Synthetic Peptide Inhibitor Research Articles

Calcium/calmodulin-dependent protein kinase II regulates hippocampal synaptic transmission

Extracellular application of protein kinase inhibitors was used to examine the role of calcium/calmodulin-dependent protein kinase II (CaM-KII) in synaptic transmission in the CA1 region of rat hippocampus. Bath application of the broad spectrum, membrane permeable kinase inhibitor H7 (250 μM) decreased excitatory synaptic responses elicited in hippocampal slices. Whereas H7 inhibits several protein kinases and has non-specific effects, several synthetic peptides have been developed as specific inhibitors of CaM-KII. Using in situ phosphorylation in hippocampal slices, we demonstrate that extracellular application of synthetic peptide inhibitors of CaM-KII preferentially suppresses the phosphorylation of synapsin I at the CaM-KII specific site. This suppression was not reversed by the application of a calcium ionophore indicating the decrease in phosphorylation does not result only from blockade of presynaptic calcium influx. Thus, it appears the peptides gain access to intracellular compartments and retain their inhibitory properties. Further, we found that extracellular application of these peptide inhibitors decreased excitatory synaptic responses elicited in the CA1 region of hippocampal slices with relative potencies consistent with their ability to block CaM-KII activity in vitro. Peptide application did not alter the input resistance of postsynaptic cells nor responses elicited by glutamate ionotophoresis. These results suggest that CaM-KII activity, possibly through phosphorylation of presynaptic synapsin I, is required for sustained synaptic transmission at mammalian synapses.

Characterization of Proteasomes Isolated from Rat Liver

Proteasomes are cylindrical particles which have a pseudohelical arrangement of subunits. On 2D-PAGE gels, rat liver proteasome preparations give rise to up to 25 proteins which are encoded by at least 16 different genes that are all members of the same family. Proteasomes are able to degrade protein substrates to acid soluble peptides. They have at least five different catalytic components which can be distinguished by the use of synthetic peptide substrates and inhibitors which have very different reactivity at the different sites. Proteasomes can undergo conformational changes when treated with various effectors of their multiple peptidase activities. They are found in the nucleus and in the cytoplasm and, in cultured cells, show changes in localization during the course of the cell cycle.

Combination of inhibition of thrombin and blockade of thromboxane A2 synthetase and receptors enhances thrombolysis and delays reocclusion in canine coronary arteries.

The efficacy of thrombolytic therapy in treating patients with acute myocardial infarction is limited by failure to achieve reperfusion in some patients, by the prolonged time required to achieve reperfusion, and by reocclusion of some coronary arteries. We designed this study to examine the effect of combined inhibition of thrombin and thromboxane synthesis and blockade of thromboxane A2 receptors in addition to tissue-type plasminogen activator (t-PA) on thrombolysis and reocclusion in an experimental canine model with coronary thrombosis. Blood flow velocity in the left anterior descending coronary artery (LAD) of 32 anesthetized mongrel dogs was monitored by a pulsed Doppler flow probe. Coronary thrombosis was induced by applying electrical stimulation to the LAD at the site where an external constrictor was used to narrow the artery. Three hours after the formation of occlusive thrombus, animals were randomly assigned to receive one of the following: 1) t-PA (80 micrograms/kg + 8 micrograms.kg-1.min-1 i.v.) and saline; 2) t-PA and hirulog, a hirudin-based synthetic peptide and specific thrombin inhibitor (2 mg/kg + 2 mg.kg-1.hr-1 i.v.); 3) t-PA and ridogrel, a combined thromboxane A2 synthetase inhibitor and receptor antagonist (5 mg/kg + 2.5 mg.kg-1.hr-1 i.v.); or 4) t-PA, hirulog, and ridogrel. Reperfusion developed in 14% (one of seven) of dogs treated with t-PA alone at an average of 86 +/- 4 minutes after treatment, in 78% (seven of nine) of dogs treated with t-PA plus hirulog at 53 +/- 11 minutes, in 13% (one of eight) of dogs treated with t-PA plus ridogrel at 85 +/- 5 minutes, and in 88% (seven of eight) of dogs treated with t-PA, hirulog, and ridogrel at 37 +/- 10 minutes (comparison of the frequency of and the time to reperfusion, both p < 0.01). Among the dogs with reestablished coronary blood flow, reocclusion developed in the one treated with t-PA alone at 36 minutes after reperfusion, in seven of the seven treated with t-PA plus hirulog at 66 +/- 15 minutes, and in two of the seven treated with t-PA, hirulog, and ridogrel at 151 +/- 21 minutes (comparison of the frequency of and time to reocclusion, both p < 0.05). Reocclusion was not detected in the one dog treated with t-PA and ridogrel or in the other five dogs treated with t-PA, hirulog, and ridogrel within 180 minutes after reperfusion. Hirulog prolonged and maintained activated clotting times at a level twice that of baseline values. Hirulog inhibited ex vivo platelet aggregation induced by thrombin, and ridogrel inhibited platelet aggregation induced by U46619, a thromboxane mimetic. Inhibition of thrombin in addition to treatment with t-PA enhances thrombolysis. A combination of inhibition of thrombin and thromboxane synthetase and blockade of thromboxane A2 receptor enhances thrombolysis and delays or may prevent reocclusion of the recanalized coronary arteries.

