Mast Cell Degranulating Peptide Research Articles

Contribution of a non-inactivating potassium current to the resting membrane potential of fusion-competent human myoblasts.

1. Using the patch-clamp technique, a new non-inactivating voltage-gated potassium current, IK(ni), was studied in cultured fusion-competent human myoblasts. 2. IK(ni) is activated at voltages above -50 mV and its conductance reaches its maximum around +50 mV. Once activated, the current remains at a steady level for minutes. 3. Reversal potential measurements at various extracellular potassium concentrations indicate that potassium ions are the major charge carriers of IK(ni). 4. IK(ni) is insensitive to potassium channel blockers such as charybdotoxin, dendrotoxins, mast cell degranulating (MCD) peptide, 4-aminopyridine (4-AP), 3,4-diaminopyridine (3,4-DAP) and apamin, but can be blocked by high concentrations of TEA and by Ba2+. 5. A potassium channel of small conductance (8.4 pS at +40 mV) with potential dependence and pharmacological properties corresponding to those of IK(ni) in whole-cell recording is described. 6. IK(ni) participates in the control of the resting potential of fusion-competent myoblasts, suggesting that it may play a key role in the process of myoblast fusion.

Involvement of the Peptide-Sensitive Channel in the Translocation of Basic Peptides into Mitochondria

The Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by several highly basic peptides. Among these peptides, the most active are pCOX IV (1-12)Y, a mitochondrial addressing peptide and dynorphin B (1-13), a peptide unrelated to mitochondrial physiology. The voltage-dependent characteristics of the block duration of the PSC induced by these peptides and the fact that these peptides are imported into mitochondria in an in vitro assay suggest the involvement of the PSC in peptide translocation into mitochondria. We have analyzed the interaction of Mast Cell Degranulating peptide (MCD), a disulfide rich basic peptide, with yeast and mammalian mitochondria. Electrophysiological experiments with native and reduced forms of this peptide (nMCD and rMCD) showed an interaction of both forms with the yeast PSC. On the other hand, only rMCD blocked the electrical activity of the bovine adrenal cortex PSC. Similarly, although both forms inhibited the import of dynorphin B (1-13) into yeast mitochondria, only rMCD inhibited this import in bovine mitochondria. The correlation between electrophysiological and biochemical data strongly suggest that dynorphin B is translocated across the outer membrane at the level of the PSC.

The Precursors of the Bee Venom Constituents Apamin and MCD Peptide Are Encoded by Two Genes in Tandem Which Share the Same 3′-Exon

From a cDNA library prepared from venom glands of worker bees, clones encoding the precursors of apamin and MCD peptide have been isolated. The cDNAs are similar at the 5'-ends and identical in their 3'-regions. Analysis of the corresponding genes has revealed the existence of six exons separated by introns rich in A + T. Starting from the 5'-end, these exons are arranged in the following order: three exons of the mast cell-degranulating (MCD) peptide precursor, two exons of the gene for the apamin precursor, and finally a 3'-exon present in both cDNAs. This suggests that the bulk of the apamin gene resides in the third intron of the MCD peptide gene. Using inverse polymerase chain reaction, a segment of genomic DNA upstream of the first exon of the MCD precursor gene was obtained. The sequence of this segment shows 81% identity to the DNA sequence preceding the first exon of the apamin gene and both contain a putative TATA box. We thus propose that the mRNA encoding the apamin precursor originates from a primary transcript which starts in the third intron of the MCD peptide gene. Both cDNAs encode unusually small precursors comprising only 46 amino acids in case of apamin and 50 in the case of the MCD peptide.

48. Correlation of endogenous noradrenaline release and neuronal inhibition in rat locus coeruleus slices by combined voltammetric and electrophysiological recording

Using electroencephalographic (EEG) recordings in freely moving rats and extracellular neuronal firing-rate recordings in hippocampal slices, we examined the effects of riluzole (RP 54274), a compound with anti-glutamate properties, against the convulsive seizures and the cellular hyperexcitability produced by the mast-cell degranulating peptide (MCD), dendrotoxin I (DTXi) and 4-aminopyridine (4-AP). I.c.v. administration of riluzole (10 nmol) prevented the seizures induced by MCD, and to a lesser extent those due to DIXi, whilst leaving 4-AP seizures unaffected. This effect was also present after oral administration of the compound (4 mg kg−1) and lasted for approximately 6 h. Electrophysiological recordings in vitro confirmed that riluzole dose dependently and reversibly abolished the sustained increase in firing rate induced by both MCD and DTXi in the hippocampus. These results indicate that the anti-epileptic spectrum of riluzole in this model has similarities with, but is not identical to, that of classical potassium channel openers, and differs from that of calcium channel blockers or other glutamate antagonists such as D(−)-2-amino-5-phosphono-valeric acid. However, since MCD releases glutamate, the preventive effect of riluzole in this model may involve direct or indirect interaction with glutamatergic processes.

