Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.     

Phosphatidylcholine activation of bacterial phosphatidylinositol-specific phospholipase C toward PI vesicles

The effect of different phospholipids on the kinetic behavior of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis toward PI vesicles has been investigated. Cosonicated PC/PI vesicles displayed enhanced hydrolysis of PI when less than 0. 20 mole fraction PC was incorporated into the vesicle; higher mole fractions of PC led to a decrease from the maximum activity mimicking surface dilution of substrate. Since the PC could affect PI-PLC binding to vesicles, the effect of separate PC vesicles on enzymatic hydrolysis of PI vesicles was examined. Separate phosphatidylcholine vesicles were found to activate PI-PLC-catalyzed cleavage of PI vesicles up to 7-fold. The activation was completely abolished when the PC vesicle was composed of cross-linked molecules. In the absence of enzyme, fluorescence resonance energy transfer studies did not detect any fusion between PI and PC vesicles if the total lipid concentration was below 2 mM. Higher total lipid concentrations (>20 mM) increased PC transfer between PC and PI vesicles, producing a PI vesicle population with small amounts of PC in the outer monolayer. This suggested that the activation of PI-PLC toward PI vesicles reflects the time scale of transfer of PC from PC vesicles to PI vesicles. Cosonicated PC/PI vesicles provide a measure of enzyme activity versus mole fraction of PC that can be used to estimate the extent of vesicle exchange or fusion between separate vesicle pools. The effects of other phospholipid vesicles on PI-PLC hydrolysis of PI were also examined; zwitterionic lipids were activators while anionic phospholipids inhibited activity. The results indicated that PC molecules in the PI interface allosterically bind to PI-PLC and help anchor enzyme in a more active conformation to the PI interface.     

Allosteric activation of phosphatidylinositol-specific phospholipase C: specific phospholipid binding anchors the enzyme to the interface

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al. (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents. This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.     

Activity of phosphatidylinositol-specific phospholipase C from Bacillus cereus with thiophosphate analogs of dimyristoylphosphatidylinositol

We examined the effects of nasal continuous positive airway pressure (CPAP) on exercise performance in patients with obstructive sleep apnea (OSA). Six patients were treated with nasal CPAP on seven successive days and underwent overnight sleep studies and multiple sleep latency test (MSLT) at the beginning and after the last day of the treatment. The subjects also performed incremental exercise testing using a bicycle ergometer followed by 0-w, 25-w, 50-w,--(3 minutes each) until maximum level. Arterial oxygen pressure, arterial carbon dioxide pressure at rest while awake, apnea/ hypopnea index, longest apnea duration, the lowest percutaneous oxygen saturation measured by a pulse oximeter and the value of MSLT were significantly improved after nasal CPAP. Moreover, maximal oxygen consumption was significantly increased from 1841 ml/min +/- 350 to 2125 ml/min +/- 351 (p < 0.05); however, other cardiorespiratory parameters did not change significantly. The improvement of exercise performance by short-term nasal CPAP treatment in OSA patients may correlate with the improvement of sleepiness.     

Increased expression of phosphatidylinositol-specific phospholipase C resistant prion proteins on the surface of activated platelets

The surface expression of prion protein (PrP(C)) on human platelets, as detected by flow cytometry with the monoclonal antibody 3F4, increased more than two-fold (4300 v 1800 molecules/platelet) after full activation. Maximal surface expression of PrP(C) occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37 degrees C. In comparison, PrP(C) on the surface of platelets, activated at 22 degrees C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrP(C) and P-selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrP(C) was translocated from internal granules to the plasma membrane during activation, as is P-selectin. Platelet PrP(C) was not removed from the surface of platelets by phosphatidylinositol-specific phospholipase C (PIPLC) treatment but was degraded by proteinase K. Platelets may serve as a useful model for following the cellular processing of PrP(C).     

Action of phosphatidylinositol-specific phospholipase Cgamma1 on soluble and micellar substrates. Separating effects on catalysis from modulation of the surface

The kinetics of PI-PLCgamma1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixed micelles were monitored by 31P NMR spectroscopy. That cIP is also a substrate (Km = approximately 15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLCgamma1 to form cIP and I-1-P with the enzyme specific activity and ratio of products (cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and other amphiphilic additives. Cleavage of both cIP and PI by the enzyme is optimal at pH 5. The effect of Ca2+ on PI-PLCgamma1 activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2+ activates the enzyme; however, high Ca2+ inhibits PI-PLCgamma1 hydrolysis of phosphoinositides (but not cIP) with the extent of inhibition dependent on pH, substrate identity (cIP or PI), substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLCgamma1 by high Ca2+ is proposed to derive from the divalent metal ion-inducing clustering of the PI and reducing its accessibility to the enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; they have no effect on cIP hydrolysis at either pH value. These different kinetic patterns are used to propose a model for regulation of the enzyme. A key hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase). However, this activation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.     

Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site. The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface [Zhou, C., et al. (1997) Biochemistry 36, 347-355; Zhou, C., et al. (1997) Biochemistry 36, 10089-10091]. In contrast to PC, anionic short-chain phospholipids with smaller headgroups [phosphatidylmethanol (PMe) and phosphatidic acid (PA)] as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis. PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate. Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities. If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site. When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site. Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC. A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.     

Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function

Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape of L. monocytogenes from the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives of L. monocytogenes containing these specific plcA mutations were found to have phenotypes similar to that of the plcA deletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread. Thus, catalytic activity of PI-PLC is required for all its intracellular functions.     

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.     

Structural mapping of the catalytic mechanism for a mammalian phosphoinositide-specific phospholipase C

The crystal structures of various ternary complexes of phosphoinositide-specific phospholipase C-delta 1 from rat with calcium and inositol phosphates have been determined at 2.30-2.95 A resolution. The inositol phosphates used in this study mimic the binding of substrates and the reaction intermediate and include D-myo-inositol-1,4,5-trisphosphate, D-myo-inositol-2,4, 5-trisphosphate. D-myo-inositol-4,5-bisphosphate, and D,1-myo-inositol-2-methylene-1,2-cycli?monophosphonate. The complexes exhibit an almost invariant mode of binding in the active site, each fitting edge-on into the active site and interacting with both the enzyme and the catalytic calcium at the bottom of the active site. Most of the active site residues do not undergo conformational changes upon binding either calcium or inositol phosphates. The structures are consistent with bidentate liganding of the catalytic calcium to the inositol phosphate intermediate and transition state. The complexes suggest explanations for substrate preference, pH optima, and ratio of cyclic to acyclic reaction products. A reaction mechanism is derived that supports general acid/base catalysis in a sequential mechanism involving a cyclic phosphate intermediate and rules out a parallel mechanism where acyclic and cyclic products are simultaneously generated.     

Activation of phosphatidylinositol-specific phospholipase C in response to HDL3 and LDL is markedly reduced in cultured fibroblasts from Tangier patients

We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.     

Flow-regulatory function of upper airway in health and disease: a unified pathogenetic view of sleep-disordered breathing

Although the Starling resistor behavior of the upper airway during sleep has been well established in health and disease, its physiological implications have not been fully appreciated. The purposes of the present communication are to reassess the current state of knowledge within the framework of the Starling resistor concept and to examine the implications of the concept on homeostatic feedback respiratory control and the pathogenesis of the sleep apnea syndrome. The main inferences drawn from the assessment include: (1) Owing to the Starling resistor properties of the upper airway and the well-organized neurochemical control mechanism, the upper airway performs important homeostatic flow regulatory function; it appropriately dampens the potentially unstable breathing during sleep and prevents the PaCO2 from falling below the apneic threshold; (2) Under certain conditions, the upper airway flow regulatory function fails to achieve appropriate dampening, leading to development of a variety of sleep-related breathing disorders that include underdamping due to overly sensitive central chemoresponsiveness and/or excessive lung to chemoreceptor transport lag--central sleep apnea; overdamping due to upper airway obstructive dysfunction--obstructive sleep apnea and/or hypopnea; and, finally, conditions with mixed features of central underdamping with coexisting collapsible upper airway; and (3) Successful treatment of these conditions requires restoration of appropriate damping. The overdamping imposed by the faulty upper airway is effectively reduced by surgical and medical approaches, and by application of nasal continuous positive airway pressure (CPAP). Reduction of PaCO2 by use of acetalzolamide and/or aminophylline reduces the plant gain, thus effectively offsetting the underdamping of central origin. Owing to the dual effect of nasal CPAP on the upper airway and respiratory pump, use of nasal CPAP can also effectively reduce the plant gain, accounting for the therapeutic effect of nasal CPAP on the central sleep apnea.     

Molecular diversity and double regulatory mechanism of activation of phospholipase C in rat brain

Whereas evidence for a G protein-dependent stimulation of phospholipase C (PLC) is abundant, reports on the inhibition of PLC through a G protein-mediated pathway have only recently begun to appear. In the present study, cerebral cortex membranes were chosen since they have a readily measurable Gpp[NH]p and Ca2+-stimulated PLC activity. Nanomolar concentrations of Gpp[NH]p, a hydrolysis-resistant GTP analogue, inhibited basal inositol 1,4,5-trisphosphate (IP3) production, with a maximum inhibition of 25% at 10 nM. Increasing the concentrations of Gpp[NH]p to over 10 nM resulted in a reversal of the inhibitory effect and onset of stimulation of IP3 production. GDPbetaS as a G protein inhibitor and U-73122 as a putative PLC-beta inhibitor had little effect on basal IP3 production at 100 microM and 1 microM, respectively. However, GDPbetaS and U-73122 completely antagonized both the inhibition and the stimulation of IP3 production produced by lower and higher concentrations, respectively, of Gpp[NH]p. Rat cortical membranes expressed a greater amount of PLC-beta1. These data suggest that PLC-beta1 isozymes may be regulated by both inhibitory and stimulatory G protein-mediated mechanisms.     

