Calcium-dependent aggregation of human serum amyloid P component: Inhibition by the cyclic 4,6-pyruvate acetal of galactose

Serum amyloid P component is a normal plasma glycoprotein which is the precursor of amyloid P component, a minor but universal constituent of amyloid deposits. When isolated human P component is exposed to free ionised Ca²⁺ it aggregates and precipitates. This phenomenon is completely inhibited by the presence of 10^?4?10^?2 M methyl 4,6-O-(1-car?yethylidene)-β-D-galactopyranoside, a recently synthesised specific ligand for amyloid P component. This observation suggests that the autoaggregation of human amyloid P component involves the Ca²⁺ dependent specific ligand binding property of P component, but does not distinguish between receptor-site-mediated and allosteric mechanisms.     

Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100.

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84--88% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the absence of F- but only 50--60% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the presence of F-. In either case, further increase in EGTA concentration did not alter the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 - 10(-5) M Ca2+, (yielding a [free Ca2+] of approximately 2 - 10(-6) M) and 5 - 10(-5) M Mn2+ or Co2+ and partially by 5 - 10(-5) M Sr2+ but not by addition of 5 - 10(-5) M Ba2+, Zn2+, Ni2+ or Fe2+. A [free Ca2+] of 7.2 - 10(-5) M markedly inhibited cyclase activity in the presence of F-. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-TETRAACETIC ACID, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concentrations of Ca2+ and Mg2+. A second component is totally Ca2+ dependent and inhibited by high concentrations of F-. Mn2+, Co2+ and Sr2+ appear to be in vitro Ca2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca2+ dependent cyclase component but a complete abolition of the NaF stimulated component.     

Somatostatin decreases voltage-gated Ca2+ currents in GH3 cells through activation of somatostatin receptor 2

The secretion of growth hormone (GH) is inhibited by hypothalamic somatostatin (SRIF) in somatotropes through five subtypes of the somatostatin receptor (SSTR1-SSTR5). We aimed to characterize the subtype(s) of SSTRs involved in the Ca2+ current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin-perforated patch clamp to record voltage-gated Ca2+ currents, using a holding potential of -80 mV in the presence of K+ and Na+ channel blockers. We first established the presence of T-, L-, N-, and P/Q-type Ca2+ currents in GH3 cells using a variety of channel blockers (Ni+, nifedipine, omega-conotoxin GVIA, and omega-agatoxin IVA). SRIF (200 nM) reduced L- and N-type but not T- or P/Q-type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+ currents to find the maximal effective concentration. Activation of SSTR2 with 10(-7) and 10(-8) M L-797,976 decreased the voltage-gated Ca2+ current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10(-7) M did not modify the voltage-gated Ca2+ current and did not affect the Ca2+ current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage-gated Ca2+ currents by SRIF, which contributes to the decrease in intracellular Ca2+ concentration and GH secretion by SRIF.     

Epidermal growth factor-induced increases in inositol trisphosphates, inositol tetrakisphosphates, and cytosolic Ca2+ in a human hepatocellular carcinoma-derived cell line

A human hepatocellular carcinoma-derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5-bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml), a rapid (10-15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 and Ins(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 20 s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.     

Isolation and characterization of two major serum proteins from the dogfish, Mustelus canis, C-reactive protein and amyloid P component

Two major serum components from the dogfish, Mustelus canis, have been isolated using affinity chromatography. Both proteins bind to an AH-Sepharose 4B-phosphorylcholine affinity matrix in the presence of Ca2+ and are eluted from the column by EDTA. Upon readdition of Ca2+ to the eluted proteins, the two proteins can be separated by passage through a column of Sepharose CL-4B. The first protein, C-reactive protein, passes through the Sepharose CL-4B column in the presence of Ca2+ whereas the second protein, serum amyloid P component, remains bound. The serum amyloid P component is then eluted from the Sepharose CL-4B in pure form by EDTA. The dogfish C-reactive protein isolated by the phosphorylcholine affinity matrix precipitates with pneumococcal C-polysaccharide and with a synthetic derivative of bovine serum albumin to which phosphorylcholine is covalently attached. The precipitation is inhibited by either EDTA or by phosphorylcholine. Dogfish C-reactive protein has a molecular weight of approximately 250,000 with dimeric subunits of Mr = 50,000. Upon addition of beta-mercaptoethanol these dimeric subunits dissociate to two identical monomeric subunits of Mr = 25,000. The protein cross-reacts immunologically with goat antisera prepared against rabbit C-reactive protein. The dogfish serum amyloid P component has a molecular weight of at least 250,000 with monomeric subunits of Mr = 25,000. Cross-reactivity of the amyloid P component with the C-reactive protein could not be shown. However, NH2-terminal sequence analysis of the first 20 amino acids showed some homology. The relationship of dogfish C-reactive protein to the C-reactive proteins in Limulus polyphemus and in rabbits and humans is discussed.     

