords: Molecular plant pathology; Sexual development; Septin; Fungus;