A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition.

A peptide designated DP-107 was synthesized containing amino acid residues 558-595 of the envelope glycoprotein gp160 of human immunodeficiency virus type 1 strain LAI (HIV-1LAI). Algorithms for secondary structure have predicted that this region of the envelope transmembrane protein should form an extended alpha-helix. Consistent with this prediction, analysis by circular dichroism (CD) indicated that, under physiological conditions, DP-107 is approximately 85% helical. The high degree of stable secondary structure in a synthetic peptide of this size suggests self-association typical of a coiled coil or leucine zipper. In biological assays, the peptide efficiently blocked virus-mediated cell-cell fusion processes as well as infection of peripheral blood mononuclear cells by both prototypic and primary isolates of HIV-1. A single amino acid substitution in the peptide greatly destabilized its solution structure as measured by CD and abrogated its antiviral activity. An analogue containing a terminal cysteine was oxidized to form a dimer, and this modification lowered the dose required for antiviral effect from 5 to about 1 microgram/ml. These results suggest that both oligomerization and ordered structure are necessary for biological activity. They provide insights also into the role of this region in HIV infection and the potential for development of a new class of antiviral agents.

Effects of spermine on water absorption, polyethylene glycol 4000 permeability and collagenase activity in rat descending colon in vivo.

1. The effects of spermine in the concentration range 0-10 mmol/l on (a) the fluid absorption, (b) the polyethylene glycol permeability, (c) the release of collagenase activity activity into the lumen and (d) the histological appearance of rat descending colon were examined. 2. Spermine (5 mmol/l) decreased fluid absorption from 48.83 +/- 2.98 (n = 7) to 23.98 +/- 2.32 (n = 6) microliters h-1 cm-2 (P < 0.01); polyethylene glycol 4000 permeability was increased from 0.030 +/- 0.001 (n = 7) to 0.047 +/- 0.003 (n = 6) cm/h (P < 0.01) and luminal collagenase activity increased from a negligible control value to 250 +/- 39 (n = 6) units/ml (P < 0.001). Spermine also caused oedema formation within the mucosal interstitial fluid, without inducing an overt breakdown of the mucosa at the luminal surface. 3. Polyamine-free dialysed seminal plasma had no effect on polyethylene glycol 4000 permeability, although it still caused a significant decrease in colonic fluid absorption from 48.83 +/- 2.98 (n = 7) (control) to 31.41 +/- 2.08 (n = 5) microliters h-1 cm-2 (P < 0.01). 4. Low-molecular-mass heparin (600 units/ml) prevented the spermine (5 mmol/l)- and whole-semen-induced increase in colonic polyethylene glycol 4000 permeability and reduced the effect of semen on fluid absorption by 63% (P < 0.001) and that of spermine by 56% (P < 0.01). 5. The Zn2+ chelator and collagenase inhibitor o-phenanthroline reduced the effect of spermine on fluid absorption and polyethylene glycol 4000 permeability by 100% (P < 0.001) and on interstitial oedema formation. o-Phenanthroline also reduced the effects of whole semen on fluid absorption (by 70%, P < 0.01) and on polyethylene glycol 4000 permeability by 95%, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes.

