Metalloendoprotease Cleavage of 18.2- and 14.1-Kilodalton Basic Proteins Dissociating from Rodent Myelin Membranes Generates 10.0- and 5.9-Kilodalton G-Terminal Fragments

Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37 degrees C; pH 7.4). After 1-3 h, electrophoresis of the membranes revealed loss of 50% of 18.2- and 14.1-kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane-associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4-kDa and minor 10.0- and 5.9-kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2- and 14.1-kDa forms of MBP were cleaved near residue 73 to produce a 10.0- and 5.9-kDa C-terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5-20% using leupeptin (20 microM) but up to 50% by dithiothreitol mM), phenylmethylsulphonyl fluoride (1 mM), and phosphoramidon (50 microM) but up to 50% by dithiothreitol (DDT, 10 mM). Only DDT and 1,10-phenanthroline substantially blocked the formation of the characteristic 10.0-and 5.9-kDa C-terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N-ethylmaleimide/leupeptin-sensitive calpains and phosphoramidon-sensitive endopeptidase 24.11.     

Zinc Ions Stabilise the Association of Basic Protein with Brain Myelin Membranes

Myelin basic protein (MBP) dissociated from brain myelin membranes when they were incubated (37 degrees C; pH 7.4) at physiological ionic strength. Zinc ions inhibited, and calcium promoted, this process. Protease activity in the membrane preparations cleaved the dissociated MBP into both small (less than 4 kilodaltons) and large (greater than 8 kilodaltons) fragments. The latter were detected, together with intact MBP, by gel electrophoresis of incubation media. Zinc ions appeared to act in two distinct processes. In the presence or absence of added CaCl2, zinc ions in the range 0.1-1 mM inhibited MBP-membrane dissociation. This process was relatively insensitive to heat and Zn2+ could be substituted by either copper (II) or cobalt (II) ions. A second effect was evident only in the presence of added calcium ions, when lower concentrations of Zn2+ (less than 0.1 mM) inhibited MBP-membrane dissociation and the accumulation of intact MBP in incubation media. This process was heat sensitive and only copper (II), but not cobalt (II), ions could replace Zn2+. To determine whether endogenous zinc in myelin membranes is bound to MBP, preparations were solubilised in buffers containing Triton X-100/2 mM CaCl2 and subjected to gel filtration. Endogenous zinc, as indicated by a dithizone-binding method, eluted with fractions containing both MBP and proteolipid protein (PLP). Thus, one means whereby zinc stabilises association of MBP with brain myelin membranes may be by promoting its binding to PLP.     

Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Solution NMR structure of an immunodominant epitope of myelin basic protein. Conformational dependence on environment of an intrinsically unstructured protein

Using solution NMR spectroscopy, three-dimensional structures have been obtained for an 18-residue synthetic polypeptide fragment of 18.5 kDa myelin basic protein (MBP, human residues Q81-T98) under three conditions emulating the protein's natural environment in the myelin membrane to varying degrees: (a) an aqueous solution (100 mM KCl pH 6.5), (b) a mixture of trifluoroethanol (TFE-d2) and water (30 : 70% v/v), and (c) a dispersion of 100 mM dodecylphosphocholine (DPC-d38, 1 : 100 protein/lipid molar ratio) micelles. This polypeptide sequence is highly conserved in MBP from mammals, amphibians, and birds, and comprises a major immunodominant epitope (human residues N83-T92) in the autoimmune disease multiple sclerosis. In the polypeptide fragment, this epitope forms a stable, amphipathic, alpha helix under organic and membrane-mimetic conditions, but has only a partially helical conformation in aqueous solution. These results are consistent with recent molecular dynamics simulations that showed this segment to have a propensity to form a transient alpha helix in aqueous solution, and with electron paramagnetic resonance (EPR) experiments that suggested a alpha-helical structure when bound to a membrane [I. R. Bates, J. B. Feix, J. M. Boggs & G. Harauz (2004) J Biol Chem, 279, 5757-5764]. The high sensitivity of the epitope structure to its environment is characteristic of intrinsically unstructured proteins, like MBP, and reflects its association with diverse ligands such as lipids and other proteins.     

