Effect of actin-binding protein on the sedimentation properties of actin.

Actin and actin-binding protein (ABP) have recently been purified from human platelet cytoskeletons (S. Rosenberg, A. Stracher, and R.C. Lucas, 1981, J. Cell Biol. 91:201-211). Here, the effect of ABP on the sedimentation of actin was studied. When ABP was added to preformed F-actin filaments, it bound until a maximum ratio of 1:9 (ABP:actin, mol:mol) was reached. however, when actin was polymerized in the presence of ABP, two and a half times more ABP was able to bind to the actin- that is, every 3.4 actin monomers were now bound by an ABP dimer. ABP was not able to induce the sedimentation of actin under nonpolymerizing conditions but was able to reduce the time and concentration of actin required for sedimentation under slow polymerizing conditions. ABP, therefore, exerts its effect of G-actin by either nucleating polymerization or by cross-linking newly formed oligomers into a more sedimentable form.

Isolation and characterization of actin and actin-binding protein from human platelets.

Human blood platelets, which are highly motile cells essential for the maintenance of hemostasis, contain large quantities of actin and other contractile proteins. We have previously introduced a method (Lucas, R. C., T. C. Detwiler, and A. Stracher, J. Cell Biol., 1976, 70(2, Pt. 2):259 a) for the quantitative recovery of the platelets' cytoskeleton using a solution containing 1% Triton X-100 and 10 mM EGTA. This cytoskeleton contains most of the platelets' actin, actin-binding protein (ABP, subunit molecular weight = 260,000), and a 105,000-dalton protein. Negative staining of this Triton-insoluble residue on an EM grid shows it to consist of branched cables of actin filaments aligned in parallel. When this cytoskeletal structure is dissolved in high-salt solutions, the actin and ABP dissociate and can subsequently be separated. Here we will present simple and rapid methods for the individual purifications of platelet actin and platelet ABP. When purified actin and ABP are recombined in vitro, they are shown to be both necessary and sufficient for the reformation of the cytoskeletal complex. The reformed structure is visualized as a complex array of fibers, which at the EM level are seen to be bundles of actin filaments. The reformation of the cytoskeleton requires only that the actin be in the filamentous form--no accessory proteins, chelating agents, divalent cations, or energy sources are necessary. In vivo, however, the state of assembly of the platelets' cytoskeleton appears to be under the control of the intracellular concentration of free calcium. Under conditions where proteolysis is inhibited and EGTA is omitted from the Triton-solubilization step, no cytoskeleton can be isolated. The ability of Ca+2 to control the assembly and disassembly of the platelets' cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation.

Muscular dystrophy: inhibition of degeneration in vivo with protease inhibitors.

The protease inhibitors leupeptin and pepstatin were used in vivo in genetically dystrophic chickens to determine their effects on the histological and biochemical changes observed in this disease. These compounds appear to delay the degeneration of muscle tissue which is characteristic of this disorder and thus may have potential therapeutic value in the treatment of muscular dystrophy.

Proteolysis of myosin during platelet storage.

Physical properties of actomyosin from either fresh or stored platelets have been compared. Actomyosin obtained from platelets after 3 days of storage contained myosin that was 60-80% degraded to myosin rod. No myosin rod was detected in fresh platelets. The platelet myosin rod is similar to the rod produced by limited proteolysis of skeletal muscle myosin.

Subunits and their interactions.

There is fairly general agreement that myosin isolated from rabbit skeletal muscle has a molecular weight of about 500,000. The higher values that have been reported apparently reflect protein aggregation related to the method of preparation. On the basis of present evidence, the myosin molecule has an elongate helical core of two f subunits (average weight about 215,000) that extend into a globular head region containing three g subunits (average weight about 20,000). Myosin may be dissociated into subunits by a number of methods. In 5 M guanidine, the myosin molecule is dissociated into f and g subunits, while at pH above 10, the g subunits are dissociated from the intact fibrous core of myosin. The dissociation of g subunits at pH 10 is accompanied by the loss of both ATPase activity and actin-binding capacity; however, the exact biological significance of the g subunits is presently uncertain. In preliminary studies, the f subunits appear to contain the sulfhydryl residues currently implicated in myosin ATPase, and there is some indication of allosteric regulation of enzymic activity.

