Yutaka Shintani

Potentiation of slow component of delayed rectifier K+ currentby cGMP via two distinct mechanisms: inhibition of phosphodiesterase 3 and activation of protein kinase G

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by intracellular guanosine 3′5′ cyclic monophosphate (cGMP) was investigated in guinea‐pig sino‐atrial (SA) node cells using the whole‐cell patch‐clamp method. When a cell was dialyzed with pipette solution containing 100 μM cGMP, IKs started to gradually increase and reached a maximum increase of a factor of 2.37±0.39 (n=4) about 10–15 min after rupture of patch membrane. Atrial natriuretic peptide (ANP, 100 nM) also potentiated IKs, consistent with intracellular cGMP‐induced enhancement of IKs. Bath application of a selective blocker of the cGMP‐inhibited phosphodiesterase (PDE3) milrinone (100 μM) enhanced IKs by a factor of 1.50±0.09 (n=4) but failed to further enhance IKs after a maximum stimulation by intracellular cGMP (100 μM), suggesting that blockade of PDE3 activity is involved in the enhancement of IKs. A potent but nonspecific PDE inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX, 100 μM) further increased IKs stimulated by 100 μM milrinone, indicating that PDE subtypes other than PDE3 are also involved in the regulation of basal IKs in guinea‐pig SA node cells. Bath application of 100 μM 8‐bromoguanosine 3′5′ cyclic monophosphate (8‐Br‐cGMP) increased IKs by a factor of 1.48±0.11 (n=5) and this stimulatory effect was totally abolished by cGMP‐dependent protein kinase (PKG) inhibitor KT‐5823 (500 nM), suggesting that the activation of PKG also mediates cGMP‐induced potentiation of IKs. These results strongly suggest that intracellular cGMP potentiates IKs not only by blocking PDE3 but also by activating PKG in guinea‐pig SA node cells.

Pro-inflammatory cytokine-induced matrix metalloproteinase-1 (MMP-1) secretion in human pancreatic periacinar myofibroblasts

Matrix metalloproteinases (MMPs) are the proteases involved in the degradation of the extracellular matrix. MMP-1 is thought to be one of the key enzymes in fibrolysis, a process closely related to tissue remodeling. In the present study, we investigated MMP-1 secretion from human pancreatic periacinar myofibroblasts in response to pro-inflammatory cytokines IL-1β and TNF-α. We also attempted to clarify the intracellular signaling pathways mediating the cytokine-induced MMP-1 secretion. MMP-1 secretion was measured by an enzyme-linked immunosorbent assay. MMP-1 molecules were analyzed by Western blotting. MMP-1 mRNA expression was evaluated by Northern blotting. IL-1β and TNF-α stimulated the MMP-1 secretion in a dose- and time- dependent manner. Ninety percent of MMP-1 was secreted as inactive form (pro-MMP-1). The effects of IL-1β and TNF-α were significantly inhibited by PD98059 MEK/ERK inhibitor). In contrast, SB203580 (p38 MAPK inhibitor), GF109203X (PKC inhibitor), and PDTC (NF-ĸB inhibitor) did not alter the MMP-1 secretion induced by IL-1β and TNF-α. These effects were also observed at the mRNA level. In conclusion, in human pancreatic periacinar myofibroblasts, MMP-1 secretion was regulated by the pro-inflammatory cytokines via the MEK/ERK cascade. Thus, human pancreatic periacinar myofibroblasts may play an important role in the remodeling of damaged pancreatic tissue in chronic pancreatitis via MMP-1 secretion.

