Unique accumulation of neuropeptides in an insect: FMRFamide-related peptides in the cockroach, Periplaneta americana.

FMRFamides belong to the most extensively studied neuropeptides in invertebrates and exhibit diverse physiological effects on different target organs, such as muscles, intestine and the nervous system. This study on the American cockroach confirms for the first time that extended FMRFamides occur in non-dipteran insects. By means of tandem mass spectrometry, these neuropeptides were structurally elucidated, and sequence information was used for subsequent cloning of the cockroach FMRFamide gene. This precursor gene encodes for 24 putative peptides and shows sufficient similarity with the Drosophila FMRFamide gene. Of the 24 peptides, 23 were detected by mass spectrometric methods; it is the highest number of neuropeptide forms shown to be expressed from a single precursor in any insect. The expression was traced back to single neurons in the thoracic ganglia. The unique accumulation of these FMRFamide-related peptides in thoracic perisympathetic organs provides the definite evidence for a tagma-specific distribution of peptidergic neurohormones in neurohaemal release sites of the insect CNS. Excitatory effects of the cockroach FMRFamides were observed on antenna-heart preparations. In addition, the newly described FMRFamides reduce the spike frequency of dorsal-unpaired median neurons and reduce the intracellular calcium concentration, which may affect the peripheral release of the biogenic amine octopamine.

The large conductance Ca2+-activated potassium channel (pSlo) of the cockroach Periplaneta americana: structure, localization in neurons and electrophysiology.

Voltage-activated, Ca2+-sensitive K+ channels (BK or maxi K,Ca channels) play a major role in the control of neuronal excitability. We have cloned pSlo, the BK channel alpha subunit of the cockroach Periplaneta americana. The amino acid sequence of pSlo shows 88% identity to dSlo from Drosophila. There are five alternatively spliced positions in pSlo showing differential expression in various tissues. A pSlo-specific antibody prominently stained the octopaminergic dorsal unpaired median (DUM) neurons and peptidergic midline neurons in Periplaneta abdominal ganglia. HEK293 cells expressing pSlo exhibit K+ channels of 170 pS conductance. They have a tendency for brief closures, exhibit subconductance states and show slight inward rectification. Activation kinetics and voltage dependence are controlled by cytoplasmic [Ca2+]. In contrast to dSlo, pSlo channels are sensitive to charybdotoxin and iberiotoxin. Mutagenesis at two positions (E254 and Q285) changed blocking efficacy of charybdotoxin. In contrast to pSlo expressed in HEK293 cells, native IbTx-sensitive K,Ca currents in DUM and in peptidergic neurons, exhibited rapid, partial inactivation. The fast component of the K,Ca current partly accounts for the repolarization and the early after-hyperpolarization of the action potential. By means of Ca2+-induced repolarization, BK channels may reduce the risk of Ca2+ overload in cockroach neurons. Interestingly, the neurons expressing pSlo were also found to express taurine, a messenger that is likely to limit overexcitation by an autocrine mechanism in mammalian central neurons.

Peptidergic modulation of insect voltage-gated Ca(2+) currents: role of resting Ca(2+) current and protein kinases A and C.

The modulation of voltage-gated Ca(2+) currents in isolated dorsal unpaired median (DUM) neurons of cockroach was investigated using whole cell patch clamp. The neuropeptide neurohormone D (NHD), a member of the adipokinetic hormone family, affected Ca(2+) currents at pico- to nanomolar concentrations. It strongly enhanced currents activating at lower depolarizations, whereas those activating at strong depolarizations were slightly attenuated. The first effect results from upregulation of a previously characterized omega-conotoxin MVIIC- and omega-agatoxin IVA-sensitive "mid/low voltage-activated" (M-LVA) Ca(2+) current. The cAMP-analogue 8-bromo-cAMP, forskolin, and the catalytic subunit of protein kinase A (PKA) mimicked the stimulating action of NHD. In addition, preincubation of neurons with the PKA inhibitor KT 5720 abolished the action of NHD. Thus NHD seems to upregulate the M-LVA current via channel phosphorylation by PKA. Activation of protein kinase C by oleoylacetylglycerol (OAG) mimicked the effect of NHD, and subsequent NHD application only enhanced the current to a moderate extent. On the other hand, inhibition of protein kinase C (PKC) by Gö 6976 abolished the NHD effect. These results indicate that also PKC, too, may play a role in the peptidergic modulation of the M-LVA Ca(2+) current. The reduction of Ca(2+) currents in the high-voltage-range is caused by the NHD-induced upregulation of a voltage-independent Ca(2+) resting current, I(Ca,R), which most probably leads to enhanced Ca(2+)-dependent inactivation of voltage-gated Ca(2+) currents. To assess the major consequences of the Ca(2+) current changes, current-clamp investigations were performed. Experiments with iberiotoxin, a specific blocker of BK-type Ca(2+)-dependent K(+) currents, and the M-LVA current-blocking omega-toxins suggested that NHD causes-via increasing Ca(2+)-dependent K(+) currents-a larger hyperpolarization of action potentials. The lowering in the action potential threshold produced by NHD, however, seems to be a direct consequence of the hyperpolarizing shift of the activation curve of total Ca(2+) current resulting from NHD-induced upregulation of the M-LVA current component.

