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PubMed1999

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors.

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.

Amino Acid Sequence↗
PubMed1997

Structure of the complex of leech-derived tryptase inhibitor (LDTI) with trypsin and modeling of the LDTI-tryptase system.

BACKGROUND: Tryptase is a trypsin-like serine proteinase stored in the cytoplasmic granules of mast cells, which has been implicated in a number of mast cell related disorders such as asthma and rheumatoid arthritis. Unlike almost all other serine proteinases, tryptase is fully active in plasma and in the extracellular space, as there are no known natural inhibitors of tryptase in humans. Leech-derived tryptase inhibitor (LDTI), a protein of 46 amino acids, is the first molecule found to bind tightly to and specifically inhibit human tryptase in the nanomolar range. LDTI also inhibits trypsin and chymotrypsin with similar affinities. The structure of LDTI in complex with an inhibited proteinase could be used as a template for the development of low molecular weight tryptase inhibitors. RESULTS: The crystal structure of the complex between trypsin and LDTI was solved at 2.0 A resolution and a model of the LDTI-tryptase complex was created, based on this X-ray structure. LDTI has a very similar fold to the third domain of the turkey ovomucoid inhibitor. LDTI interacts with trypsin almost exclusively through its binding loop (residues 3-10) and especially through the sidechain of the specificity residue Lys8. Our modeling studies indicate that these interactions are maintained in the LDTI-tryptase complex. CONCLUSIONS: The insertion of nine residues after residue 174 in tryptase, relative to trypsin and chymotrypsin, prevents inhibition by other trypsin inhibitors and is certainly responsible for the higher specificity of tryptase relative to trypsin. In LDTI, the disulfide bond between residues 4 and 25 causes a sharp turn from the binding loop towards the N terminus, holding the N terminus away from the 174 loop of tryptase.

Chymases↗
PubMed2003

Major kinetic traps for the oxidative folding of leech carboxypeptidase inhibitor.

The leech carboxypeptidase inhibitor (LCI) is a 66-amino acid protein, containing four disulfides that stabilize its structure. This polypeptide represents an excellent model for the study and understanding of the diversity of folding pathways in small, cysteine-rich proteins. The pathway of oxidative folding of LCI has been elucidated in this work, using structural and kinetic analysis of the folding intermediates trapped by acid quenching. Reduced and denatured LCI refolds through a rapid, sequential flow of one- and two-disulfide intermediates and reaches a rate-limiting step in which a mixture of three major three-disulfide species and a heterogeneous population of non-native four-disulfide (scrambled) isomers coexist. The three three-disulfide intermediates have been identified as major kinetic traps along the folding pathway of LCI, and their disulfide structures have been elucidated in this work. Two of them contain only native disulfide pairings, and one contains one native and two non-native disulfide bonds. The coexistence of three-disulfide kinetic traps adopting native disulfide bonds together with a significant proportion of fully oxidized scrambled isomers shows that the folding pathway of LCI features properties exhibited by both the bovine pancreatic trypsin inhibitor and hirudin, two diverse models with extreme folding characteristics. The results further demonstrate the large diversity of disulfide folding pathways.

Amino Acid Sequence↗
PubMedDate not supplied

A model of a segmental oscillator in the leech heartbeat neuronal network.

We modeled a segmental oscillator of the timing network that paces the heartbeat of the leech. This model represents a network of six heart interneurons that comprise the basic rhythm-generating network within a single ganglion. This model builds on a previous two cell model (Nadim et al., 1995) by incorporating modifications of intrinsic and synaptic currents based on the results of a realistic waveform voltage-clamp study (Olsen and Calabrese, 1996). Due to these modifications, the new model behaves more similarly to the biological system than the previous model. For example, the slow-wave oscillation of membrane potential that underlies bursting is similar in form and amplitude to that of the biological system. Furthermore, the new model with its expanded architecture demonstrates how coordinating interneurons contribute to the oscillations within a single ganglion, in addition to their role of intersegmental coordination.

Animals↗
PubMed2002

Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea).

