Trifluoperazine inhibition of contraction in permeabilized skeletal, cardiac and smooth muscles

To gain insights into the mechanism of the central helix of calmodulin and troponin-C in the Ca2(+)-regulation of force development in striated and smooth muscles, the present study was made of the TFP induced inhibition of contraction, and of the uptake of these proteins by skinned fibers. Calmodulin was four-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac muscles despite the differences in their troponin-C isoforms. Also, the results were comparable between fast-twitch fiber, when calmodulin was exchanged for troponin-C to act on TnI, and smooth muscle, where calmodulin acts on myosin light chain kinase. These findings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sites in the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFP-independent domain in troponin-C that binds TnI.     

Influence of heparins on inositol 1,4,5-trisphosphate-induced calcium mobilization in permeabilized human platelets

Heparin has been shown to prevent inositol 1,4,5-trisphosphate (IP3) binding to its receptor and to inhibit IP3-induced calcium mobilization in a variety of cells. Heparin added to whole blood at a concentration of 1 U/ml prevented thrombin-induced secretion of granule contents and irreversible aggregation of platelets. Heparin (2-15 kDa) had no inhibitory effect on IP3-induced calcium mobilization in Fura 2-loaded, saponin (10-15 micrograms/ml)-permeabilized platelets. None of the commercially available heparin preparations can induce inhibition of agonist-induced calcium mobilization in intact platelets because they are not cell permeant. Mild saponin treatment makes the membrane permeable to IP3, but restricts the action of heparins. Recent observations suggesting heparin's affinity to IP3 binding sites will be of clinical interest if effective cell permeant analogs can be developed.     

Formation and composition of a graded estuarine shell bed

The surface bed of a mudflat in the Ythan Estuary, Aberdeenshire, consists of mud and sandy mud overlying broken-shell material, the whole bed being graded. Production of shell fragments by bird predation and their subsequent incorporation in the bed due to the activities of a burrowing fauna are considered. The graded bed represents a stable situation in the mudflat area. Diagenesis of shells within the bed varies with species, Mytillus being prone to disintegration into fine needles of carbonate.     

Calcium-induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells

The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.     

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique.

"Whole-cell" patch recordings using nystatin permeabilization were made from single human platelets during application of agonists from a "puffer" pipette. In platelets clamped near the resting potential and bathed in Na+ saline, 40 microM ADP activated a transient inward current within tens of milliseconds. At -73 mV the current lasted between 0.1 and 1 s and had a peak of between 13 and 31 pA in different cells. Ion substitution experiments indicated that the channel is permeable to Na+,K+, and Ba2+ and presumably also to Ca2+, but is not permeable to Cl-. The single channel conductance was 15 pS (near the resting potential) in nominally Ca(2+)-free saline and 11 picosiemens in BaCl2 saline. Thrombin, at 1 unit/ml, did not elicit detectable currents during a 3-s application in platelets bathed in 1 mM Ca2+, Na+ saline. Under the same conditions, in fura-2-loaded cells, thrombin-evoked Ca2+ entry (monitored by Mn2+ quench) was detectable after a delay of 1.4 s. This suggests that early thrombin-evoked Ca2+ entry occurs via small conductance channels, below the resolution of the patch clamp technique, or by an electroneutral pathway. The ADP-evoked channel has the requisite speed of activation to account for the rapid Ca2+ influx observed during stopped-flow studies of agonist-evoked changes in [Ca2+]i.     