Calcium-induced calcium release in neurosecretory insect neurons: fast and slow responses.

The dynamics of intracellular free Ca(2+)([Ca(2+)](i)) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca(2+) channels caused a steep increase in [Ca(2+)](i). Depolarizations lasting for < 100ms led to Ca(2+) release from intracellular stores as is indicated by the finding that the rise of [Ca(2+)](i) was greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca(2+)current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca(2+) influx enhanced in this way again caused a rise of [Ca(2+)](i) sensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca(2+)responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca(2+) responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca(2+) rises after having been treated with NHD. Thus, the variability of Ca(2+) signals might be caused by different filling states of Ca(2+) stores, and the resting Ca(2+) current seems to represent a source to fill empty Ca(2+) stores. In line with this notion, block of the endoplasmic Ca(2+) pump by thapsigargin (1 microM) produced either no or largely varying Ca(2+) responses. The Ca(2+) signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca(2+) release channels. Block of mitochondrial Ca(2+) uptake with carbonyl cyanide m -chlorophenylhydrazone (CCCP, 1 microM) completely abolished cADPR-induced Ca(2+) signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca(2+) uptake pathways and that some of these pathways involve mitochondria.

Non-synaptic ion channels in insects--basic properties of currents and their modulation in neurons and skeletal muscles.

Insects are favoured objects for studying information processing in restricted neuronal networks, e.g. motor pattern generation or sensory perception. The analysis of the underlying processes requires knowledge of the electrical properties of the cells involved. These properties are determined by the expression pattern of ionic channels and by the regulation of their function, e.g. by neuromodulators. We here review the presently available knowledge on insect non-synaptic ion channels and ionic currents in neurons and skeletal muscles. The first part of this article covers genetic and structural informations, the localization of channels, their electrophysiological and pharmacological properties, and known effects of second messengers and modulators such as neuropeptides or biogenic amines. In a second part we describe in detail modulation of ionic currents in three particularly well investigated preparations, i.e. Drosophila photoreceptor, cockroach DUM (dorsal unpaired median) neuron and locust jumping muscle. Ion channel structures are almost exclusively known for the fruitfly Drosophila, and most of the information on their function has also been obtained in this animal, mainly based on mutational analysis and investigation of heterologously expressed channels. Now the entire genome of Drosophila has been sequenced, it seems almost completely known which types of channel genes--and how many of them--exist in this animal. There is much knowledge of the various types of channels formed by 6-transmembrane--spanning segments (6TM channels) including those where four 6TM domains are joined within one large protein (e.g. classical Na+ channel). In comparison, two TM channels and 4TM (or tandem) channels so far have hardly been explored. There are, however, various well characterized ionic conductances, e.g. for Ca2+, Cl- or K+, in other insect preparations for which the channels are not yet known. In some of the larger insects, i.e. bee, cockroach, locust and moth, rather detailed information has been established on the role of ionic currents in certain physiological or behavioural contexts. On the whole, however, knowledge of non-synaptic ion channels in such insects is still fragmentary. Modulation of ion currents usually involves activation of more or less elaborate signal transduction cascades. The three detailed examples for modulation presented in the second part indicate, amongst other things, that one type of modulator usually leads to concerted changes of several ion currents and that the effects of different modulators in one type of cell may overlap. Modulators participate in the adaptive changes of the various cells responsible for different physiological or behavioural states. Further study of their effects on the single cell level should help to understand how small sets of cells cooperate in order to produce the appropriate output.