Antagonism by glibenclamide of the effect of morphine in hippocampal preparations

We previously showed that morphine lowered the affinity of Ca 2+ antagonist binding and subsequently enhanced field potentials in hippocampal preparations. In the present study, the effect of various K + channel antagonists on these actions of morphine was studied. Higher K d value of [ 3H]nitrendipine binding was obtained for membranes prepared from slices treated with morphine. Concomitant treatment of slices with morphine and tetramethylammonium (TMA) or glibenclamide attenuated the effect of morphine. Apamin and mast cell-degranulating (MCD) peptide were without effect on morphine-induced change in [ 3H]nitrendipine binding. In those experiments, no change in concentration of binding sites was observed. Glibenclamide reduced the morphine enhancement of field potentials. These results suggested the regulation of Ca 2+ channels by morphine through K + channel opening.

Mast cell degranulating (MCD) peptide analogs with reduced ring structure.

Mast cell degranulating (MCD) peptide, a component of bee venom, is a 22 amino acid peptide with two disulfide bridges. In this first structure-activity study of MCD peptide, three analogs were synthesized and tested: two analogs shortened by omitting sequences 6-10 and 8-13, respectively, and one analog lacking the disulfide bridge between cysteine residues 5 and 19. These analogs were synthesized by solid-phase methods and were compared to MCD peptide in two assays for inflammation: histamine release from mast cells and superoxide anion release from neutrophils. All three analogs produced histamine release, although with only about one fifth of the activity of MCD peptide. Superoxide anion-releasing activity, however, did not parallel histamine release. MCD peptide did not release superoxide anion, while the 6-10 and 8-13 deletion analogs were strong and weak stimulants, respectively, of this anion. CD spectra showed that the secondary structures of the three analogs were very similar to that of MCD peptide, so that a change in secondary structure cannot completely explain the changes in releasing activities. Charge differences between the two deletion analogs and MCD peptide may explain some of the differences in activity. This is the first demonstration that the various activities of MCD peptide can be separated, and provides a lead through which the purported antiinflammatory activity of MCD peptide may possibly be explored in the future.

K + channel involvement in induction of synaptic enhancement by mast cell degranulating (MCD) peptide

A bee venom, mast cell degranulating peptide (MCD), which induces long-term potentiation (LTP) of synaptic transmission in hippocampal slices, was found to possess multiple functions. They include (1) binding and thereby inhibiting a voltage-dependent K +-channel in brain membranes, (2) incorporation in a lipid bilayer to form voltage-dependent and cation-selective channels by itself, and (3) activation of a pertussis toxin (Ptx)-sensitive GTP-binding proteins. In this study, we prepared several derivatives and analogues of MCD and investigated which function is more closely related to the induction of LTP. Another bee venom, apamin, formed ion channels in a lipid bilayer which were indistinguishable from those formed by MCD. D-MCD, an optical isomer of MCD, activated a Ptx-sensitive GTP-binding protein. However, these peptides did not induce LTP in the hippocampal slices. A snake venom, dendrotoxin-I (DTX-I), bound to the same K +-channels as MCD and did induce LTP. These results suggest that the most potent aspect of MCD involved in LTP inducibility is its interaction with the voltage-dependent K +-channel.

Swapping of functional domains in voltage-gated K + channels

Functionally significant properties of domains in the amino acid sequence of potassium (K+) channel-forming proteins have been investigated by constructing chimeric K+ channels. The N-terminal domain of ShA2 channels was responsible for the fast inactivation (IKA) and also determined a shift in the threshold of activation whereas the membrane domain determined the timecourse of slow inactivation. The binding site for dendrotoxin (DTX), but not for mast cell degranulating peptide (MCDP), is completely located on the loop between the membrane spanning segments S5 and S6 in RCK1 channels. A certain part of this region which has recently been designated as a narrow part of the pore was found to be not responsible for the differences in the single-channel current amplitude between RCK4 and RCK2 K+ channels. Interchange of the C-terminal domain did not influence activation or inactivation of the channels.

