Carl B. Baron

Rate-limiting energy-dependent steps controlling oxidative metabolism-contraction coupling in rabbit aorta.

1. We tested the hypotheses that coupling between oxidative metabolism and force in noradrenaline (NOR)‐activated rabbit aorta is controlled (a) by an energy‐dependent step or steps in receptor‐operated coupling mechanisms upstream to myosin light chain (MLC) kinase, or (b) by energy limitation of MLC kinase‐mediated phosphorylation of the MLC or actin‐activated myosin ATPase. 2. Oxidative energy production was rapidly inhibited by decreasing organ bath PO2 to less than 30 mmHg. There was no difference, comparing KCl‐ or NOR‐induced force, in the rates of decrease of [PCr] (phosphocreatine) or [ATP] following inhibition of oxidative energy production. (In this report we use the term [PCr] and [ATP] to indicate mean tissue values). Initial rates of decrease in [PCr] and [ATP] following inhibition of oxidative energy production were 0.05 mM/min and 0.06 mM/min, respectively. 3. Despite similar decreases in mean tissue [PCr] and [ATP], relaxations of KCl‐induced contractions following inhibition of oxidative energy production were markedly delayed and were blunted compared to relaxations seen during NOR‐induced contractions. The threshold mean tissue [PCr] and [ATP] for relaxation during KCl stimulation were 0.25 and 0.60 to 0.80 mM, respectively. During NOR stimulation, threshold values of [PCr] and [ATP] were 0.50 mM and 0.80 mM, respectively. Mean tissue [PCr] and [ATP] levels at 50% relaxation of KCl‐induced force were less than 0.1 mM and 0.1 mM, respectively. Fifty per cent relaxation of NOR‐induced force occurred at [PCr] and [ATP] values of 0.35 mM and 0.65 mM, respectively. 4. MLC phosphorylation levels decreased during relaxation of NOR force evoked by inhibition of oxidative energy production. There was no change in the level of MLC phosphorylation following inhibition of oxidative energy production in KCl‐contracted muscle even at mean tissue [PCr] and [ATP] lower than values associated with decreases in MLC phosphorylation during relaxations of NOR‐induced force. 5. Oxygen‐induced redevelopment of force during NOR exposure was not dependent on extracellular Ca2+. Mean tissue [PCr] increased prior to onset of O2‐evoked force redevelopment. Increases in MLC phosphorylation were seen at the time of onset of force redevelopment. 6. Oxidative metabolism‐contraction coupling during NOR‐stimulation seems not to be due to energy limitation of the MLC kinase reaction or actin‐activated myosin ATPase. Data suggest the rate‐limiting step is an energy‐dependent reaction in receptor‐operated coupling mechanisms upstream to MLC kinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of two Copper Reagents for Detection of Saturated and Unsaturated Neutral Lipids by Charring Densitometry

Abstract The intensity of staining of each of four classes of neutral lipids (monoacylglycerol, diacylglycerol, triacylglycerol and fatty acid methyl ester) is shown to be dependent on the number or amount of carbon-carbon double bonds (C=C) when using 3% cupric acetate in 8% phosphoric acid. In contrast, staining with 10% cupric sulfate in 8% phosphoric acid is relatively Independent of the number or amount of C=C.

[39] The isolation of bacterial membrane ATPase and nectin☆

Publisher Summary The ATPase localized in the plasma membrane of Streptococcus faecalis is believed to function in the energized uptake of K + ions and amino acids. As described in this chapter, the enzyme can be isolated as a soluble homogeneous protein on a fairly large scale by the procedure devised by Schnebli. This procedure is based on the smaller scale isolation methods developed earlier by Abrams s and Abrams and Baron. A notable feature of the isolation procedure is that the dissociation of the ATPase from the membrane is accomplished under very mild conditions and without the aid of such agents as detergents or ultrasound. To detach the membrane-bound enzyme, protoplast membrane ghosts are washed repeatedly, first with solutions containing high salt and Mg 2+ and then with solutions at low ionic strength containing no Mg 2+ . This series of washes eventually leads to a spontaneous abrupt release of the ATPase from the membranes along with some other membrane proteins.