The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.     

Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+ concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349-357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+ channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+ channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.     

Sustained and transient calcium currents in horizontal cells of the white bass retina

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch-clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15-60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent.     

Co-expression of metabotropic glutamate receptor 7 and N-type Ca(2+) channels in single cerebrocortical nerve terminals of adult rats

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in cerebrocortical nerve terminals of adult rats, the inhibition of glutamate release is mediated by mGluR7. In this preparation, the major component of glutamate release is supported by P/Q-type Ca2+ channels (72.7%). However, mGluR7 selectively reduced the release component that is associated with N-type Ca2+ channels (29.9%). Inhibition of P/Q channels by mGluR7 is not masked by the higher efficiency of these channels in driving glutamate release when compared with N-type channels. Thus, activation of mGluR7 failed to reduce the release associated with P/Q channels when the extracellular calcium concentration, ([Ca2+]o), was reduced from 1.3 to 0.5 mm. Through Ca2+ imaging, we show that Ca2+ channels are distributed in a heterogeneous manner in individual nerve terminals. Indeed, in this preparation, nerve terminals were observed that contain N-type (31.1%; conotoxin GVIA-sensitive) or P/Q-type (64.3%; agatoxin IVA-sensitive) channels or that were insensitive to these two toxins (4.6%). Interestingly, the great majority of the responses to l-AP4 (95.4%) were observed in nerve terminals containing N-type channels. This specific co-localization of mGluR7 and N-type Ca2+-channels could explain the failure of the receptor to inhibit the P/Q channel-associated release component and also reveal the existence of specific targeting mechanisms to localize the two proteins in the same nerve terminal subset.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Effects of nitrates and calcium channel blockers on Ca2+-ATPase in the microsomal fraction of porcine coronary artery smooth muscle cells

The effects of the antianginal drugs nitroglycerin, nicorandil, diltiazem, verapamil and nicardipine on the activity of calcium-stimulated magnesium-dependent ATPase (Ca2+-ATPase) were investigated in the microsomal fraction from porcine coronary artery smooth muscle cells. Two discrete Ca2+-dependent ATPase components were observed: [1] a high affinity component, which was a specific Ca2+-ATPase, [with a half saturation constant for Ca2+ (Km) of 0.44 microM, and maximum velocity (Vmax) of 124.3 pmol of phosphate (Pi) released/micrograms of protein/30 min]: [2] a low affinity component in which Ca2+ could be replaced by Mg2+ without loss of its activity. Nitroglycerin and nicorandil (1 microM and 10 microM) both stimulated the activity of the Ca2+-ATPase significantly [142 +/- 12 (mean +/- standard error), and 137 +/- 10% of the control with nitroglycerin, and 152 +/- 17 and 135 +/- 20% with nicorandil] at a Ca2+ concentration of 0.3 microM. Diltiazem, verapamil and nicardipine did not cause significant stimulation. Nitroglycerin and nicorandil (1 microM), significantly decreased the Km for Ca2+ from the control value of 0.44 +/- 0.06 microM to 0.26 +/- 0.03 and 0.22 +/- 0.03 microM, respectively. Nitroglycerin and nicorandil may dilate coronary arteries by stimulating this Ca2+ extrusion pump enzyme through reduction of intracellular Ca2+ in smooth muscle cells.     

Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) regulates intracellular Ca2+ by mobilizing Ca2+ from a non-mitochondrial store. We have investigated the effects of Ca2+ on the binding of [32P]Ins (1,4,5)P3 to permeabilized rat hepatocytes and a liver plasma membrane-enriched fraction. Increasing the free Ca2+ concentration in the medium from 0.1 nM to 0.7 microM increased the capacity of a high affinity binding component (KD = 2-3 nM) in permeabilized cells by a factor of 10. If the membrane fraction was preincubated at 37 degrees C before binding was measured at 4 degrees C, all of the Ins(1,4,5)P3 receptors were transformed to a low affinity state (KD = 65 +/- 12 nM, Bmax = 3.1 +/- 0.1 fmol/mg, n = 4). When 0.7 microM of Ca2+ was added, the receptors were totally transformed to a high affinity state (KD = 2.8 +/- 0.4 nM, Bmax = 2.7 +/- 0.4 fmol/mg, n = 4). The EC50 of the Ca2(+)-induced interconversion of the Ins(1,4,5)P3 receptor was 140 nM. This Ca2(+)-induced transformation of the Ins(1,4,5)P3 receptor from a low affinity to a high affinity state was associated with an inhibition of the Ins(1,4,5)P3-induced Ca2+ release in permeabilized hepatocytes. These data suggest that the Ins(1,4,5)P3-dependent hormones, by increasing the intracellular Ca2+ concentration, induce a reversible transformation of the receptor from its low affinity state, coupled to the Ca2+ release, to a desensitized high affinity state. Transformation of the receptor may play a role in the oscillatory release of Ca2+ observed in single isolated hepatocytes.     