Mast cell degranulating (MCD) peptide and its optical isomer activate GTP binding protein in rat mast cells

The MCD peptide in bee venom induces degranulation in mast cells. The internal calcium concentration of mast cells increased and remained high following MCD stimulation. This calcium increase was blocked by pertussis toxin (Ptx) treatment, suggesting that MCD peptide activates Ptx-sensitive G-protein. Even in the absence of external calcium in the incubation medium, the calcium concentration increased by MCD treatment. but soon returned to the original level. D-MCD, the optical isomer of the MCD peptide, also increased the internal calcium concentration through a Ptx-sensitive pathway. We suggest that cationic clusters at one side of the surface are more important in activating the G-protein than the α-helix conformation.

MCD peptide and dendrotoxin I activate c-fos and c-jun expression by acting on two different types of K + channels. A discrimination using the K + channel opener lemakalim

Mast cell degranulating (MCD) peptide and Dendrotoxin I (DTXI), two potent hyperexcitability-inducing toxins acting on voltage-dependent potassium channels, induce the expression of both c-fos and c-jun mRNA in i.c.v. treated rats. The distribution of c-fos and c-jun expression has been analyzed by in situ hybridization. The expression is particularly high in the cerebral cortex and hippocampus for both toxins. However differences of expression between MCD and DTXI-treated animals have been observed in hypothalamus and thalamic and amygdaloid nuclei. Moreover, brain areas such as cerebellum which have high amounts of binding sites for both MCD and DTXI do not show any induction of c-fos and c-jun. Lemakalim, a K+ channel opener, prevents the MCD-induced activation of both 'immediate-early genes' in all brain areas but is unable to inhibit the induction of c-fos and c-jun induced by DTXI. These two toxins which are generally believed--from molecular approaches--to act on the same voltage-dependent K+ channel, clearly act in vivo on two distinct classes of channels.

Evidence for the interaction of mast cell-degranulating peptide with pertussis toxin-sensitive G proteins in mast cells

K channel blocker properties have been reported for mast cell-degranulating peptide (MCD) in the central nervous system, but its action mechanism in mast cells remains unknown. We studied the effect of MCD on the membrane potential of rat peritoneal mast cells using the fluorescent probe bis-oxonol. Unexpectedly, MCD induced a decrease in bis-oxonol fluorescence, in a rapid and then a slower phase, suggesting hyperpolarization of mast cells. Other K +-channel blockers, tetraethylammonium and 4-aminopyridine, did not significantly modify the bis-oxonol fluorescence and did not alter the effect of MCD. The late phase of bis-oxonol fluorescence decrease was inhibited by ouabain and by potassium deprivation, whereas histamine release was not affected. The first phase of putative hyperpolarization induced by MCD coincided with histamine release and with the generation of inositol polyphosphates. Prior treatment of the cells with pertussis toxin inhibited these effects of MCD. MCD stimulated the GTPase activity of purified G proteins (G 0/G i) in a concentration-dependent manner. These results indicate that the effect of MCD on mast cells is unrelated to K − channels but that it is relevant to the activation of pertussis toxin-sensitive G proteins leading to the activation of phospholipase C. A direct interaction of MCD with G proteins is proposed, which, unlike mastoparan, does not require positive cooperativity.

Direct activation of GTP-binding proteins by venom peptides that contain cationic clusters within their alpha-helical structures

Direct interactions of venom peptides that contained a cysteine-stabilized α-helical motif within their internal molecules with αβγ-trimeric GTP-binding proteins (G proteins) were studied in reconstituted phospholipid vesicles. Mast cell-degranulating (MCD) peptide stimulated the steady-state rate of GTP hydrolysis catalyzed by the reconstituted G proteins. Synthetic D-MCD peptide, the optical isomer of MCD peptide, was also effective in the activation of G proteins as L-MCD peptide. The stimulations by L- and D-peptides were both abolished in G proteins that had been ADP-ribosylated by pertussis toxin. Charybdotoxin also stimulated, though slightly, the GTPase activity of G proteins. Such a stimulation was, however, not observed upon the incubation of G proteins with other venom peptides such as apamin, sarafotoxin and endothelin. Thus, in comparison of the amino acid sequences of their venom peptides, the extent of the activation of G proteins appeared to be correlated with the number of basic amino acid residues around the α-helix. These results suggest that cationic clusters at one side of the α-helical surface are more important in the direct activation of G proteins than a specific, α-helical structure.

Mast Cell Degranulating Peptide Increases the Frequency of Spontaneous Miniature Postsynaptic Currents in CA3 Rat Hippocampal Neurons.