In the present paper the chromosome complement (n = 13; 2n = 26) of the common leech Haemopis sanguisuga (L.) (Annelida: Hirudinea: Hirudinidae) was analyzed using banding techniques and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal clusters (18S-28S rDNA) in the pericentromeric region of one large metacentric chromosome pair; this region, which consisted of heterochromatin rich in GC base pairs, was preferentially stained by silver nitrate (Ag-NOR). The (TTAGGG)n telomeric probe was hybridized with the termini of nearly all chromosomes, whereas the (GATA)n probe did not label any chromosome areas.

Animals↗
PubMed1999

Possible roles of extracellular matrix and cytoskeleton in leech body wall muscles.

Round circomyarian fibres of leeches are peculiar helical muscles. The fibres are characterized by a lack of junctions, being separated by a thick extracellular matrix, and by scarce end-plates. Even so, the fibres grouped in units show the same degree of contraction. Biochemical, immunocytochemical and ultrastructural studies were performed in order: (a) to demonstrate the presence in the extracellular matrix of fibronectin, collagen type IV and laminin and in the cytoskeleton of desmin and alpha-actinin; (b) to show the possible link of extracellular matrix with the scaffold of intermediate filaments; (c) to evaluate how the extracellular matrix can play a role in the transduction of a signal during contraction-relaxation-superelongation phases.

Journal Article↗
PubMed1996

Hirudins: from leeches to man.

Hirudins are a group of highly homologous polypeptides from the medicinal leech, Hirudo medicinalis. They have an extremely high and specific affinity for thrombin and are consequently potent anticoagulants. They inhibit platelet aggregation induced by thrombin and efficiently inhibit thrombin when it is bound to a fibrin clot. Recombinant hirudins have now been produced and have been used to show a wide range of effects on thrombosis in animal models. They are particularly effective against venous-type thrombi and have greater effects than heparins on the platelet-rich thrombi in arteries. Recombinant hirudins are currently undergoing clinical trails in deep venous thrombosis and acute coronary syndrome and as an adjunct to thrombolysis in myocardial infarction. Results from pilot trials indicate promising advantages over the currently used antithrombotic agents. Larger clinical trails have been initiated and we await with great interest their imminent publication.

Amino Acid Sequence↗
PubMed2004

Role of kinetic intermediates in the folding of leech carboxypeptidase inhibitor.

The oxidative folding and reductive unfolding pathways of leech carboxypeptidase inhibitor (LCI; four disulfides) have been characterized in this work by structural and kinetic analysis of the acid-trapped folding intermediates. The oxidative folding of reduced and denatured LCI proceeds rapidly through a sequential flow of 1-, 2-, 3-, and 4-disulfide (scrambled) species to reach the native form. Folding intermediates of LCI comprise two predominant 3-disulfide species (designated as III-A and III-B) and a heterogeneous population of scrambled isomers that consecutively accumulate along the folding reaction. Our study reveals that forms III-A and III-B exclusively contain native disulfide bonds and correspond to stable and partially structured species that interconvert, reaching an equilibrium prior to the formation of the scrambled isomers. Given that these intermediates act as kinetic traps during the oxidative folding, their accumulation is prevented when they are destabilized, thus leading to a significant acceleration of the folding kinetics. III-A and III-B forms appear to have both native disulfides bonds and free thiols similarly protected from the solvent; major structural rearrangements through the formation of scrambled isomers are required to render native LCI. The reductive unfolding pathway of LCI undergoes an apparent all-or-none mechanism, although low amounts of intermediates III-A and III-B can be detected, suggesting differences in protection against reduction among the disulfide bonds. The characterization of III-A and III-B forms shows that the former intermediate structurally and functionally resembles native LCI, whereas the III-B form bears more resemblance to scrambled isomers.

Amino Acid Sequence↗
PubMed1958

The fine structure of the adipose cell of the leech Glossiphonia complanata.

