Co-distribution of calmodulin-dependent protein kinase II and inositol trisphosphate receptors in an apical domain of gastrointestinal mucosal cells.

The Type 3 inositol 1,4,5-trisphosphate (InsP3) receptor is expressed at high levels in gastrointestinal tissues. This receptor has 16 potential phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaM kinase II). To determine if the Type 3 InsP3 receptor is likely to be a physiologic substrate for CaM kinase II, localizations of the Type 3 InsP3 receptor and CaM kinase II were compared in tissues of the gastrointestinal tract. Cellular and subcellular localizations were determined by immunofluorescence microscopy in rat intestine, pancreas, and stomach, and in isolated rabbit gastric glands. Both proteins were found in the apical region of intestinal enterocytes, pancreatic acinar cells, and gastric parietal, chief, and surface mucous cells. CaM kinase II was found throughout the entire intracellular canalicular F-actin domain of parietal cells, whereas the type 3 InsP3 receptor was restricted to the neck region. Thus, in several gastrointestinal tissues the Type 3 InsP3 receptor is specifically localized to a portion of the apical cytoskeletal domain in which resides the calcium-responsive effector CaM kinase II.

Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells.

We have previously shown that a 222-kDa polypeptide co-immunoprecipitates together with the type-I myoinositol 1,4,5-trisphosphate receptor (IP3R) in WB rat liver epithelial cell extracts, when the immunoprecipitation is carried out with a type-I isoform specific antibody (Joseph, S. K. (1994) J. Biol. Chem. 269, 5673-5679). Utilizing isoform-specific antibodies raised to unique sequences within the COOH-terminal region of IP3 receptors, we now report that the co-immunoprecipitating 222-kDa polypeptide is the type-III IP3R isoform and that type-III IP3R antibodies (Abs) can co-immunoprecipitate the type-I IP3R isoform. Co-immunoprecipitation of IP3R isoforms was not due to cross-reactivity of the antibodies for the following reasons: (a) on immunoblots the type-III antibodies did not cross-react with type-I IP3R and vice versa; (b) inclusion of the COOH-terminal type-III peptide had no effect on the ability of type-I IP3R Ab to co-immunoprecipitate the type-III IP3R but blocked the ability of type-III IP3R Ab to coimmunoprecipitate the type-I isoform; and (c) crude hepatocyte lysates contain undetectable amounts of type-III IP3R, and immunoprecipitation with type-III IP3R Ab does not co-immunoprecipitate any other isoforms. However, type-I and type-II IP3R isoforms were co-immunoprecipitated by their respective antibodies in hepatocyte lysates. Sucrose density gradient analysis of WB cell lysates indicated that the co-immunoprecipitating fraction is exclusively located at the density expected for tetrameric receptors, suggesting that co-immunoprecipitation was not a reflection of the nonspecific aggregation of IP3R isoforms. Phosphorylation of either type-I or type-III immunoprecipitates by protein kinase A indicated that only the type-I IP3R could be phosphorylated in vitro. Fractionation of WB cell membranes and immunofluorescence studies showed that the type-I and type-III isoforms have very similar sub-cellular localizations. We conclude that the WB cell contains both type-I and type-III IP3R isoforms and that a proportion of these receptors exist as heterotetramers.

Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave trigger zone of pancreatic acinar cells.

Agonist-induced cytosolic Ca2+ (Ca2+i) signals begin as apical-to-basal Ca2+i waves in pancreatic acinar cells and in other polarized epithelia. However, the basis of this polarized Ca2+i signaling pattern is unknown. Here we use immunocytochemistry to demonstrate that the type 3 inositol trisphosphate receptor is localized to the extreme apex of pancreatic acinar cells, the region which corresponds to the trigger zone from which Ca2+i signals originate in this cell type (Kasai, H., Li, Y.X., and Miyashita, Y. (1993) Cell 74, 669-677). We also show that inositol trisphosphate-mediated Ca2+ release induces amylase release from permeabilized pancreatic acini. Since Ca2+i signals begin by inositol trisphosphate-mediated Ca2+ release, these findings suggest that localization of the type 3 inositol trisphosphate receptor to the trigger zone is responsible for the generation of apical-to-basal Ca2+i waves, and that this organization may be important for regulating apical exocytosis in pancreatic acinar cells.