Localization of a thrombin-sensitive calcium pool in platelets.

Electron microscopic x-ray microprobe analysis of pyroantimonate precipitates in platelets fixed in osmium tetroxide-pyroantimonate revealed calcium localization in the nucleoids of alpha-granules. This pool of calcium had largely disappeared within 10 sec after stimulation of platelets by thrombin. Such a rapid change suggests that this calcium pool may have a regulatory role in stimulus-response coupling.

Calpain inhibitors as therapeutic agents in nerve and muscle degeneration.

It seems plausible to hypothesize that in all forms of neurodegeneration or other forms of tissue degeneration, a common pathway exists that, when deciphered, could lead to our understanding of a variety of diseases that result in tissue necrosis, as well as offer potential for therapeutic intervention. In recent years progress toward elucidating this common pathway has been accelerated through the studies of a number of laboratories, including our own, on the role of the protease calpain in this process. Thus, in a variety of disorders, such as stroke, spinal cord injury, traumatic nerve injury, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, muscular dystrophy, cataract formation, unregulated calpain proteolysis, initiated via dysregulation of calcium ion homeostasis, participates in the pathogenesis and is a potentially unifying mechanistic event. In order to demonstrate the feasibility of the approach we have taken in using the calpain inhibitor leupeptin as a therapeutic agent, I will describe two areas of research in which we have been engaged over the past 20 years. One is our long-standing interest in muscular dystrophy. The other is of more recent vintage, and involves the use of calpain inhibitors to protect sensory hair cells and spiral ganglion neurons from damage associated with acoustic trauma, this latter in collaboration with Dr. R. Salvi at SUNY-Buffalo and Dr. A. Shulman at SUNY-Downstate.

Leupeptin protects sensory hair cells from acoustic trauma.

Calpains, a family of calcium activated proteases, promote the breakdown of cellular proteins, kinases, phosphatases and transcription factors. Calpain inhibitors attenuate some neurodegenerative processes in certain cell types. Here we show that leupeptin, a potent calpain inhibitor, protects the sensory hair cells in the inner ear from acoustic overstimulation (48 h, 100 or 105 dB SPL, octave band noise at 4 kHz). Acoustic overstimulation caused a significant increase in calpain immunolabeling in the sensory epithelium suggesting a possible role in noise-induced cochlear degeneration. Infusion of leupeptin into the inner ear significantly reduced the amount of sensory cell loss from acoustic overstimulation. However, leupeptin did not protect against hair cell loss from the ototoxic drug, carboplatin.

Calpain inhibitors protect auditory sensory cells from hypoxia and neurotrophin-withdrawal induced apoptosis.

Inhibitors of calpain have been shown to protect nerve growth factor (NGF)-deprived ciliary ganglion neurons and hypoxic cortical neurons. Calpains have been identified in the cochlea and are active during ischemic injury. Since apoptosis can be initiated by loss of neurotrophic support, hypoxia, and ototoxins (e.g., cisplatin, CDDP), the role of calpain inhibitors under these conditions was examined in auditory hair cells and neurons. Dissociated spiral ganglion neuron (SGN) cell cultures and organ of Corti explants from P3 rats were used to test the efficacy of calpain inhibitors as otoprotective molecules. Our results indicate that calpain inhibitor I, calpain inhibitor II, and leupeptin all provided significant protection of SGNs against neurotrophin-withdrawal and hypoxia-induced apoptosis. The increase in neuronal survival ranged from 2.16 to 2.31 times greater than in untreated neurotrophin-withdrawn SGN cell cultures. BOC-Asp(Ome)-Fluoromethyl Ketone (B-D-FMK), a general caspase inhibitor, increased neuronal survival 2.16 times more. Neuronal survival rates were from 1.88 to 2.27 times greater than in untreated, hypoxic neurons and hair cell survival rates were from 1.98 to 2.03 times greater than untreated, hypoxic organ of Corti explants. However, protection of auditory hair cells and neurons from CDDP-induced damage (10 and 6 micrograms/ml, respectively) was limited with any of these calpain inhibitors. Apoptotic pathways initiated by neurotrophin-deprivation and ototoxic stress (e.g., CDDP) have been shown to be different. Our results agree with this finding, with neurotrophin-withdrawal and hypoxia, but not CDDP damage-induced apoptosis being calpain-dependent.