Ionized and bound calcium inside isolated sarcoplasmic reticulum of skeletal muscle and its significance in phosphorylation of adenosine triphosphatase by orthophosphate.

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5--15 mM) on ice gives calcium loads of 50--60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid], but almost completely released by ionophore X-537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non-saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half-saturation constant of 5,8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 microliter/mg protein, respectively. Calcium-dependent phosphorylation of sarcoplasmic reticulum calcium-transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium-dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively. It is further suggested that the saturation of the low-affinity calcium-binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium-dependent phosphorylation than in calcium-independent phosphorylation from orthophosphate. Maximum calcium-dependent phosphoprotein formation at 20 degrees C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.     

Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments

Experiments were performed with isolated human myelin membrane preparations to analyse factors that may modulate association of myelin basic protein (MBP) with the membranes and could contribute to demyelinating processes. Transfer of membranes (5 mg protein ml-1) at 37 degrees C and pH 7.4 from a hypotonic medium, in which they were relatively stable, to one of physiological ionic strength produced three major effects: (1) initial dissociation of MBP from the membranes by a nonenzymatic process that was doubled in the presence of millimolar Ca2+/Mg2+; (2) within 10 min, the appearance in the medium of three major MBP fragments (14.4, 10.3, and 8.4 kilodaltons); and (3) progressive acidification of dissociated MBP and its fragments, probably due to deamidation. Between 1 and 6 h a steady state was apparent in which protein equivalent to 15% of the MBP originally bound to the membranes was found in the medium. The three major MBP fragments formed two-thirds of this solubilised material and appeared metabolically stable for 24 h. The kinetics of peptide formation suggested that dissociated, rather than membrane-bound, MBP was cleaved by myelin-associated neutral proteases. Two-dimensional electrophoresis and immunoblotting using two monoclonal antibodies indicated that proteolysis occurred in the vicinity of residues 35 and 75. Evidence was also obtained for removal of C-terminal arginines and relatively rapid deamidation in the C-terminal half of MBP. These modifications of MBP might also occur if extracellular fluid gained access to the compacted cytoplasmic space of the myelin sheath.     

The use of gel filtration to follow conformational changes in proteins. Conformational flexibility of bovine myelin basic protein.

The hydrodynamic behavior of bovine myelin basic protein was studied by gel filtration through Sephadex G-100 under conditions which included variations in pH from 2 to 12, variations in ionic strength from 0.01 to 1.5 M at pH 2 and from 0.1 to 2 M at pH 7, and variations in guanidinium chloride concentration from 0 to 6 M. A number of well characterized compact globular proteins were subjected to the same conditions for comparison. Compact globular proteins showed major conformational transitions due to acid, alkali, and guanidinium chloride denaturation and, possibly, minor transitions as well. Myelin basic protein behaved like a flexible linear polyelectrolyte, expanding continuously between pH 11 and pH 2 to 3 at ionic strength 0.1 M and contracting continuously with increase in ionic strength at pH 2 and at pH 7 to the point of salting-out. Relatively low concentrations of guanidinium chloride (approximately 0.5 M) were sufficient to cause the basic protein to expand. With increasing concentration of the denaturant the molecule continued to expand, but in a noncooperative manner. These results demonstrated the lack of significant intramolecular stabilization in the protein.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Calcium binding by subcellular fractions of bovine adrenal medulla