Primary structure, ligand binding, and localization of the human type 3 inositol 1,4,5-trisphosphate receptor expressed in intestinal epithelium.

The second messenger, inositol 1,4,5-trisphosphate (InsP3) transduces many hormonal signals which regulate Ca(2+)-dependent processes in the intestinal epithelium. To study the receptors for InsP3 (InsP3Rs), which function as intracellular Ca2+ channels, cDNA clones encoding InsP3Rs were isolated from a human colon adenocarcinoma cell line, HT29. The majority of clones encoded the type 3 InsP3R, the product of the ITPR3 gene on chromosome 6, for which only a 147-amino-acid fragment was known previously (Ozcelik, T., Sudhof, T. C., and Francke, U. (1991) Cytogenet. Cell Genet. Abstr. 58, 1880; Sudhof, T. C., Newton, C. L., Archer, B. T., III, Ushkaryov, Y. A., and Mignery, G. A. (1991) EMBO J. 10, 3199-3206). The complete sequence of the type 3 InsP3R polypeptide (2,671 amino acids) is described here. Primary structure analysis indicates a pattern of conserved and variable regions which is characteristic of the InsP3R family. A 250-kDa protein (SDS-PAGE) which specifically binds InsP3 is immunoprecipitated by affinity-purified antibodies raised against a COOH-terminal fusion protein. Transient expression in COS-7 cells of a polypeptide comprising the NH2-terminal 750 amino acids establishes that the ligand-binding domain is localized to this region. Lysates from transfected COS-7 cells bind InsP3 with high affinity (Kd = 151 nM) compared with other inositol phosphates (InsP3 >> Ins 1,3,4,5-P4 > InsP6 > Ins 1,4-P2 >> Ins 1-P). Immunocytochemical localization in the intestine reveals expression in crypt and villus epithelial cells, but not in cells of the lamina propria, submucosa, or muscularis layers. The subcellular distribution and appearance of staining is consistent with localization on the endoplasmic reticulum, with the highest concentration of staining occurring adjacent to the apical brush border of villus cells.

Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic cardiac valves.

Bioprosthetic valves fail frequently because of pathological mineralization, a process that begins in cell remnants of the glutaraldehyde (GLUT) fixed tissue. Other pathological cardiovascular calcification and physiological mineralization in skeletal/dental tissues are both largely initiated in cell-derived membranous structures (often called "matrix vesicles"), and the enzyme alkaline phosphatase (AP) likely has an important function in the pathogenesis of mineral nucleation. This study tested the hypothesis that AP might also be present in and contribute to calcification of bioprosthetic valves. AP activity of fresh and GLUT-treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Following 24 hours in 0.6% HEPES-buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained in GLUT-treated tissue relative to that of fresh tissue (Vmax, 24 vs. 45 mumol reaction product/min/mg tissue protein, respectively), although binding was somewhat reduced (Km, 1.9 X 10(3) vs. 1.4 X 10(3) microM substrate, respectively). Enzyme reaction product was demonstrated in both fixed and fresh tissue by light microscopic histochemical studies, confirming the biochemical results. Reaction product was noted along membranes of vascular endothelial cells and interstitial fibroblasts, the sites of early calcific deposits in bioprosthetic valves, by ultrastructural examination of GLUT-treated tissue. We conclude that GLUT-treated bovine pericardium retains much of the hydrolytic activity of AP, an enzyme associated with normal skeletal and pathological cardiovascular and noncardiovascular mineralization, and suggest that further examination of the mechanistic role of this enzyme may stimulate new approaches for slowing or preventing calcification of bioprosthetic tissue.

Effect of delay between tissue harvest and glutaraldehyde pretreatment on mineralization of bovine pericardium used in bioprosthetic heart valves.