Treatment of experimental autoimmune myasthenia gravis in rabbits with leupeptin, a protease inhibitor.

We injected 12 New Zealand white rabbits intraperitoneally with 15 mg/kg Leupeptin on alternative days for about 4 months. After 1 week of Leupeptin treatment, they were challenged with purified acetylcholine receptor (AChR) from Torpedo californica in Freund's complete adjuvant. All control animals died within 60 days. Six animals treated with Leupeptin did not develop EAMG in spite of repeated AChR injections. Three animals developed clinical signs of EAMG after 65 days. The clinical course was short in the one that survived and prolonged in the 2 that finally died. All animals (Leupeptin-treated and controls) had circulating anti-AChR antibodies. Among the survivors, titers were slightly lower and EMG repetitive stimulation tests were normal. Leupeptin (0.02-200 mM) did not prevent curaremimetic [3H]toxin binding to AChR in membranes or in solution, nor dissociate AChR-toxin-antibody complexes. Immune response to antigens other than receptor remained intact in Leupeptin-treated animals. Leupeptin was not toxic at the doses given. The mechanism of this protection is not well understood. Leupeptin seems to decelerate the turnover rate of AChR induced by anti-AChR antibodies and/or to decrease the complement-mediated immune attack against the muscle end-plate.

Calcium-induced degeneration of the cytoskeleton in monkey and human peripheral nerves.

Biopsy specimens of human and monkey peripheral nerves, when incubated in calcium containing media, showed a loss of neurofilaments and microtubules with replacement by granular debris. Cytoskeletal structures remained intact when incubated in calcium-free media. Disruption of neurofilaments and microtubules in calcium containing media was inhibited by the thiol protease inhibitor, leupeptin. Similar incubations of excised Pacinian corpuscles revealed evidence of early terminal axon degeneration in the presence of calcium. These data substantiate the hypothesis that neural cytoskeletal degradation in primates and in man is calcium-mediated.

Enhancement of neuromuscular recovery after nerve repair in primates.

This investigation describes the use of the calcium-activated protease inhibitor, leupeptin, as an adjunctive therapy to the microsurgical repair of median nerves in a primate model. Our results indicate that leupeptin facilitates morphological recovery in denervated thenar muscles and in distal sensory and mixed motor-sensory nerve trunks and functional recovery measured by motor nerve conduction velocity. Toxicological testing of leupeptin showed that, when administered at a dose of 12 mg/kg, intramuscularly, once daily, haematological and clotting profiles were not adversely affected.

Calpain Inhibitors as Neuroprotective Agents in Neurodegenerative Disorders.

It seems plausible to hypothesize that in all forms of neurodegeneration or other forms of tissue degeneration, a common pathway exists which when deciphered could lead to our understanding of a variety of diseases which result in tissue necrosis as well as offer potential for therapeutic intervention. A relatively recent interest has been our preliminary studies on the role of neurodegeneration in hearing loss and tinnitus, particularly that associated with noise. These studies grew out of a collaboration emanating from early discussions with Professor Abraham Shulman of the State University of New York, Health Science Center at Brooklyn, Department of Otolaryngology, and Dr Richard J. Salvi, of the Center for Communication Disorders and Sciences, Hearing Research Laboratories, State University of New York at Buffalo. Further studies in this very promising area of research are continuing for noise induced hearing loss protection and tinnitus control. A brief review of calpain is presented.

The effects of leupeptin on cochlear blood flow, auditory sensitivity, and histology.