Effects of synthetic serine protease inhibitors on proliferation and collagen synthesis of human pancreatic periacinar fibroblast-like cells

Protease inhibitors are currently used as therapeutic agents for chronic pancreatitis in Japan. We previously reported that human pancreatic periacinar fibroblast-like cells (hPFCs) could be cultured from isolated pancreatic acini, and those are thought to play a crucial role in pancreatic fibrosis correlating with platelet-derived growth factor (PDGF) and transforming growth factor &bgr;1 (TGF-&bgr;1) (Pancreas 1997;14:373–82). The present study was designed to examine the effects of synthetic serine protease inhibitors (FOY-007 and FOY-305) on proliferation and collagen synthesis of hPFCs under cytokine stimulation. The cell proliferation and collagen synthesis were evaluated using assays of [3H]-thymidine incorporation and procollagen type I c-terminal peptide (PIP), and [14C]-proline incorporation to de novo synthesized collagen, respectively. The cell proliferation stimulated by PDGF was inhibited by the application of FOY-007 dose dependently (1–100 &mgr;M) and FOY-305 at 100 &mgr;M. FOY-007 attenuated the collagen synthesis and PIP production stimulated by TGF-&bgr;1 dose dependently, but FOY-305 inhibited only PIP production. Both protease inhibitors demonstrated no effect on the proliferation and collagen synthesis of hPFCs when they were not stimulated by PDGF or TGF-&bgr;1. Thus, serine protease inhibitors act on hPFCs to diminish the effects of PDGF on proliferation and the effects of TGF-&bgr;1 on collagen synthesis.

Macrolipasemia in Crohn's disease

A 38-year-old male patient who had been treated for Crohns disease was found to have serum lipase activity that was persistently increased - 10-fold above the normal upper limit. He was diagnosed with chronic pancreatitis based on slightly elevated elastase-1 level and retrograde pancreatography showing slight dilatation of the main pancreatic duct. Therefore, the hyperlipasemia was thought to be due to pancreatitis. However, the serum amylase and trypsin was not increased at any time, and no serious findings suggestive of pancreatitis were detected on morphologic examination. Thus, there were discrepancies between the serum lipase activity and other laboratory and clinical findings. Exclusion chromatography of the patients serum suggested macromolecular lipase, and further immunologic testing including affinity chromatography, enzyme-linked immunosorbent assay, and immunoprecipitation assay showed that serum lipase was bound to immunoglobulin GK. Therefore, the hyperlipasemia was caused by immunoglobulin-linked lipase, termed “macrolipasemia”. Macrolipasemia has rarely been reported, and this is the first reported case of macrolipasemia accompanied by Crohns disease.

A case of toxic shock-like syndrome presenting with serious hypoproteinaemia because of a protein-losing gastroenteropathy

Abstract. Tasaki K, Sasaki M, Bamba M, Shintani Y, Andoh A, Tsujikawa T, Koyama S, Fujiyama Y, Bamba T (Shiga University of Medical Science, Shiga, Japan). A case of toxic shock‐like syndrome presenting with serious hypoproteinaemia because of a protein‐losing gastroenteropathy (Case report). J Intern Med 2001; 250: 174–179.

Proliferative effect of phospholipase A2 on rat periacinar fibroblastoid cells of the pancreas

We have shown previously that rat pancreatic periacinar fibroblastoid cells (PFCs) can be cultured from isolated pancreatic acini. In the present study, immunocytochemical examination of the PFC extracellular matrix was performed using antibodies against prolyl hydroxylase α and β subunits, types I, III, and IV collagen, fibronectin, and laminin. The PFC content of α-smooth muscle actin and platelet-derived growth factor (PDGF) receptor were studied by immunoblotting. We demonstrated that PFCs synthesized extracellular matrix and expressed α-smooth muscle actin and PDGF receptors. These results suggested that PFCs resemble myofibroblasts and may play a critical role in pancreatic fibrosis. Conversely, pancreatic-type phospholipase A2, (P-PLA2.), one of the pancreatic digestive enzymes, has been shown to induce DNA synthesis of Swiss 3T3 fibroblasts. To determine whether this enzyme is involved in pancreatic fibrosis, we studied P-PLA2s proliferative and chemotactic effects on PFCs as well as its digestive activity. The proliferative and chemotactic effects were investigated using 3H-thymidine incorporation and a chemotactic assay, respectively. P-PLA2 had both proliferative and chemotactic effects. P-PLA2 is considered a growth factor for PFCs and is implicated in pancreatic fibrosis.