Mast cell degranulating peptide (MCDP) is a neurotoxic agent isolated from bee venom. It produces a long-term potentiation in the hippocampus. We now report that MCDP, at nanomolar concentrations, induces a reduction of a transient voltage-dependent potassium current (ID) in CA3 rat pyramidal neurons and a persistent (>30 min) enhancement of the frequency of spontaneous miniature excitatory and inhibitory postsynaptic currents (m.e.p.s.c.s. and m.i.p.s.c.s.). M.e.p.s.c.s. and m.i.p.s.c.s. were recorded in the presence of bicuculline (30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), respectively. The increased frequency of m.e.p.s.c.s. (408 +/- 60%) and m.i.p.s.c.s. (583 +/- 553%) was independent of the reduction of ID because 4-aminopyridine (4-AP, 30 microM - 2 mM) blocked ID but had no effects on m.e.p.s.c.s. and m.i.p.s.c.s. In the presence of the calcium channel blocker manganese (3 mM), MCDP still enhanced the frequency of m.e.p.s.c.s. (326 +/- 162%). It is concluded that MCDP augments the release of excitatory and inhibitory transmitter by an action, which is independent of calcium influx, through voltage-dependent channels.

Riluzole prevents hyperexcitability produced by the mast cell degranulating peptide and dendrotoxin I in the rat

Using electroencephalographic (EEG) recordings in freely moving rats and extracellular neuronal firing-rate recordings in hippocampal slices, we examined the effects of riluzole (RP 54274), a compound with anti-glutamate properties, against the convulsive seizures and the cellular hyperexcitability produced by the mast-cell degranulating peptide (MCD), dendrotoxin I (DTX i) and 4-aminopyridine (4-AP). I.c.v. administration of riluzole (10 nmol) prevented the seizures induced by MCD, and to a lesser extent those due to DIX i, whilst leaving 4-AP seizures unaffected. This effect was also present after oral administration of the compound (4 mg kg −1) and lasted for approximately 6 h. Electrophysiological recordings in vitro confirmed that riluzole dose dependently and reversibly abolished the sustained increase in firing rate induced by both MCD and DTX i in the hippocampus. These results indicate that the anti-epileptic spectrum of riluzole in this model has similarities with, but is not identical to, that of classical potassium channel openers, and differs from that of calcium channel blockers or other glutamate antagonists such as D(−)-2-amino-5-phosphono-valeric acid. However, since MCD releases glutamate, the preventive effect of riluzole in this model may involve direct or indirect interaction with glutamatergic processes.

Long-term enhancement of synaptic transmission by synthetic mast cell degranulating peptide and its localization of binding sites in hippocampus

A bee venom, mast-cell-degranulating (MCD) peptide, was synthesized by stepwise formation of the two disulfide bridges. This synthetic MCD peptide induced long-term potentiation (LTP) in the CA1 region of a hippocampus slice at concentrations ranging from 10 −7 to 10 −5 M. The potentiating effect of MCD was not lost by biotinylation of its N-terminus, and thus it became possible to investigate localization of its binding site at a cellular level in the hippocampus slice at a LTP-inducible concentration (1 μM). We found that even after brief application of N-biotinyl-MCD (1 μM) to perfusate, its presence was observed in pyramidal cell bodies in the CA1 and CA3 regions, but not in granule cells in the dentate gyrus of the hippocampus slice. This observation does not coincide with the localization of its high-affinity binding sites revealed by 125I-MCD binding, suggesting that another type(s) of MCD binding site, such as low-affinity binding sites, are present in the hippocampus.

Mast cell degranulating peptide induces the expression of the c-fox proto-oncogene in hippocampus

The intrahippocampal injection of the mast cell degranulating (MCD) peptide, a bee venom component acting on the K + channel, results in the appearance of the proto-oncogene c-fos mRNA in the ipsi- and contralateral hippocampus of the treated animals without generating convulsions. This MCD-induced transcriptional event is discussed in terms of cellular plasticity since MCD peptide is known to induce long-term potentiation.

A K+ channel in Xenopus nerve fibres selectively blocked by bee and snake toxins: binding and voltage‐clamp experiments.