The two distinct types of cytoplasm seen with the light microscope in the adipose cell of the leech Glossiphonia complanata have been identified in the electron microscope image of this cell. One of these, the basophil cytoplasm, contains many well oriented, paired membranes which are much more clearly evident when calcium ions are added to the fixative. The membranes sometimes appear as concentric arrays of lamellae and are thought to represent sections through a phospholipide-containing body. The paired membranes and the concentric lamellae have granules attached to them and resemble in size and structure the membranes of the endoplasmic reticulum encountered in many mammalian cells. Small dense cytoplasmic particles are present throughout the cell; they may be ferritin molecules, derived from the breakdown of haemoglobin taken in as food. On the basis of a previous histochemical study and the present electron microscope investigation, it is suggested that these paired membranes are similar to the organized type of mammalian ER and the results seem to confirm the belief that these membranes are composed of layers of phospholipoprotein together with attached particles of ribonucleoprotein.

Adipocytes↗
PubMed1970

Monosynaptic chemical and electrical connexions between sensory and motor cells in the central nervous system of the leech.

The synaptic connexions that underlie three different segmental shortening reflexes have been traced by recording intracellularly from individual sensory and motor nerve cells in the C.N.S. of the leech. The fourteen sensory cells involved in these reflexes respond specifically to one of three modalities: touch, pressure, or noxious stimuli applied to the skin. All three types of sensory neurone give rise to excitatory synaptic potentials in two large motoneurones. Each of these motor cells provides excitatory innervation to the longitudinal muscle fibres of the opposite side of the segment. The mechanism of synaptic transmission is, however, different for each type of sensory cell.1. An impulse in a sensory cell that responds to touch gives rise to a short-latency depolarizing potential in the large longitudinal motoneurones by way of an electrical synapse. This junction rectifies so that excitation can spread in only one direction (from the sensory to the motor cell), whereas a hyperpolarizing potential can pass only in the opposite direction.2. The synaptic potential evoked in the motoneurone by an action potential in a sensory cell responding to noxious stimuli can be attributed to the action of a chemical transmitter agent and has different properties: the post-synaptic potential arises after a delay of about 2-4 msec, is abolished by high concentrations of Mg, and enhanced by high concentrations of Ca. Several lines of evidence show that this connexion is monosynaptic.3. The synaptic potential following an impulse in a pressure cell is produced by both chemical and electrical synaptic mechanisms. Rectification, similar to that described for the touch cell, also occurs at this electrical synapse.4. One or more impulses in any one of the fourteen mechanoreceptor cells in the ganglion can initiate impulses in the large longitudinal motoneurones to produce a shortening of the segment. The contraction is abolished by blocking impulse initiation in the motoneurones.5. The arborizations of the sensory cells and the motoneurone within the neuropile have been studied histologically after injecting a fluorescent dye. Their processes are intertwined in a highly complex manner so that the sites of the synaptic junctions cannot be determined with the resolutions so far achieved. Nevertheless, taken together the histological and the electrical results support the idea that individual cells are connected in a stereotyped pattern and operate by distinctive mechanisms.6. These findings provide a basis for studying the functional role of chemical and electrical synaptic mechanisms in these pathways.

Action Potentials↗
PubMed2005

Role for calcium signaling and arachidonic acid metabolites in the activity-dependent increase of AHP amplitude in leech T sensory neurons.

Previous studies have revealed a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) neurons of the leech Hirudo medicinalis. The firing of T cells is characterized by an AHP, which is mainly due to the activity of the Na+/K+ ATPase. Low-frequency repetitive stimulation of T neurons leads to a robust increment of the AHP amplitude, which is correlated with a synaptic depression between T neuron and follower cells. In the present study, we explored the molecular cascades underlying the AHP increase. We tested the hypothesis that this activity-dependent phenomenon was triggered by calcium influx during neural activity by applying blockers of voltage-dependent Ca2+ channels. We report that AHP increase requires calcium influx that, in turn, induces release of calcium from intracellular stores so sustaining the enhancement of AHP. An elevation of the intracellular calcium can activate the cytosolic isoforms of the phosholipase A2 (PLA2). Therefore we analyzed the role of PLA2 in the increase of the AHP, and we provide evidence that not only PLA2 but also the recruitment of arachidonic acid metabolites generated by the 5-lipoxygenase pathway are necessary for the induction of AHP increase. These data indicate that a sophisticated cascade of intracellular signals links the repetitive discharge of T neurons to the activation of molecular pathways, which finally may alter the activity of critical enzymes such as the Na+/K+ ATPase, that sustains the generation of the AHP and its increase during repetitive stimulation. These results also suggest the potential importance of the poorly studied 5-lipoxygenase pathway in forms of neuronal plasticity.