Our previous studies implicated the involvement of protein kinase-A in the inhibitory effects of isoproterenol and relaxin on oxytocin-stimulated phosphoinositide turnover in rat myometrium. To understand the possible mechanisms involved, the properties and regulation of phospholipase-C (PLC) in purified myometrial plasma membranes from estrogen-primed rats were studied. The PLC activity measured with exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate was Ca2+ dependent. The nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate stimulated PLC activity with a ED50 of 1.6 microM and shifted the calcium dependence curve to the left. Guanosine 5'-(3-O-thio)triphosphate-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis was inhibited by activation of endogenous and exogenous cAMP-dependent protein kinase (PKA). The effects of endogenous and exogenous PKA were significantly reversed by IP20, a potent synthetic peptide inhibitor of PKA. In the presence of [gamma-32Pi]ATP and exogenous PKA, 32Pi was incorporated in an IP20-sensitive manner into major bands at approximately 17,000, 20,000-24,000, 33,000, 38,000, 40,000-44,000, and other higher mol wt. These data indicate that one or more GTP-binding proteins mediate activation of membrane-bound PLC in rat myometrium. Phosphorylation of one or more membrane-associated proteins by PKA may regulate myometrial PLC activity and play a role in the inhibitory effects of isoproterenol and relaxin.

Inactivation of the Ba2+ current in dissociated Helix neurons: voltage dependence and the role of phosphorylation.

The rate of inactivation of the voltage-dependent Ba2+ current in dissociated neurons from the snail Helix aspersa was found to be modulated by phosphorylation. Conditions were chosen such that the most likely mechanism of inactivation of the Ba2+ current was a voltage-dependent/calcium-independent inactivation process. If adenosine-triphosphate (ATP) was not included in the patch electrode filling solution, or if alkaline phosphatase was added, the Ba2+ current rapidly ran down and the rate of inactivation greatly increased with time. Dialysis with either ATP gamma S or the phosphatase inhibitor okadaic acid (OA) either enhanced the amplitude or greatly reduced the rate of run-down of the Ba2+ current (depending upon the presence of ATP), as well as reducing the rate of inactivation. However, dialysis with either the catalytic subunit of the cyclic-adenosine-mono-phosphate-dependent protein kinase (cAMP-PK), a synthetic peptide inhibitor of this enzyme, or staurosporine (a potent inhibitor of protein kinase C), did not have any significant effect on the amplitude or kinetics of the Ba2+ current. Surprisingly, dialysis with a peptide inhibitor (CKIP) of the Ca2+/calmodulin-dependent protein kinase II (Ca(2+)-CaM-PK) significantly reduced the rate of inactivation of this current. These results suggest that phosphorylation may exert its effect by modulating the gating properties of the Ca2+ channels.

Thrombin inhibition enhances tissue-type plasminogen activator-induced thrombolysis and delays reocclusion.

The objectives of this study were to test the hypotheses that thrombin inhibitors 1) enhance tissue-type plasminogen activator (t-PA)-induced coronary thrombolysis and 2) prevent or delay coronary artery reocclusion. Seventy-one dogs developed occlusive coronary thrombi after introducing a copper coil into the left anterior descending coronary artery (LAD). Coronary blood flows were monitored by an externally positioned pulsed Doppler flow probe. t-PA was given with or without heparin at different times after LAD occlusions. In some experiments, hirugen, a synthetic hirudin-based peptide and specific thrombin inhibitor, was given as 4 mg/kg i.v. bolus and 3 mg.kg-1.h-1 i.v. infusion at 30 min after LAD occlusion with t-PA and a bolus of heparin. Thrombolysis times were significantly shorter in t-PA- and heparin-treated dogs than in dogs treated with t-PA alone. Reocclusion times were significantly longer in t-PA- and heparin-treated dogs than in dogs treated with t-PA alone. Continuous heparin infusions prolonged reocclusion times to greater than 180 min in all treated dogs. The addition of hirugen to t-PA plus one bolus heparin prolonged reocclusion times to 90 +/- 6 min in dogs with 30-min thrombi. Thus heparin enhances t-PA-induced thrombolysis and delays reocclusion. Addition of a specific thrombin inhibitor, such as hirugen, to heparin enhances its effect on delaying reocclusion.