Significantly more calcium per gram protein was found in a relatively pure granule fraction isolated from fresh bovine adrenal medulla than in predominantly mitochondrial fractions isolated from the same tissue. Sixty-four and 55% of the calcium associated with chromaffin granule and mitochondrial fractions, respectively, was released into the supernatant upon lowering the tonicity of the medium. The per cent calcium released by this procedure was significantly greater for granules than for mitochondria (p < 0.05). The amount of calcium per gram protein released into the supernatant also was greater in granule fractions than in mitochondrial fractions (p < 0.05). These data, coupled with a previous report that 10^?3 M EDTA does not markedly decrease the calcium content of whole granules, indicate that the excess calcium of the granule fractions relative to the mitochondrial fractions is maintained within the particles of that fraction. The functional significance of the relatively large amount of calcium in chromaffin granules is not clear. The presence of 150 mM sodium chloride or potassium chloride decreases calcium binding by granule or mitochondrial fragments incubated in 2.2 mM calcium chloride in 0.2 M Tris, pH 7, by about 50%. EDTA, 10^?3 M, removes all but a small residual of the calcium associated with the granule or mitochondrial fragments whereas lowering the concentration of Tris increases calcium binding to about the same extent in both these subcellular fractions. The calcium-binding properties of granule and mitochondrial fragments therefore appear to be quantitatively and qualitatively similar. Inhibition of catecholamine release by relatively high concentrations of sodium may be explained by competitive inhibition of calcium binding. Calcium binding by granule fragments decreases with an increase in hydrogen ion concentration.     

Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane

Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding.     

Racemization of Individual Aspartate Residues in Human Myelin Basic Protein

Human myelin basic protein (MBP), a long-lived brain protein, undergoes gradual racemization of its amino acids, primarily aspartic acid and serine. Purified protein was treated at neutral pH with trypsin to yield peptides that were separated by HPLC using a C18 column. Twenty-nine peptides were isolated and analyzed for amino acid composition and aspartate racemization. Each aspartate and asparagine in the protein was racemized to a different extent, ranging from 2.2 to 17.1% D isomer. When the racemization was examined in terms of the beta-structure model of MBP, a correlation was observed in which six aspartate/asparagine residues assumed to be associated with myelin membrane lipids showed little racemization (2.2-4.9% D isomer), whereas five other aspartate residues were more highly racemized (9.9-17.1% D isomer). Although the observed aspartate racemization may be related to steric hindrance by neighboring residues and/or the protein secondary structure, interaction of aspartates with membrane lipids may also be a major factor. The data are compatible with a model in which each MBP molecule interacts with adjacent cytoplasmic layers of myelin membrane through a beta-sheet on one surface and loops and helices on the other surface, thereby stabilizing the myelin multilamellar structure.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.     

Interactions of the 18.5 kDA isoform of myelin basic protein with a2+-calmodulin: In vitro studies using gel shift assay

The interactions of the 18.5 kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using glutaraldehyde or dithiobis[succinimidylpropionate] (DSP) cross-linking, and SDS-polyacrylamide gel electrophoresis. The following forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a recombinant murine product (rmMBP), and their fragments generated by digestion with cathepsin D (EC 3.4.23.5). In physiological buffers (10 mM HEPES-NaOH, pH 7.4, 5 mM CaCl₂, 0.0035% glutaraldehyde; or 50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 1 mM CaCl₂, 0.0035% DSP), MBP and CaM interacted primarily in a 1:1 molar ratio, consistent with previous studies that used 6 M urea, i.e. denaturing conditions. Moreover, the appearance of higher-order bands (not previously observed) suggested that the mechanism of interaction of the two proteins involved a series of relatively complex equilibria, resulting in 2:1 ratios of MBP to CaM. This observation would explain the cooperativity of association inferred from fluorescence studies [13]. Our results demonstrated further that the interaction involved the C-terminal domain of MBP, again in a primarily 1:1 molar ratio with CaM, consistent with our identification of a CaM-binding motif at the C-terminus.     

Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme

Two different molecular species of protein methylases I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23), one specific for myelin basic protein (MBP) and the other for histone, have been purified from calf brain to near homogeneity, as discerned by nondenaturing polyacrylamide gel electrophoresis. Although both methylases share some common properties, such as utilization of S-adenosyl-L-methionine as the methyl donor and methylation of protein-bound arginine residues, they are distinctly different from each other in molecular weight and in catalytic, as well as the immunological, properties. The MBP-specific protein methylase I (approximately 500 kDa) methylates MBP preferentially (Km = 2 X 10(-7) M) and histone to a much lesser extent (Km = 1 X 10(-4) M), while the histone-specific methylase I (approximately 275 kDa) methylates histone only. Both methylases exhibit two major subunit bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 100 and 72 kDa for the MBP-specific and 110 and 75 kDa for the histone-specific. At 0.5 mM p-chloromercuribenzoate, about 50% of the MBP-specific enzyme remained as active, while most of the histone-specific enzyme activity was lost. In 2 mM guanidine HCl, approximately 90% of the former enzyme activity remained while nearly complete inactivation of the latter enzyme was observed. The enzymes also exhibited quite different inactivation profiles toward high temperature (45-65 degrees C); MBP-enzyme was stable up to 50 degrees C and was rapidly inactivated at higher temperatures with an inflection point at about 57 degrees C. However, under the identical conditions, histone-enzyme was inactivated progressively and linearly in the same temperature range. Finally, Western immunoblot analysis of polyclonal antibodies directed against either enzyme exhibited no cross-reactivity with the other.     