Animals↗
PubMed2006

Graded inhibitory synaptic transmission between leech interneurons: assessing the roles of two kinetically distinct low-threshold Ca currents.

In leeches, two pairs of reciprocally inhibitory heart interneurons that form the core oscillators of the pattern-generating network for heartbeat possess both high- and low-threshold (HVA and LVA) Ca channels. LVA Ca current has two kinetically distinct components (one rapidly activating/inactivating, ICaF, and another slowly activating/inactivating, ICaS) that mediate graded transmission, generate plateau potentials driving burst formation, and modulate spike-mediated transmission between heart interneurons. Here we used different stimulating protocols and inorganic Ca channel blockers to separate the effects of ICaF and ICaS on graded synaptic transmission and determine their interaction and relative efficacy. Ca2+ entering by ICaF channels is more efficacious in mediating release than that entering by ICaS channels. The rate of Ca2+ entry by LVA Ca channels appears to be as critical as the amount of delivered Ca2+ for synaptic transmission. LVA Ca currents and associated graded transmission were selectively blocked by 1 mM Ni2+, leaving spike-mediated transmission unaffected. Nevertheless, 1 mM Ni2+ affected homosynaptic enhancement of spike-mediated transmission that depends on background Ca2+ provided by LVA Ca channels. Ca2+ provided by both ICaF and ICaS depletes a common pool of readily releasable synaptic vesicles. The balance between availability of vesicles and Ca2+ concentration and its time course determine the strength of inhibitory transmission between heart interneurons. We argue that Ca2+ from multichannel domains arising from ICaF channels, clustered near but not directly associated with the release trigger, and Ca2+ radially diffusing from generally distributed ICaS channels interact at common release sites to mediate graded transmission.

Action Potentials↗
PubMed2006

Spike-mediated and graded inhibitory synaptic transmission between leech interneurons: evidence for shared release sites.

Inhibitory synaptic transmission between leech heart interneurons consist of two components: graded, gated by Ca2+ entering by low-threshold [low-voltage-activated (LVA)] Ca channels and spike-mediated, gated by Ca2+ entering by high-threshold [high-voltage-activated (HVA)] Ca channels. Changes in presynaptic background Ca2+ produced by Ca2+ influx through LVA channels modulate spike-mediated transmission, suggesting LVA channels have access to release sites controlled by HVA channels. Here we explore whether spike-mediated and graded transmission can use the same release sites and thus how Ca2+ influx by HVA and LVA Ca channels might interact to evoke neurotransmitter release. We recorded pre- and postsynaptic currents from voltage-clamped heart interneurons bathed in 0 mM Na+/5 mM Ca2+ saline. Using different stimulating paradigms and inorganic Ca channel blockers, we show that strong graded synaptic transmission can occlude high-threshold/spike-mediated synaptic transmission when evoked simultaneously. Suppression of LVA Ca currents diminishes graded release and concomitantly increases the ability of Ca2+ entering by HVA channels to release transmitter. Uncaging of Ca chelator corroborates that graded release occludes spike-mediated transmission. Our results indicate that both graded and spike-mediated synaptic transmission depend on the same readily releasable pool of synaptic vesicles. Thus Ca2+, entering cells through different Ca channels (LVA and HVA), acts to gate release of the same synaptic vesicles. The data argue for a closer location of HVA Ca channels to release sites than LVA Ca channels. The results are summarized in a conceptual model of a heart interneuron release site.

Action Potentials↗
PubMed1995

Stimulus-evoked changes of extra- and intracellular pH in the leech central nervous system. I. Bicarbonate dependence.