Reactivation of Ca2+-dependent cytoplasmic contraction in permeabilized cell models of the heliozoon Echinosphaerium akamae

Permeabilized cell models of the large heliozoon Echinosphaerium akamae were prepared by treatment with 100 mM EGTA or 1% Triton X-100. When > 10(-6) M Ca(2+) was added to the EGTA-permeabilized cells, axopodial cytoplasm became contracted and several swellings were formed along the axopodial length. Axonemal microtubules remained intact, while higher concentration of Ca(2+) (> 10(-4) M) induced microtubule disassembly and complete breakdown of the axopodia. In Triton-permeabilized cells, cytoplasmic contraction and relaxation of the cell body were induced repeatedly by successive addition and removal of Ca(2+). The contraction did not require ATP, and was not inhibited by cytochalasin B. Electron microscopy showed, in EGTA-permeabilized axopodia, contractile tubules became granulated by the addition of Ca(2+). From these observations, it is strongly suggested that Ca(2+)-dependent granulation of the contractile tubules is responsible for the axopodial contraction.     

Thrombin induces serotonin secretion and aggregation independently of inositol phospholipids hydrolysis and protein phosphorylation in human platelets permeabilized with saponin

We have observed that the addition of Ca2+ to platelets, permeabilized with saponin, promotes a drastic dephosphorylation of proteins and polyphosphoinositides without inducing platelet responses. Subsequent addition of thrombin could promote secretion of serotonin and aggregation in the absence of phospholipase C-induced breakdown of the inositol phospholipids and protein phosphorylation. This information indicates that activation of saponized platelets by thrombin is independent of the formation of second messengers derived from the phospholipase C-induced breakdown of the inositol phospholipids. The implications of this result for intact platelets are discussed.     

Modes of inhibitory action of 4 beta-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets

The tumor-promoting phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC50 vs thrombin = 0.5 nM, greater than 90% inhibition at 10 nM. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 nM PMA. A non-tumor-promoting phorbol ester, 4-O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated AA release, but not GTP gamma S- and AIF4(-)-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A2 by PMA pretreatment is discussed.     

Role of platelets in contraction of canine tracheal muscle elicited by PAF in vitro

To elucidate mechanisms of platelet-activating factor (PAF)-induced contraction, we studied the effect of PAF on 203 canine tracheal smooth muscle (TSM) strips from 45 dogs in vitro in the presence and absence of platelets. PAF (10(-11) to 10(-7) M) alone caused no contraction of TSM even in the presence of airway epithelium. In the presence of 2 x 10(5) platelets/microliter, PAF was an extremely potent contractile agonist (threshold 10(-11) M). This response was inhibited by the PAF antagonist, CV-3988 (10(-6) M), and reversed by the serotonin antagonist, methysergide (EC50 = 3.7 +/- 0.79 x 10(-9) M). Neither atropine nor chlorpheniramine (10(-9) to 10(-6) M) attenuated the response to PAF + platelets. In the presence of platelets, 10(-7) M PAF caused an increase in perfusate concentration of serotonin from 0.93 +/- 0.037 x 10(-8) to 1.7 +/- 0.046 x 10(-8) M (P less than 0.001). Tachyphylaxis, previously demonstrated to be irreversible, was shown to be a platelet-dependent phenomenon; contraction could be repeated in the same TSM after addition of fresh platelets. We demonstrate that PAF-induced contraction of canine TSM is caused by the release of cellular intermediates such as serotonin from platelets. We also demonstrate the site of PAF-induced tachyphylaxis in airway smooth muscle contraction.     

Formation and metabolism of inositol 1,4,5-trisphosphate in human platelets.

1. myo-[3H]Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], when added to lysed platelets, was rapidly converted into [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], which was in turn converted into [3H]inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. 2. Labelling of platelets with [32P]Pi, followed by h.p.l.c., was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymic and non-enzymic techniques. 3. Ins(1,4,5)P3 was formed rapidly, and reached a maximum at about 4 s. It was also rapidly degraded, and was no longer detectable after 30-60 s. 4. Formation of Ins(1,3,4,5)P4 was almost as rapid as that of Ins(1,4,5)P3, and it remained detectable for a longer time. 5. Ins(1,3,4)P3 was formed after an initial lag, and this isomer reached its maximum, which was 10-fold higher than that of Ins(1,4,5)P3, at 30 s. 6. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than Ins(1,4,5)P3 are responsible for the sustained maintenance of a high concentration of intracellular Ca2+. It is proposed that either Ins(1,3,4)P3 or Ins(1,3,4,5)P4 may also be Ca2+-mobilizing agents.     