Smooth muscle sarcolemma-associated phospholipase C-β2; agonist-evoked translocation

About 25% of the total cellular PLCβ2 content was found to be associated with a sarcolemmal fraction (SARC) isolated from unstimulated porcine trachealis smooth muscle. SARC‐associated PLCβ2 was located within two compartments, a detergent‐extractable compartment and a nondetergent extractable compartment. SARC PLCβ2 was measured after extraction with 0.6 M KCl; therefore, PLCβ2 was not bound solely by electrostatic forces within either of these compartments. PLCβ2 was shown to translocate from cytosol to SARC during a 20‐sec activation of intact muscle with a muscarinic agonist, carbachol (CARB); i.e., cytosolic total PLCβ2 content decreased significantly to 73 ± 7% of control and SARC total PLCβ2 content increased to 180 ± 15% of control value. This translocation was maintained at 5 min of CARB. CARB‐evoked translocation occurred into the detergent‐extractable SARC fraction, and PLCβ2 content in this fraction increased 300% compared with that in unstimulated muscle. After CARB, SARC PLCβ2 content accounted for >50% of total cellular PLCβ2 content. CARB‐evoked increase in PLC activity in SARC paralleled the increase in PLCβ2 content. CARB‐induced translocations of PLCβ2 from the cytosol to SARC were of a similar magnitude as occurred with phorbol ester‐induced translocations of PKCα. J. Cell. Physiol. 171:271–283, 1997.

Inositol 1,4,5-trisphosphate compartmentalization in tracheal smooth muscle

Pool sizes of inositol phosphate species in myo-[3H]inositol-labeled porcine tracheal smooth muscle were determined under three conditions: (a) unstimulated; (b) stimulated with carbachol; (c) atropine-relaxed from a carbachol contraction. In unstimulated muscle, the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content was 14 pmol/100 nmol lipid P1. This is equivalent to a mean [Ins(1,4,5)P3] of about 3 microM (in total cellular water), a level about 30-fold in excess of that required for Ca2+ release from Ins(1,4,5)P3-sensitive sarcoplasmic reticulum (SR). Pool sizes of breakdown products of Ins(1,4,5)P3 were relatively small or absent in unstimulated muscle, suggesting that, under this condition, Ins(1,4,5)P3 was sequestered and had limited access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. During carbachol stimulation, the Ins(1,4,5)P3 pool did not increase while those of other mono-, di-, and trisphosphate isomers increased over 10-fold. Subsequent atropine-induced relaxation resulted in a partial depletion (40%) of total tissue Ins(1,4,5)P3. Decreases in Ins(1,4,5)P3 were paralleled by decreases in Ins(1,4)P2 and Ins(1,3,4)P3. During contraction a portion of total tissue Ins(1,4,5)P3 has access to Ins(1,4,5)P3 3-kinase and 5-phosphatase and to Ins(1,4,5)P3-sensitive SR, though during antagonist-induced relaxation access to Ins(1,4,5)P3-sensitive SR for Ca2+ release is restricted. Data are consistent with a mechanism by which a large pool of Ins(1,4,5)P3 present in the unstimulated state in a sequestered compartment can contribute in activated muscle to increases in [Ins(1,4,5)P3] in a nonsequestered compartment, controlling SR Ca2+ release.

Polyamines, PI(4,5)P2, and actin polymerization

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol‐4‐phosphate 5‐kinases (PI(4)P5K), enzymes that convert phosphatidylinositol‐4‐phosphate to phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha‐difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 ± 3.5% and 81.5 ± 4.0% of control, respectively. Accurately reversing DFMO‐evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD‐sensitive PI(4)P5K. PUT and SPM were not involved in DFMO‐evoked changes in cellular PI(4,5)P2 contents. In DFMO‐treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 ± 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO‐evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO‐differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD‐sensitive PI(4,5)P2 pools, probably formed via SPD‐sensitive PI(4)P5K, that likely control actin polymerization. J. Cell. Physiol. 209: 405–412, 2006.

Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase

In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375–383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8–15), phosphatidylinositol (4)‐phosphate 5‐kinase plays a critical role in the regulation of membrane concentrations of phosphatidylinositol (4,5)‐bisphosphate and formation of inositol (1,4,5)‐trisphosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular phosphatidylinositol (4)‐phosphate 5‐kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton‐associated phosphatidylinositol (4)‐phosphate 5‐kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton‐associated enzyme, studied using phosphatidylinositol (4)‐phosphate substrate concentrations that produced phosphatidylinositol 4,5‐bisphosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2–0.4 mM, increased phosphatidylinositol (4)‐phosphate 5‐kinase activity two to four times compared to controls run without spermine. The EC50 for spermine‐evoked increases in activity was 0.17 ± 0.02 mM. Spermine‐evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton‐associated phosphatidylinositol (4)‐phosphate 5‐kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm − 10−8 to 10−5 M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine‐activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton‐bound phosphatidylinositol (4)‐phosphate 5‐kinase. J. Cell. Physiol. 177:161–173, 1998.

ALDOLASE A INS(1,4,5)P3-BINDING DOMAINS AS DETERMINED BY SITE-DIRECTED MUTAGENESIS

We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1, 4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P(3) binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P(3) binding, increased EC(50) values for Fru(1,6)P(2)-evoked release of bound Ins(1,4,5)P(3) and increased K(i) values for Ins(1,4, 5)P(3)-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P(3). Computer-simulated docking of Ins(1,4,5)P(3) in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P(3) to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P(2) binding. Results indicate that Ins(1,4,5)P(3) binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P(3) and Fru(1, 6)P(2) for binding. Although Ins(1,4,5)P(3) binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4, 5)P(3)-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P(3) in other proteins.

Mechanical and Biochemical Events During Hypoxia-Induced Relaxations of Rabbit Aorta

Hypoxic relaxation of norepinephrine contractions of isolated rabbit aorta is rapid, whereas relaxation of KCl contractions is slower and blunted. The data given here suggest that with receptor-evoked contractions of rabbit aorta, the energy-limitation of ATP-dependent K+ channels and other sarcolemmal channels, myosin light chain kinase, and actin-activated myosin ATPase are probably not involved in oxidative energy-contraction coupling. The data strongly support the hypothesis that the rate limiting, energy-dependent step is upstream to myosin light chain kinase, which is 50% inhibited at an ATP concentration of about 0.5 mM. This energy-dependent step may be in the inositol phospholipid transduction system, as we have previously postulated (Coburn et al., 1988). In contrast the energy-limited reaction during KCl contractions appears to be the actin-activated myosin ATPase which is 50% inhibited at a mean ATP concentration of about 0.1 mM.

SMOOTH MUSCLE ALDOLASE C-BOUND INOSITOL 1,4,5-TRISPHOSPHATE STUDIED IN VITRO UNDER PHYSIOLOGICAL CONDITIONS

Our goal was to quantitate inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding to aldolase C tetramer (aldolase4) and its displacement by inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) under conditions which approximated the in vivo state. Anions were found to have major effects. Decreasing [KCl] from 100 to 10mM, at 0 degrees C and pH 7.0, increased maximal Ins(1,4,5)P3 binding to 1.0 to 2.4mol per mol aldolase4. At 10 and 30mEq/l [Cl-], an additional high affinity site was detected (Kds = 0.43 and 0.86 microM, respectively). Increasing concentrations of other anions (SO42-, propanoate-, HCO3-, acetate-) also inhibited binding, but effects would be minimal at concentrations of these anions present in the cytoplasm of living cells. Ins(1,3,4)P3 displacement of aldolase C-bound Ins(1,4,5)P3 was sensitive to [Cl-]; at 30mEq/l [Cl-] and 37 degrees C, Ins(1,3,4)P3 released 20% of bound Ins(1,4,5)P3 at concentrations of 100nM. Changing temperature from 0 to 37 degrees C increased Kds for Ins(1,4,5)P3 binding. Changes in free [Ca2+], [Mg2+], [Na+] and [K+] and changes in osmolality had no effect on Ins(1,4,5)P3 binding to aldolase C. In vivo Ins(1,4,5)P3-aldolase4 binding at 30mEq/l [Cl-] and 37 degrees C were calculated for different [Ins(1,4,5)P3]free over the range 0.2 to 1.0 microM. For different cytoplasmic [Ins(1,4,5)P3]free. Ins(1,4,5)P3 binding to aldolase4 was sufficient, if acutely released, to nearly double cytoplasmic [Ins(1,4,5)P3]free. We proposed a schema whereby release of aldolase C-bound Ins(1,4,5)P3 evoked by Ins(1,3,4)P3 amplifies effects of phospholipase C-formed Ins(1,4,5)P3.