Protein kinase C and phospholipase C: bilayer interactions and regulation

In animal models, calcium antagonists (Ca-A) administered before ischemia and reperfusion reduced myocardial necrosis, attenuated postischemic contractile dysfunction, and reduced tissue calcium. In 753 patients with acute myocardial infarction (AMI), we examined if use of Ca-A at the onset of symptoms (n = 127 patients) reduced infarct size as estimated from peak creatine kinase (CKmax) and lactate dehydrogenase (LDmax) activities. The study had an observational exposed/nonexposed design, and both crude and adjusted effects were investigated. Crude effects: In the restricted cohort of patients not receiving thrombolytic treatment (thr- pts; n = 411 patients), CKmax and LDmax were lower in Ca-A+ patients than in Ca-A- patients, being 643 versus 887 U/l (2 p = 0.004) and 708 versus 867 U/l (2 p = 0.005), respectively. When using log (CKmax) and log (LKmax) as outcomes, the same results were found (2 p = 0.002). More of the restricted cohort of the pts used Ca-A in the lower quartiles of CKmax and LDmax (p for linear trend = 0.005 and 0.004 for CKmax and LDmax, respectively). Adjusted effects: Thrombolysis was an effect modifier of the association between Ca-A and peak enzyme levels. In thr-pts, the coefficients of Ca-A were negative and borderline significant for log (CKmax; 2 p = 0.088) and negative and highly significant for log (LDmax; 2 p = 0.010) when adjusting for confounders. The present observational study indicates that the use of a Ca-A at the onset of AMI reduces infarct size, as estimated from CKmax and LDmax activities.     

Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl(alpha 1-->6)-D-myo-inositol, an essential fragment of GPI anchors

Numerous proteins on the external surface of the plasma membrane are anchored by glycosylated derivatives of phosphatidylinositol (GPI), rather than by hydrophobic amino acids embedded in the phospholipid bilayer. These GPI anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) to release a water-soluble protein with an exposed glycosylinositol moiety and diacylglycerol, which remains in the membrane. We have previously determined the crystal structure of Bacillus cereus PI-PLC, the enzyme which is widely used to release GPI-anchored proteins from membranes, as free enzyme and also in complex with myo-inositol [Heinz, D.W., Ryan, M. Bullock, T.L., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863]. Here we report the refined 2.2 A crystal structure of this enzyme complexed with a segment of the core of all GPI anchors, glucosaminyl(alpha 1-->6)-D-myo-inositol [GlcN-(alpha 1-->6)Ins ]. The myo-inositol moiety of GlcN(alpha 1-->6)Ins is well-defined and occupies essentially the same position in the active site as does free myo-inositol, which provides convincing evidence that the enzyme utilizes the same catalytic mechanism for cleavage of PI and GPI anchors. The myo-inositol moiety makes several specific hydrogen bonding interactions with active site residues. In contrast, the glucosamine moiety lies exposed to solvent at the entrance of the active site with minimal specific protein contacts. The glucosamine moiety is also less well-defined, suggesting enhanced conformational flexibility. On the basis of the positioning of GlcN(alpha 1-->6)Ins in the active site, it is predicted that the remainder of the GPI-glycan makes little or no specific interactions with B. cereus PI-PLC. This explains why B. cereus PI-PLC can cleave GPI anchors having variable glycan structures.     

Families of phosphoinositide-specific phospholipase C: structure and function

A large number of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC isozymes have been found in a broad spectrum of organisms and although they have common catalytic properties, their regulation involves different signalling pathways. A number of recent studies provided an insight into domain organisation of PI-PLC isozymes and contributed towards better understanding of the structural basis for catalysis, cellular localisation and molecular changes that could underlie the process of their activation.     

Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.     

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.     

Pyrimidinoceptor-mediated activation of phospholipase C and phospholipase A2 in RAW 264.7 macrophages

Electron microscopy of toad (Bufo marinus) muscle fixed without relaxing after a single eccentric contraction at muscle lengths greater than optimum showed over-stretched half-sarcomeres in sufficient numbers to account for more than half of the imposed stretch. Such sarcomeres were absent in another muscle fixed without relaxing after an isometric contraction at the same length and largely absent in a third muscle that underwent eccentric contraction at muscle lengths less than optimum. This provides direct evidence in support of the hypothesis that lengthening of muscles at long length involves lengthening of a few half sarcomeres to beyond filament overlap, while most half sarcomeres are extended much less than in proportion to muscle extension.