Modulation of the Effector Functions of Cytolytic T-Lymphocytes with Synthetic Peptide Inhibitors of Protein Kinases

The hypothesis was tested that it is possible to influence cellular responses of intact cells using synthetic peptide substrates, pseudosubstrates, and inhibitors of protein kinases. Using cytotoxic T-cells (CTL), we demonstrate here that some basic amino acid-containing synthetic peptide substrates of protein kinases [e.g., of cGMP-dependent protein kinase (peptide PKQ-S), synthetic peptide inhibitor of cGMP-dependent protein kinase (peptide PKG-I), and peptide corresponding to the tyrosine phosphorylation site in pp60src (peptide RR-src)] were strongly inhibitory in T-cell receptor (TCR) and T-cell growth factor, interleukin 2 (IL-2)-triggered proliferation of CTL. These peptides also inhibited other cellular responses of CTL. Peptides which contain basic amino acids, but do not have substrate specificity determinants for protein kinase, were not inhibitory. The inhibition with peptides is not due to their toxicity, since no cell death was observed by the trypan blue exclusion test and by lactate dehydrogenase release. Use of the granule exocytosis assay provided opportunities to clarify the mechanism of the peptide action. Tested peptides inhibited not only cell-surface ligand-induced CTL activation, but also affected cell-surface receptor-independent CTL activation (granule exocytosis and γ-interferon secretion) induced by the synergistic action of the protein kinase C activator (PMA) and ionophore A23187. It was found that minor changes in amino acid composition or amino acid position in the synthetic peptides dramatically change their ability to affect lymphocytes. Pretreatment with peptide PKQ-S at 37 °C, but not at 22 or 4 °C, for at least 90 min, followed by washing, was sufficient to observe inhibition of CTL function (a small effect was observed even after 60 min of pretreatment). Results of the peptide binding and of intracellular localization studies support the view that studied peptides exert their effect after gaining access to the cell interior. Percoll gradient fractionation of CTL disrupted by nitrogen cavitation revealed that the largest proportion of radiolabel was in cytosol. The requirement for the relatively high concentration of peptides needed to demonstrate their effects may reflect their partial degradation by peptidases. The presented data support the model in which specific features of amino acid composition allow for the peptide internalization and interference with cellular functions.

Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling

Computer modeling of the three-dimensional structure of an enzyme, based upon its primary sequence alone, is a potentially powerful tool to elucidate the function of enzymes as well as design specific inhibitors. The cercarial (larval) protease from the blood fluke Schistosoma mansoni is a serine protease hypothesized to assist the schistosome parasite in invading the human circulatory system via the skin. A three-dimensional model of the protease was built, taking advantage of the similarity of the sequence of the cercarial enzyme to the trypsin-like class of serine proteases. A large hydrophobic S-1 binding pocket, suspected from previous kinetic studies, was located in the model and confirmed by new kinetic studies with both synthetic peptide substrates and inhibitors. Unexpected structural characteristics of the enzyme were also predicted by the model, including a large S-4 binding pocket, again confirmed by assays with synthetic peptides. The model was then used to design a peptide inhibitor with 4-fold increased solubility, and a series of synthetic inhibitors were tested against live cercariae invading human skin to confirm that predictions of the model were also applicable in a biologic assay.