Peptidergic modulation of an insect Na(+) current: role of protein kinase A and protein kinase C.

The modulation of voltage-gated Na(+) currents in isolated somata of dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana was investigated using the patch-clamp technique. The neuropeptide Neurohormone D (NHD), which belongs to the family of adipokinetic hormones, reversibly reduced the Na(+) current in concentration-dependent manner (1 pM to 10 nM). At 10 nM, NHD caused an attenuation of the maximum of current-voltage (I-V) relation for peak currents by 23 +/- 6%. An analysis of NHD action on current kinetics in terms of the Hodgkin-Huxley formalism revealed that NHD reduces the time constant of inactivation, whereas steady-state activation and inactivation as well as the time constant of activation were not affected. In addition, NHD prolonged the recovery from inactivation. The cAMP analogue 8-bromo-cAMP, forskolin, and the catalytic subunit of protein kinase A mimicked the action of NHD. Furthermore, preincubation of cells with the protein kinase A inhibitor KT 5720 abolished the action of NHD. Thus NHD seems to modify the Na(+) current via channel phosphorylation by protein kinase A. Activation of protein kinase C by oleoylacetylglycerol (OAG) also reduced the Na(+) current, but it did not occlude the action of NHD. On the other hand, inhibition of protein kinase C by chelerythrine or Gö 6976 did not essentially impair the NHD effects.

Ca2+ resting current and Ca2+-induced Ca2+ release in insect neurosecretory neurons.

The effect of a Ca2+ resting current on spiking and intracellular Ca2+ concentration [Ca2+]i of isolated cockroach dorsal unpaired median neurons was investigated by combining microfluometry (Fura2) with patchclamping. The resting current was reduced by 10 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 10 microM mefenamic acid, which caused slower spiking of cells. Up-modulation of the resting current by neurohormone D (NHD, 100 fM to 10 pM) transiently increased [Ca2+]i. This Ca2+ transient was largely reduced by the ryanodine receptor antagonist dantrolene (10 microM) but it was not reduced by the IP3-receptor antagonist heparin which indicates Ca2+-induced Ca2+ release. The NHD-effect on [Ca2+]i was strongly attenuated by NPPB and mefenamic acid. Thus, NHD caused Ca2+-induced Ca2+ release by enhancing the Ca2+ resting current.

omega-Toxins affect Na+ currents in neurosecretory insect neurons.

omega-toxins specifically block certain Ca2+ channels in mammalian neurons as well as in dorsal unpaired median neurons isolated from the cockroach Periplaneta americana. In these cockroach neurons both the P/Q-type blockers omega-agatoxin IVA and omega-conotoxin MVIIC but not the N-type Ca2+ channel blocker omega-conotoxin GVIA affected fast Na+ currents sensitive to tetrodotoxin and veratridine. Both omega-toxins enhanced Na+ current decay and thus decreased the amplitudes of the peak currents. They also led to a slower recovery from inactivation. Toxin effects developing within a few min were ot removed upon washing. They were not use-dependent. The description of the effect of omega-conotoxin MVIIC on current kinetics in terms of the Hodgkin-Huxley model revealed that steady-state parameters were not affected whereas the time constant of inactivation was considerably reduced. Under control conditions, the inactivation time constant is similar to the time constant of recovery from inactivation. The toxin-induced increase of the latter time constant and the decrease of the inactivation time constant indicate that inactivation can no longer be described by first-order kinetics.

Ca2+ currents in central insect neurons: electrophysiological and pharmacological properties.