1. We have measured the effect of repetitive electrical nerve root stimulation on the extracellular potassium activity (aKe) and the extracellular pH (pHe) and intracellular pH (pHi) in segmental ganglia of the leech Hirudo medicinalis with double-barreled K(+)- and pH-sensitive microelectrodes. To investigate the influence of CO2/HCO3-, we compared the stimulus-evoked changes in aKe, pHe, and pHi in the presence and absence of 5% CO2-24 mM HCO3- in the saline. 2. An electrical nerve root stimulation at 20-30 Hz for 1 min caused a rapid increase of 1.11 +/- 0.79 (SD) mM in aKe, followed by an aKe undershoot of 0.17 +/- 0.15 mM when the stimulation was discontinued (n = 6). aKe transients were not significantly affected by CO2/HCO3-. 3. In 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered, nominally CO2/HCO3(-)-free saline, low stimulus intensities or stimulus durations up to a few seconds resulted in a fast alkaline pHe transient. This alkalinization was followed by a larger and longer-lasting extracellular acidification when the stimulation was intensified and prolonged. A stimulation at 20 Hz, 5 V for 1 min caused an average alkaline shift of 0.083 +/- 0.055 pH units, followed by an acidosis of 0.079 +/- 0.038 pH units (n = 63). A change from 5 mM HEPES-buffered saline to 20 mM HEPES-buffered saline attenuated the stimulus-evoked pHe transients by 50-60%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Stimulus-evoked changes of extra- and intracellular pH in the leech central nervous system. II. Mechanisms and maintenance of pH homeostasis.

1. We have studied extracellular pH (pHe) and intracellular pH (pHi) changes evoked by repetitive electrical side nerve stimulation (20 Hz, 1 min) in segmental ganglia of the leech Hirudo medicinalis using double-barreled, pH-sensitive microelectrodes to elucidate the involvement of neurotransmitters, of carbonic anhydrase, and of active acid/base transport on the extracellular H+ homeostasis. In saline buffered with 5% CO2-24 mM HCO3-, the stimulation induced a small and brief alkalinization followed by an acidification in the extracellular spaces (ECS), whereas neurons acidified and glial cells alkalinized (see previous paper). 2. Blocking synaptic transmitter release by superfusion with 20 mM Mg2+ saline (CO2/HCO3(-)-free) led to a reversible reduction of both activity-induced pHe changes by approximately 90% and to a complete suppression of the intracellular acidification of neurons. After application of the glutamate/kainate receptor blocker 6-cyano-7-dinitroquinozaline-2,3-dione (CNQX, 50 microM) to CO2/HCO3(-)-free saline, the stimulus-evoked pHe changes were reversibly reduced. The gamma-aminobutyric acid-A (GABAA) receptor antagonist picrotoxin (50 microM) led to an amplification of the extracellular alkalinization in the presence of CO2/HCO3-. Bath application of the excitatory transmitter agonists carbachol or kainate to CO2/HCO3(-)-free saline induced biphasic alkaline-acid transients in the ECS; the inhibitory transmitters GABA and serotonin had no detectable effects on the pHe (saline buffered with CO2/HCO3-).(ABSTRACT TRUNCATED AT 250 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Recombinant leech antiplatelet protein prevents collagen-mediated platelet aggregation but not collagen graft thrombosis in baboons.

Leech antiplatelet protein (LAPP) is a specific inhibitor of collagen-induced human platelet aggregation and adhesion to collagen under static conditions. Recombinant LAPP (rLAPP) and L-366,763 (acetylated-Cys-Asn-Pro-Arg-Gly-Asp-Cys-NH2), a peptidyl fibrinogen receptor antagonist, were evaluated in an anesthetized baboon thrombosis model using a collagen-coated graft segment of an arteriovenous shunt to elicit thrombus formation. Animals were randomized to receive systemic intravenous administration of rLAPP (100 micrograms.kg-1 x min-1; n = 5), L-366,763 (8.5 micrograms.kg-1 x min-1; n = 3), or saline (n = 3). Despite complete and selective inhibition of type I collagen-induced ex vivo aggregation of platelets, rLAPP had no significant effect on the rate or the extent of 111-In-labeled platelet deposition onto the collagen graft and no effect on template bleeding time. In contrast, L-366,763 completely prevented platelet deposition, maintained blood flow, and significantly prolonged bleeding time at the dosage that inhibited ex vivo aggregation in response to all agonists studied. In this study, the absence of an antithrombotic benefit of rLAPP contrasted sharply with the efficacy of the fibrinogen receptor antagonist. These results demonstrate that specific inhibition of collagen-mediated platelet aggregation alone is not sufficient to prevent platelet-dependent thrombosis in this baboon model.