Interaction of troponin I and troponin C: Use of the two-dimensional nuclear magnetic resonance transferred nuclear overhauser effect to determine the structure of the inhibitory troponin I peptide when bound to skeletal troponin C

We have used two-dimensional 1H nuclear magnetic resonance spectroscopy to determine the structure of the synthetic inhibitory peptide Nα-acetyl TnI(104–115) amide bound to calcium-saturated skeletal troponin C (TnC). Conformational changes in the peptide induced by the formation of the troponin I (TnI) peptide-TnC complex were followed by the study of the transferred nuclear Overhauser effect, a technique that allows one to determine the structure of a ligand bound to a macromolecule. The structure of the bound TnI peptide reveals an amphiphilic α-helix, distorted around the two central proline residues. The central bend in the peptide functions to bring the residues on the hydrophobic face into closer proximity with each other, thereby forming a small hydrophobic pocket. The hydrophilic, basic residues extend off the opposite face of the peptide. Hydrophobic surfaces on TnC that become exposed upon binding of calcium are involved in the binding of the TnI peptide, but electrostatic interactions also contribute to the strength of the interaction. The role of amphiphilic helices in the targeting of calcium-binding proteins such as troponin C will be discussed.

Prostaglandin E 1 activates a chloride current in Jurkat T lymphocytes via cAMP‐dependent protein kinase

Patch-clamp studies have identified a cAMP-dependent Cl- conductance in lymphocytes that is defectively regulated in cystic fibrosis. In this study we used 125I efflux and whole-cell patch-clamp studies to investigate whether prostaglandin E1 (PGE1), an agonist that generates intracellular cAMP in Jurkat T lymphocytes, activates a Cl- conductance. Stimulation of T cells by externally applied PGE1 stimulated 125I efflux and activated a slowly developing membrane current. When external and internal Cl- were about equal, the current reversed at about zero mV, but when external Cl- was lowered from 157 to 7 mM the reversal potential shifted 75 mV in the positive direction, demonstrating that the current carrier was Cl-. In addition, the current was blocked by 10 microM 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), a potent Cl- channel blocker. A membrane-permeable cAMP analog mimicked the effect of PGE1, whereas intracellular application of a cAMP antagonist Rp-cAMP blocked the effect of PGE1. Addition of purified catalytic subunit of cAMP-dependent protein kinase (PKA) plus ATP to the recording pipette also activated a similar current, whereas internally applied Walsh inhibitor, the synthetic peptide inhibitor of PKA, blocked the PGE1 effect. These results suggest that PGE1, acting through PKA, activates a Cl- current in Jurkat T cells.

Insulin-activated protein kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the p70 S6 kinase.

p70 S6 kinase, a major insulin-mitogen-activated ribosomal S6 protein kinase in mammalian cells, is activated by phosphorylation of multiple Ser/Thr residues on the enzyme polypeptide. A synthetic peptide, corresponding to a 37-residue segment from the carboxyl-terminal tail of the kinase which resembles the sequence phosphorylated in S6, acts as a competitive inhibitor of p70 S6 kinase without itself being phosphorylated by the enzyme. This synthetic peptide is phosphorylated by an array of protein kinases which are rapidly activated by insulin. Thus, these sequences of p70 S6 kinase constitute a potential autoinhibitory pseudosubstrate site, whose phosphorylation is catalyzed by candidate upstream-activating protein kinases.

Hirulog peptides with scissile bond replacements resistant to thrombin cleavage

Using the natural protein hirudin as a model, a novel class of synthetic peptide inhibitors were recently designed. These inhibitors, ‘hirulogs’, retain the carboxy terminal Hir 53–64 domain that interacts with the anion binding exosite of thrombin, connected via an oligoglycyl spacer unit to a catalytic site-directed moiety modeled on the sequence [D]Phe-Pro-Arg-X. The scissile Arg-X bond bond of substrate-like inhibitors has been modified to the proteolytic-resistant functions as β-homo amino acids Argψ[CH 2CONH] X ( 2) and reduced bond analogues Argψ[CH 2N]X ( 3). Both classes of compounds demonstrate inhibition of thrombin amidolytic activity, and this active-site inhibition is highly sensitive to the P 1′ residue X. Thus these hirulog deriviatives are resistant to thrombin proteolysis while maintaining substrate-like interactions with the active center. Finally, hirulog derivatives with non-cleavable replacements of the scissile bond are found to be effective anticoagulant agents.