There is concern that delayed glutaraldehyde treatment of bioprosthetic tissue could potentiate calcification by autolytic generation of mineralization nuclei. This study investigated the effects on mineralization of variable delays between harvest of bovine pericardium and initial glutaraldehyde treatment, using tissue implanted subcutaneously in rats for 21 days. Susceptibility to mineralization increased statistically but only modestly with delays to 34 h. This suggests that mineralization will not be significantly inhibited by rapid treatment of otherwise properly handled tissue and that clinically important prevention of calcification will require more dramatic means.

Phosphatase enzyme activity is retained in glutaraldehyde treated bioprosthetic heart valves.

Calcification of bioprosthetic valves, which frequently causes their failure, begins in cell remnants analogous to matrix vesicles of physiologic mineralization. Because the enzyme alkaline phosphatase (AP) is important in normal skeletal mineralization, the authors hypothesized that AP also might be present in bioprosthetic valve tissue and thereby contribute to calcification. AP activity of fresh and glutaraldehyde (GLUT) treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. After 24 hrs in 0.6% HEPES buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained relative to that of fresh tissue (Vmax: 24 vs 45 microM reaction product/min/mg tissue protein, respectively), although binding was moderately reduced (KM: 1900 vs 1400 microM substrate, respectively). Light microscopic histochemistry suggested cell oriented AP activity. Ultrastructural examination of GLUT treated tissue demonstrated reaction product along membranes of vascular endothelial cells and fibroblasts, the sites of early calcific deposits in bioprosthetic valves. Thus, AP hydrolytic activity is largely preserved following GLUT treatment of bovine pericardium. These results indicate that the widely held view that GLUT eliminates all metabolic activities of bioprosthetic tissue is inaccurate and suggests that examination of the role of AP and other phosphatases may stimulate approaches for inhibiting calcification.

Cholinergic action on the heart of the leech, Hirudo medicinalis.

Experiments were performed to determine the role of acetylcholine (ACh) in neuromuscular transmission in the heart of the leech Hirudo medicinalis. Superfused or iontophoretically applied ACh rapidly depolarized both isolated heart muscle cells and muscle cells in isolated hearts in a dose-dependent manner. the depolarization was associated with a conductance increase of the muscle membrane that had a reversal potential of -9 mV. Eserine potentiated the response to superfused ACh, reducing the threshold from 10(-6) to 10(-8) mol l-1. Acetylcholinesterase was localized histochemically to be in the immediate area of neuromuscular terminals. Superfused nicotinic agonists mimicked the effects of ACh, while superfused nicotinic antagonists reversibly blocked the iontophoretic response of heart muscle fibres to ACh. 5 X 10(-7) mol l-1 curare, 5 X 10(-5) mol l-1 nicotine and 1 X 10(-4) mol l-1 atropine reduced the iontophoretic response to half its original amplitude. Alpha-bungarotoxin did not block the response of heart muscle cells to iontophoretically applied ACh. Curare was used to determine whether the neurones that innervate the heart-HE motor neurones and HA modulatory neurone--use ACh as a neuromuscular transmitter. The fast depolarizing component of the HE cell's neuromuscular transmission was reversibly blocked by 10(-4) mol l-1 curare, while the HA cell's modulatory effects on the heart were apparently unaffected by 10(-4) mol l-1 curare. Our results indicate that heart muscle cells have nicotinic acetylcholine receptors that open in the presence of ACh, thereby increasing membrane conductance. The HE motor neurone is probably cholinergic and engages these receptors in its neuromuscular transmission, while the HA modulatory neurone is probably not cholinergic.

Neuronal mapping: a photooxidation reaction makes Lucifer yellow useful for electron microscopy.

Irradiation Lucifer yellow-filled neurons with intense blue light in the presence of 3,3'-diaminobenzidine produces an electron-opaque osmiophilic polymer within the injected cells. This technique is valuable when cobalt or horseradish peroxidase injections are difficult or when a second intracellular marker is needed to demonstrate neuronal contacts.