The purpose of this study was to evaluate the long-term safety of administering leupeptin (1 mg/ml in Hank's Balanced Salt Solution) to the round window membrane by investigating its effects on cochlear blood flow, auditory sensitivity (i.e., auditory brainstem response), and cochlear histology. A comparison of baseline and posttreatment measurements of cochlear blood flow and mean arterial blood pressure in guinea pigs revealed no significant changes. Auditory brainstem response measurements revealed no significant changes in auditory threshold shifts when compared to controls at the 2-, 4-, 6-, and 8-week time points. Furthermore, poststudy surface preparations of the organs of Corti and cytocochleograms from leupeptin-treated ears and controls revealed no significant hair cell losses. These data suggest that the prolonged administration of leupeptin (1 mg/ml at a rate of 0.5 microliter/hr for 8 weeks) to the round window membrane is not ototoxic. This study may serve as a basis for future clinical trials of leupeptin administration for the prevention or treatment of noise-induced hearing loss and the management of tinnitus.

Protecting the Inner Ear from Acoustic Trauma.

Calcium activated proteases, or calpains, play an important role in neurodegeneration. In some cases, neural degeneration can be significantly reduced by leupeptin, a potent calpain inhibitor. To determine if leupeptin could protect against noise-induced hearing loss and hair cell loss, we infused leupeptin into scala tympani of one cochlea before, during and after a 14-day exposure to a 100 dB SPL, octave band noise centered at 4.0 kHz. Hearing loss, assessed with the auditory evoked response, was less in the leupeptin-treated ear than in the control ear during the early stage of recovery from acoustic trauma. In addition, hair cell loss in the leupeptin-treated ear was significantly less than in the control ear. These preliminary results suggest that leupeptin may protect against noise-induced hearing loss.

Platelet membrane-actin interaction.

In order to investigate how human platelet membranes associate with cytoskeletal proteins, purified membranes were extracted with 0.6 M KI (potassium iodide) followed by extraction with 1% octyl glucoside. The depleted membranes contain a significant amount of actin (5% of the actin that was originally present in the purified membranes) which is resistant to extraction by 0.6 M KI. We have examined how this actin interacts with the membrane skeletal fraction and find that the actin is not associated with the membrane directly or indirectly through any of the major transmembrane glycoproteins (GpIb, GpIIb, and GpIIIa), or any known specific linker proteins such as actin binding protein (ABP), alpha-actinin, or spectrin. The results of an analysis of the membrane skeletal preparation using nonreducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) does indicate, however, that the actin, along with other proteins, exists in the form of two high molecular weight complexes. We have examined the effect of potassium iodide-octyl glucoside (KI-OG) extracted membranes upon (1) the polymerization of rabbit muscle G-actin, and (2) the actin activated Mg(2+)-dependent ATPase activity of rat skeletal muscle myosin. Based on the results of these experiments we have concluded that the actin is available for interaction with exogenously added proteins and that it might possibly be in a filamentous form. These results are compelling considering the role that the cytoskeleton is thought to play in the physiological functioning of the platelet.

Purification and characterization of a calcium binding protein with "synexin-like" activity from human blood platelets.

A calcium binding protein of Mr = 54,000 has been isolated from human blood platelets. This protein has been shown to enhance Ca2+-induced aggregation of phosphatidylserine liposomes, suggesting that it may be a member of a recently recognized class of binding proteins known to interact with phospholipids and the membrane in a Ca2+ dependent manner. Because of the "synexin-like" activity of this protein it may be involved in Ca2+ regulated platelet secretion as well as cell motility.

Expression in Escherichia coli and phosphorylation with cAMP-dependent protein kinase of the N-terminal region of human endothelial actin-binding protein.

The N-terminal region of human endothelial actin-binding protein was subcloned and expressed in the pT 7-7/E. coli BL 21 (DE 3) system. This peptide was efficiently expressed in E. coli as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by western analysis using a monoclonal antibody raised against this part of the molecule. As predicted by the amino acid sequence this truncated protein (residues 21-1569), corresponding to 164 KD and containing the actin-binding domain near the amino-terminus, could be phosphorylated by cAMP-dependent protein kinase unmasking a phosphorylation site which is not apparent in the native ABP protein.