Mechanisms of endothelin-1-induced contraction in pulmonary arteries from chronically hypoxic rats

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.     

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.     

Calcium binding proteins of junctional sarcoplasmic reticulum: detection by 45Ca ligand overlay

In skeletal muscle, the junctional sarcoplasmic reticulum (JFM) plays a crucial role in excitation-contraction coupling and Ca2+ release. In the present report, the sarcoplasmic reticulum (SR) was fractionated into longitudinal SR (LSR), terminal cisternae (TC), and JFM. Each fraction had a unique protein profile as detected by SDS-polyacrylamide gel electrophoresis as well as specific Ca2+ binding proteins as judged by 45Ca ligand overlay of nitrocellulose blots. Ca2+ binding proteins of LSR were the Ca2+ ATPase (Mr of 115K), an 80K polypeptide, and the intrinsic glycoprotein (Mr of 160K); Ca2+ binding proteins of JFM were polypeptides with the following Mr values: 350K and 325K (feet components), 200K, 170K, a doublet of 140K, 118K, 65K (calsequestrin), and 52K. Measurements of Ca2+ binding to SR fractions by equilibrium dialysis indicated that 8-17 nmol Ca2+/mg of protein was specifically bound. After EDTA extraction of calsequestrin, JFM still bound Ca2+ (5-6 nmol/mg of protein), suggesting the existence of specific Ca2+ binding sites. The Ca2+ binding sites of Ca2+-gated Ca2+ release channels might be on two JFM polypeptides (Mr's of 350K and 170K) which are putative channel constituents (F. Zorzato, A. Margreth, and P. Volpe (1986) J. Biol. Chem. 261, 13252-13257).     

Phosphorylation of p36 in vitro with pp60src. Regulation by Ca2+ and phospholipid

P36 is a major substrate of the tyrosine protein kinases. P36 isolated from bovine intestine was used in phosphorylation reactions with pp60src. Phosphorylation was stimulated 3-5-fold by Ca2+, however the Km was the same (2.5 microM) at high or low Ca2+. Although the level of free Ca2+ needed for this enhanced phosphorylation was 10(-4)-10(-3) M, phosphatidylserine shifted the Ca2+ sensitivity to the 10(-6)-10(-5) M range. Independent evidence suggested that p36 interacts directly with liposomes containing phosphatidylserine. This raises the possibility that p36, like c-kinase, is a Ca2+-activated, phospholipid-dependent protein.     

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.     

Human serum amyloid P component, a circulating lectin with specificity for the cyclic 4,6-pyruvate acetal of galactose. Interactions with various bacteria.

Serum amyloid P component (SAP), a normal plasma glycoprotein, has recently been shown to have Ca2+-dependent binding specificity for methyl 4,6-O-(1-carboxyethylidene)-beta-D-galactopyranoside (MO beta DG) [Hind, Collins, Renn, Cook, Caspi, Baltz & Pepys (1984) J. Exp. Med. 159, 1058-1069]. SAP was found to bind in vitro to Klebsiella rhinoscleromatis, the cell wall of which is known to contain this particular cyclic pyruvate acetal of galactose. SAP also bound in similar amounts (approx. 6000 molecules per organism) to group A Streptococcus pyogenes, but very much less was taken up on Xanthomonas campestris, which contains the 4,6-cyclic pyruvate acetal of mannose. No SAP bound to Escherichia coli, which contains the 4,6-cyclic pyruvate acetal of glucose, or to Streptococcus pneumoniae type 4, which contains the 2,3-cyclic pyruvate acetal of alpha- rather than beta-galactopyranoside, or to other organisms (Streptococcus agalactiae, Staphylococcus aureus and Staphylococcus epidermidis), the carbohydrate structures of which are less well characterized. Binding of SAP to those organisms which it did recognize was completely inhibited or reversed by millimolar concentrations of free MO beta DG. SAP, a human plasma protein, thus behaves as a lectin and may be a useful probe for its particular specific ligand in the cell walls of bacteria and other organisms.     

Noncyclooxygenase metabolites of arachidonic acid amplify the vasopressin-induced Ca2+ signal in glomerular mesangial cells by releasing Ca2+ from intracellular stores

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.