1. The effects of mast cell degranulating peptide (MCDP), a toxin from the honey bee, and of dendrotoxin (DTX), a toxin from the green mamba snake, were studied in voltage-clamp experiments with myelinated nerve fibres of Xenopus. 2. MCDP and DTX blocked part of the K+ current. About 20% of the K+ current, however, was resistant to the toxins even in high concentrations. In Ringer solution half-maximal block was reached with concentrations of 33 nM-MCDP and 11 nM-DTX. In high-K+ solution the potency of both toxins was lower. beta-Bungarotoxin (beta-BuTX), another snake toxin, also blocked part of the K+ current, but was less potent than MCDP and DTX. 3. Tail currents in high-K+ solution were analysed and three K+ current components were separated according to Dubois (1981 b). Both MCDP and DTX selectively blocked a fast deactivating, slowly inactivating K+ current component which steeply activates between E = -60 mV and E = -40 mV (component f1). In concentrations around 100 nM, MCDP and DTX blocked neither the slow K+ current (component s) nor the fast deactivating, rapidly inactivating K+ current which activates between E = -40 mV and E = 20 mV (component f2). Similar results could be derived from K+ outward currents in Ringer solution. In high-K+, IC50 of MCDP for component f1 was 99 nM, whereas it was 7.6 microM for f2. Corresponding values for DTX are 68 nM and 1.8 microM. 4. Binding studies with nerve fibre membranes of Xenopus reveal high-affinity binding sites for 125I-labelled DTX (KD = 22 pM in Ringer solution and 81 pM in high-K+ solution). 125I-labelled DTX can be displaced from its sites completely by unlabelled DTX, toxin I (black mamba toxin), MCDP, and partially by beta-BuTX. 5. Immunocytochemical staining demonstrates that binding sites for DTX are present in nodal and paranodal regions of the axonal membrane. 6. The axonal membrane of motor and sensory nerve fibres is equipped with three types of well-characterized K+ channels and constitutes so far the best preparation to study MCDP- and DTX-sensitive K+ channels with electrophysiological and biochemical methods.

Long-term potentiation of synaptic transmission by synthetic mast cell degranulating(MCD) peptide.

A bee venom, mast cell degranulating peptide (MCD), was synthesized by stepwise formation of the two distinct disulfide bridges. This synthetic peptides with mast cell degrahulating activity MCD induced long-term potentiation (LTP) in the CAl region of hippocampus, while other peptides with mast cell degranulating activity apamin and mastoparan did not. This and other results using derivatives of the synthetic MCD suggest that a rigid structure is required for the LTP induction by MCD. Two antagonists of the N-methyl-D-aspartate (NMDA) receptor, AP5 and MK801, did not block the MCD induced-LTP. Moreover, the efficacy of the synaptic transmission in the slice which had been maximally activated by tetanic stimulation was further potentiated by MCD treatment. These results indicate that two quite distinct pathways for LTP formation exist in the CAl region of the hippocampus.

Mechanism of Induction of Long Term Potentiation by the Mast Cell Degranulating Peptide

The properties of Long Term Potentiation (LTP), which is a sustained enhancement of the synaptic response produced by a brief train of electrical stimulation, and its mechanisms are briefly reviewed. Bath application of the mast cell degranulating peptide (MCD) - a peptide isolated from bee venom- also produces in hippocampal slices LTP which is indistinguishable from that produced by a train of electrical stimulation (no change in postsynaptic cell properties, APV sensitive). In vivo push-pull experiments indicate that this LTP is not associated with a sustained enhancement of release of glutamate and aspartate, the two principle transmitters of the hippocampus, but there is a delayed enhanced release of proteins in the extracellular space which may contribute to the maintenance of the potentiation.

Analogies and differences in the mode of action and properties of binding sites (localization and mutual interactions) of two K + channel toxins, MCD peptide and dendrotoxin I

Both the bee venom toxin, mast cell degranulating (MCD) peptide, and the snake toxin, dendrotoxin I (DTX I) induce epileptiform activity and paroxystic seizures after intracerebroventricular (i.c.v.) injection to rats. Although many of the properties of the two toxins, which are blockers of the same K + channel, appear to be very similar, a number of differences have been found. (1) Induced seizures have an hippocampal origin for MCD and two different origins, situated in the cortex and in the limbic system, for DTX I. (2) A first i.c.v. administration of DTX I desensitizes against a second ipsilateral injection of the same peptide as we had previously observed for MCD. However no cross-desensitization was observed between the two different toxins. (3) The number of high affinity (K d= 41 pM) binding sites for 125I-DTX I in synaptic membranes is about 5 times higher than the number of high affinity 125I-DTX I affinity (K d= 158 pM) binding sites for 125I-MCD. (4) Autoradiographic analysis of the distribution of high affinity binding sites has been compared to our previous analysis of high affinity 125I-MCD binding sites. High levels of high affinity binding sites for both toxins seem to be localized in synaps -rich areas. However high affinity binding sites for the two toxins are not always co-localized. Analysis of the mutual interactions between DTX I and MCD binding sites has revealed the presence of classes of low affinity binding sites for MCD. In most areas of the brain, a large proportion of high affinity binding sites for DTX I is allosterically related to low affinity binding for MCD.