Alpha-granule secretion from alpha-toxin permeabilized, MgATP-exposed platelets is induced independently by H+ and Ca2+

In order to better understand granule release from platelets, we developed an alpha-toxin permeabilized platelet model to study alpha-granule secretion. Secretion of alpha-granules was analyzed by flow cytometry using P-selectin as a marker for alpha-granule release. P-selectin surface expression occurred when platelets were permeabilized in the presence of Ca2+. Responsiveness to Ca2+ was lost 30 min after permeabilization but could be reconstituted with MgATP. Alpha-toxin-permeabilized, MgATP-exposed platelets also degranulated within a pH range of 5.4-5.9 without exposure to and independent of Ca2+. ATP, GTP, CTP, UTP, and ITP supported Ca2+-induced alpha-granule secretion, while H+-induced alpha-granule secretion occurred only with ATP and GTP. Both Ca2+- and H+-induced alpha-granule secretion required ATP hydrolysis. Kinase inhibitors blocked both Ca2+- and H+-induced secretion. These data suggest that alpha-granule secretion in this permeabilized platelet system shares many characteristics with granule secretion studied in other permeabilized cell models. Furthermore, these results show that H+ can trigger alpha-granule release independent of Ca2+.     

Formation of diacyl- and alkylacylphosphatidylcholine by the membranes of human platelets

We have investigated the distribution and fatty acid preference of two acyl-CoA transferase activities in a human platelet mixed membrane fraction and in well-characterised surface and intracellular membrane subfractions prepared from it by high-voltage free-flow electrophoresis. One transferase inserts long-chain unsaturated fatty acids into 1-acyllysophosphatidylcholine (1-acyl-LPC) and the other into lyso-platelet-activating factor (LPAF). Both transferase activities were approx. 4-fold enriched in the intracellular membranes with respect to their specific activities in the mixed membranes. The surface membrane activities were correspondingly depleted. Using 1-acyl-LPC as the acceptor, all the intracellular membrane preparations showed transferase preference for the CoA ester of 8,11,14-eicosatrienoic acid. In contrast when LPAF was the acceptor the CoA esters of linoleic and arachidonic acid were the preferred donors.     

Cytosol-dependent peroxisomal protein import in a permeabilized cell system

Using streptolysin-O (SLO) we have developed a permeabilized cell system retaining the competence to import proteins into peroxisomes. We used luciferase and albumin conjugated with a peptide ending in the peroxisomal targeting sequence, SKL, to monitor the import of proteins into peroxisomes. After incubation with SLO-permeabilized cells, these exogenous proteins accumulated within catalase-containing vesicles. The import was strictly signal dependent and could be blocked by a 10-fold excess of peptide containing the SKL-targeting signal, while a control peptide did not affect the import. Peroxisomal accumulation of proteins was time and temperature dependent and required ATP hydrolysis. Dissipation of the membrane potential did not alter the import efficiency. GTP-hydrolyzing proteins were not required for peroxisomal protein targeting. Depletion of endogenous cytosol from permeabilized cells abolished the competence to import proteins into peroxisomes but import was reconstituted by the addition of external cytosol. We present evidence that cytosol contains factors with SKL-specific binding sites. The activity of cytosol is insensitive to N- ethylmaleimide (NEM) treatment, while the cells contain NEM-sensitive membrane-bound or associated proteins which are involved in the import machinery. The cytosol dependence and NEM-sensitivity of peroxisomal protein import should facilitate the purification of proteins involved in the import of proteins into peroxisomes.     

Formation, transport, contraction, and disassembly of stress fibers in fibroblasts

Swiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum-deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video-enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive manner at the cytoplasm-membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10-20 microns/h. When serum-deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post-stimulation.