Biochemistry and Pharmacology of ICI 200,880, a Synthetic Peptide Inhibitor of Human Neutrophil Elastase

ICI 200,880 is representative of a new chemical class of inhibitors of human neutrophil elastase (HNE). The compound demonstrated competitive kinetics vs HNE with a Ki value of 5.0 x 10(-10) M. The selectivity of ICI 200,880 for HNE versus a variety of enzymes ranged from 150- (relative to porcine pancreatic elastase [PPE]) to greater than 360,000-fold in favor of HNE. In pharmacokinetic studies ICI 200,880 displayed a long retention time when administered directly to the lung and was rapidly eliminated when administered intravenously. Aerosol pretreatment of hamsters with ICI 200,880 before intratracheal administration of HNE produced a long-lasting inhibition of enzyme-induced increases in lung weight, total lavageable red cells, and total lavageable white cells. Subcutaneous administration of either 50 or 100 mumol/kg (twice/day) of ICI 200,880 for 14 or 28 days prevented the time-dependent increase in alveolar diameter produced by a single intratracheal dose of PPE when compound dosing was initiated 24 hours following the enzyme. Treatment of hamsters with ICI 200,880 using the same protocol and doses for 8 weeks prevented the destructive lesion induced by a single intratracheal dose of HNE. It is concluded that ICI 200,880 has biochemical, pharmacokinetic, and pharmacologic profiles that make it a useful therapeutic agent for understanding the role of HNE in various diseases. ICI 200,880 is presently being evaluated in man.

Pseudomonas aeruginosa alkaline proteinase might share a biological function with plasmin

Pseudomonas aeruginosa alkaline proteinase, which is a zinc-dependent bacterial endopeptidase, preferentially hydrolyzed Boc-Val-Leu-Lys-methylcoumarylamide (MCA) which was originally designed as a specific substrate of plasmin, a plasma serine proteinase. The hydrolytic capacity was resistant to tosyl-lysine chloromethylketone at a concentration as high as 1 mM, but was blocked by a treatment with metal chelator such as o-phenanthroline at the concentration of 5 mM. Kinetic parameters of the amidolytic reaction were K m = 21 μM, k cat = 0.067 s −1 and k cat/ K m = 3190 M −1 s −1. A synthetic peptide inhibitor which bore a possible ligand for zinc atom at the car☐y terminal was designed. This inhibitor, Ac-Val-Leu-Lys-4-mercaptoanilide, blocked the amidolytic activity of the pseudomonal alkaline proteinase in a competitive manner with the dissociation constant ( K i) value of 24 μM. The results imply that P. aeruginosa alkaline proteinase must be an unusual zinc-dependent ‘C (COOH)-type’ endopeptidase, which hydrolyzes the peptide bond of certain amino acid residues at the car☐yl group side by specific recognition, like serine- and cysteine-proteinases. In comparison, P. aeruginosa elastase which is a typical ‘N (NH 2)-type’ metalloproteinase did not hydrolyze all of the commercially available peptide-MCA substrates tested at the present study. P. aeruginosa alkaline proteinase also hydrolyzed natural substrates of plasmin, such as fibrin and fibrinogen, with similar specific activities to plasmin. The susceptible subunits of fibrinogen were the A-alpha and B-beta ones, in this order. P. aeruginosa alkaline proteinase also exhibited an anti-coagulant activity in human plasma attributed to the direct fibrinogenolytic function. Such potential anti-coagulant capacity of the P. aeruginosa alkaline proteinase might explain, at least partly, the most characteristic pathologic feature of the P. aeruginosa septicemia, hemorrhagic lesions with lacking thrombi (Fetzer, A.E. et al. (1967) Am. Rev. Respirat. Dis. 96, 1121–1130).

Synthetic peptide inhibitors of complement serine proteases—III. Significant increase in inhibitor potency provides further support for the functional equivalence hypothesis

Synthetic peptides based on functionally equivalent (as defined by similar patterns of chemically equivalent amino acids) serine protease inhibitor (serpin) C-terminal sequences inhibit both classical and alternative pathways of complement activation. Inhibition was also found with hybrid peptides consisting of the cleavage site of one serpin (antithrombin III, α-1-antitrypsin, or antichymotrypsin) attached to the short and long functionally equivalent protease binding cores of the other two serpins. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to the long binding core of antithrombin III was selective in inhibiting the classical pathway with no effect on the alternative pathway at a concn of 10 μM. Extension of the functional equivalence hypothesis has produced inhibitors of complement activation named generic and generic +, whose sequences differ by 77% or 87%, respectively, from those of all three serpin sequences. A hybrid peptide composed of the antithrombin III cleavage site attached to the generic peptide is an inhibitor of complement activation at 500 nM, the most potent inhibitor found in this study.