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37°C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.     

Phorbol Ester-Mediated Stimulation of Phospholipase D Activity in Sciatic Nerve from Normal and Diabetic Rats

Evidence for the presence of phospholipase D activity in sciatic nerve was obtained by incubation of 32P-prelabeled nerve segments in the presence of ethanol and measurement of [32P]phosphatidylethanol (PEth) formation expressed as a fraction of total phospholipid radioactivity. PEth synthesis was enhanced with increasing concentrations of ethanol (100 mM-2 M). 4-beta-Phorbol dibutyrate (100 nM-1 microM) stimulated PEth formation up to twofold in a time- and dose-dependent manner. The stimulatory effect evoked by 100 nM phorbol ester was completely abolished by Ro 31-8220 (compound 3), a selective protein kinase C inhibitor. Efforts to identify the phospholipid precursor of PEth were unsuccessful, suggesting this product arises from a small discrete precursor pool. On subcellular fractionation of nerve, the ratio of basal and 4-beta-phorbol dibutyrate-stimulated phospholipase D activity recovered in a myelin-enriched fraction, compared with a nonmyelin fraction, was 0.5 when results are expressed as a percentage of total phospholipid radioactivity. This ratio rises to 1.2 if the results are calculated assuming only phosphatidylcholine and phosphatidylethanolamine are potential precursors. The results suggest that myelin is a major locus of phospholipase D activity. Nerve from streptozotocin-induced diabetic and control animals displayed the same basal phospholipase D activity, but the enzyme in diabetic nerve was stimulated to a greater extent by a suboptimal concentration of 4-beta-phorbol dibutyrate. These results support the conclusion that protein kinase C modulates phospholipase D activity in nerve and suggest that in diabetic nerve the enzyme activation mechanism may possess increased sensitivity.     

Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C

Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).     

Ionic mechanisms of histamine-induced responses in guinea pig intracardiac neurons

Histamine, released from mast cells, can modulate the activity of intrinsic neurons in the guinea pig cardiac plexus. The present study examined the ionic mechanisms underlying the histamine-induced responses in these cells. Histamine evokes a small membrane depolarization and an increase in neuronal excitability. Using intracellular voltage recording from individual intracardiac neurons, we were able to demonstrate that removal of extracellular sodium reduced the membrane depolarization, whereas inhibition of K+ channels by 1 mM Ba2+, 2 mM Cs+, or 5 mM tetraethylammonium had no effect. The depolarization was also not inhibited by either 10 microM Gd3+ or a reduced Cl- solution. The histamine-induced increase in excitability was unaffected by K+ channel inhibitors; however, it was reduced by either blockage of voltage-gated Ca2+ channels with 200 microM Cd2+ or replacement of extracellular Ca2+ with Mg2+. Conversely, alterations in intracellular calcium with thapsigargin or caffeine did not inhibit the histamine-induced effects. However, in cells treated with both thapsigargin and caffeine to deplete internal calcium stores, the histamine-induced increase in excitability was decreased. Treatment with the phospholipase C inhibitor U73122 also prevented both the depolarization and the increase in excitability. From these data, we conclude that histamine, via activation of H1 receptors, activates phospholipase C, which results in 1) the opening of a nonspecific cation channel, such as a transient receptor potential channel 4 or 5; and 2) in combination with either the influx of Ca2+ through voltage-gated channels or the release of internal calcium stores leads to an increase in excitability.