Amino Acid Sequence↗
PubMed1996

Membrane responses evoked by organic buffers in identified leech neurones

Using single-electrode current-clamp and two-electrode voltage-clamp, different identified neurones of the leech (Hirudo medicinalis L.) were shown to hyperpolarize, or to exhibit outward currents (usually >2 nA), during bath application of the organic buffers Mops, Pipes and Mes (10 mmol l-1). Tris and Hepes had little or no effect on the membrane properties of neurones when they were added to a bathing saline buffered by CO2-HCO3- at a constant pH of 7.4. Outward currents evoked by buffers obtained from two suppliers, Roth and Sigma, were not significantly different, except in the case of Pipes. Steady-state currents evoked by Pipes supplied by Roth were eightfold larger than steady-state currents evoked by Pipes from Sigma. A transient current peak always present in responses evoked by Pipes from Roth was never present in responses evoked by Pipes from Sigma. Outward currents evoked by buffers were due to a conductance increase and appeared to be carried by Cl-. In low-Cl- saline, hyperpolarizations evoked by Mops in Retzius cells were reduced. When Cl- was injected into heart motor neurones, the hyperpolarizations evoked by Mops reversed. Tetraethylammonium (TEA+) injected into heart interneurones did not block Mops-evoked hyperpolarizations. Mops-evoked outward currents in Retzius cells were partially blocked by d-tubocurarine and bicuculline methiodide, and the latter also partially blocked Mops-evoked hyperpolarizations in HE cells. Since d-tubocurarine partially blocked acetylcholine-evoked Cl- currents in Retzius cells and bicuculline methiodide partially blocked carbachol-evoked hyperpolarizations in HE cells, Mops appears to act on the cholinergic receptors of these neurones by mediating a Cl- conductance.

Journal Article↗
PubMed1996

Inhibition of voltage-dependent Ca2+influx by extracellular ATP in salivary cells of the leech Haementeria ghilianii

The effects of extracellular ATP on intracellular free Ca2+ concentration ([Ca2+]i) and depolarization-induced elevations of [Ca2+]i were investigated in salivary cells of the leech Haementeria ghilianii using the fluorescent Ca2+ indicator Fura-2. Simultaneously, the membrane potential was monitored or controlled by voltage-clamp. The cell membrane was depolarized either by transient elevations of the extracellular K+ concentration ([K+]o) to 90 mmol l-1 or by depolarizing steps under voltage-clamp. The resulting transient elevations of [Ca2+]i (Ca2+ transients) could be repeatedly elicited with little variability in amplitude. Ca2+ transients were completely inhibited by 2 mmol l-1 Ni2+ or in Ca2+-free saline. The transients are, therefore, dependent on Ca2+ influx from the external medium through voltage-gated Ca2+ channels. The Ca2+ influx was rapidly and reversibly inhibited by extracellular application of ATP. The effect was dose-dependent with a threshold concentration below 10(-7) mol l-1. A 50 % reduction in the amplitude of Ca2+ transients was obtained by application of 1­2 µmol l-1 ATP or ATP-gamma-S (apparent IC50, 1.6 µmol l-1 ATP) and Ca2+ transients were almost completely inhibited by 30­100 µmol l-1 ATP. Resting [Ca2+]i, the resting membrane potential and membrane potential changes induced by 90 mmol l-1 [K+]o were not affected by ATP. Adenosine (10 µmol l-1) did not affect resting [Ca2+]i, the resting membrane potential or membrane potential changes induced by 90 mmol l-1 [K+]o and had little effect on Ca2+ transients. Suramin, an antagonist of vertebrate P2 receptors, was without effect on the inhibitory actions of ATP. We conclude that activation of a suramin-insensitive purinoceptor by ATP inhibits Ca2+ influx through voltage-gated Ca2+ channels in the salivary cells of Haementeria ghilianii.

Journal Article↗