Identification of the Secreted Neutral Proteases from Anisakis simplex

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.

Serotonin Induces a Cyclic AMP-Mediated Outwardly Rectifying Slow K+ Current in a Single Identified Snail Neuron.

In the F2 neuron of the parietal ganglion of the snail Helix aspersa either bath or iontophoretic application of serotonin (5-HT) induces an outward current. This current has a long latency (10 - 60 s) and a slow time course, a 500 ms iontophoretic application of 5-HT evoking a response lasting 3 - 5 min. This slow outward current reverses at -80 mV, a value equal to EK. After doubling the extracellular K+ concentration the reversal potential of the 5-HT response is shifted by 19 mV, as predicted by the Nernst equation. The I-V curves reveal that the 5-HT-induced slow outward current is outwardly rectifying. This 5-HT response is blocked by intracellular Cs+ and tetraethylammonium (TEA+) and by extracellular TEA+ and Ba2+, but is not affected by the removal of extracellular Ca2+ or the intracellular injection of ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetra-acetic acid (EGTA). These results indicate that the outwardly rectifying slow outward current induced by 5-HT in the F2 neuron is carried by K+ and is Ca2+-independent. In the single isolated F2 neuron, 5-HT induces a 2.5-fold stimulation of the adenylate cyclase activity. In addition, both the intracellular injection of 3',5'-adenosine monophosphate (cAMP) and the application of forskolin mimic the effect of 5-HT on the F2 neuron. The phosphodiesterase inhibitor isobutylmethylxanthine also induces a slow outward current and potentiates the 5-HT response. The intracellular injection of a synthetic 20-residue peptide inhibitor of the cAMP-dependent protein kinase blocks the slow outward K+ current induced by 5-HT. These results show that in the F2 neuron, 5-HT elicits a slow K+ current via the stimulation of adenylate cyclase, an increase in intracellular cAMP and the activation of the cAMP-dependent kinase which probably phosphorylates a population of outwardly rectifying K+ channels or some protein/s associated with these channels.

Beta-Adrenergic modulation of K+ current in human T lymphocytes

The whole-cell voltage-clamp technique was employed to study the beta-adrenergic modulation of voltage-gated K+ currents in CD8+ human peripheral blood lymphocytes. The beta-receptor agonist, isoproterenol, decreased the peak current amplitude and increased the rate of inactivation of the delayed rectifier K+ current. In addition, isoproterenol decreased the voltage dependence of steady-state inactivation and shifted the steady-state inactivation curve to the left. Isoproterenol, on the other hand, had no significant effect on the steady-state parameters of current activation. The isoproterenol-induced decrease in peak current amplitude was inhibited by the beta-blocker propranolol. Bath application of dibutyryl cAMP (1 mM) mimicked the effects of isoproterenol on both K+ current amplitude and time course of inactivation. Furthermore, the reduction in the peak current amplitude in response to isoproterenol was attenuated when PKI5-24 (2-5 microM), a synthetic peptide inhibitor of cAMP-dependent protein kinase, was present in the pipette solution. The increase in the rate of inactivation of the K+ currents in response to isoproterenol was mimicked by the internal application of GTP-gamma-S (300 microM) and by exposure of the cell to cholera toxin (1 microgram/ml), suggesting the involvement of a G protein. These results demonstrate that the voltage-dependent K+ conductance in T lymphocytes can be modulated by beta-adrenergic stimulation. The effects of beta-agonists, i.e., isoproterenol, appear to be receptor mediated and could involve cAMP-dependent protein kinase as well as G proteins. Since inhibition of the delayed rectifier K+ current has been found to decrease the proliferative response in T lymphocytes, the beta-adrenergic modulation of K+ current may well serve as a feedback control mechanism limiting the extent of cellular proliferation.