Ca2+ currents in dorsal unpaired median (DUM) neurons isolated from the fifth abdominal ganglion of the cockroach Periplaneta americana were investigated with the whole cell patch-clamp technique. On the basis of kinetic and pharmacological properties, two different Ca2+ currents were separated in these cells: mid/low-voltage-activated (M-LVA) currents and high-voltage-activated (HVA) currents. M-LVA currents had an activation threshold of -50 mV and reached maximal peak values at -10 mV. They were sensitive to depolarized holding potentials and decayed very rapidly. The decay was largely Ca2+ dependent. M-LVA currents were effectively blocked by Cd2+ median inhibiting concentration (IC50 = 9 microM), but they also had a remarkable sensitivity to Ni2+ (IC50 = 19 microM). M-LVA currents were insensitive to vertebrate LVA channel blockers like flunarizine and amiloride. The currents were, however, potently blocked by omega-conotoxin MVIIC (1 microM) and omega-agatoxin IVA (50 nM). The blocking effects of omega-toxins developed fast (time constant tau = 15 s) and were fully reversible after wash. HVA currents activated positive to -30 mV and showed maximal peak currents at + 10 mV. They were resistant to depolarized holding potentials up to -50 mV and decayed in a less pronounced manner than M-LVA currents. HVA currents were potently blocked by Cd2+ (IC50 = 5 microM) but less affected by Ni2+ (IC50 = 40 microM). These currents were reduced by phenylalkylamines like verapamil (10 microM) and benzothiazepines like diltiazem (10 microM), but they were insensitive to dihydropyridines like nifedipine (10 microM) and BAY K 8644 (10 microM). Furthermore, HVA currents were sensitive to omega-conotoxin GVIA (1 microM). The toxin-induced reduction of currents appeared slowly (tau approximately 120 s) and the recovery after wash was incomplete in most cases. The dihydropyridine insensitivity of the phenylalkylamine-sensitive HVA currents is a property the cockroach DUM cells share with other invertebrate neurons. Compared with Ca2+ currents in vertebrates, the DUM neuron current differ considerably from the presently known types. Although there are some similarities concerning kinetics, the pharmacological profile of the cockroach Ca2+ currents especially is very different from profiles already described for vertebrate currents.

Octopamine modulates ionic currents and spiking in dorsal unpaired median (DUM) neurons.

Modulatory effects of octopamine on ionic currents and spiking in isolated cockroach dorsal unpaired median neurons were investigated by means of the chopped voltage-/current-clamp and the patch-clamp technique. Octopamine increased the spiking frequency at concentrations < or = 10 microM and reduced it at > 10 microM. It enhanced a low voltage-activated Ca2+ current at 1 to 100 microM. At concentrations up to 10 microM two components of Ca2+-activated K+ current were potentiated. At > 10 microM octopamine in addition reduced a high voltage-activated Ca2+ current and the Ca2+-activated K+ current. A membrane permeant cAMP-analogue imitated the effects obtained at high octopamine concentration. Octopamine and cAMP in addition reduced a depolarizing resting current at both low and high concentration. Possible mechanisms and physiological significance are discussed for the opposite effects of octopamine observed at low versus high concentrations.

Ca2+ currents in cockroach neurones: properties and modulation by neurohormone D.

In dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana two types of Ca2+ currents were found: a transient one activated by lower depolarizations (LVA) and a sustained one activated by higher voltage jumps (HVA). Both currents were strongly reduced by 100 microM Cd2+; Ni2+ was less effective. The HVA currents were insensitive to dihydropyridines, but sensitive to phenylalkylamines. The LVA component was sensitive to omega-conotoxin GVIA and SK&F96365 whereas the HVA component was insensitive to both blockers. The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), is known to increase the spiking frequence in these DUM cells. We demonstrate that NHD increases the LVA current component and decreases the HVA current component. These effects of NHD can explain the previously found NHD-induced stimulation of Ca2+ dependent K+ currents.

Neurohormone D induces ionic current changes in cockroach central neurones.

The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), increases the frequency of spontaneous activity in dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana. The increase in spike frequency is accompanied by changes in the shape and the amplitude of the single action potentials, e.g. a more pronounced afterhyperpolarization. Effects of NHD on membrane currents were investigated in these DUM cells with whole-cell voltage-clamp measurements. A voltage-independent Ca2+ current flowing at the resting potential (ICa,R) was found. NHD, at nanomolar concentrations, enhanced this ICa,R in a concentration-dependent manner. 0.1 mM Cd2+ markedly reduced ICa,R and in this case ICa,R was hardly potentiated by NHD. In the presence of NHD a fast activating Ca(2+)-dependent K+ current sensitive to charybdotoxin and to low concentrations of tetraethylammonium was augmented. The enhanced afterhyperpolarization of action potentials can be accounted for by the increase in the Ca(2+)-dependent K+ current. The changes of the membrane currents induced by NHD are discussed with respect to further effects on the spike pattern and in relation to the previously described mode of action of AKH-peptides in other preparations.

Neurohormone D increases the intracellular Ca2+ level in cockroach neurones through a Cd(2+)-sensitive Ca2+ influx.

The modulating effect of the octapeptide neurohormone D (NHD) on the intracellular calcium level [Ca2+]i of neurones from the dorsal midline of the cockroach terminal ganglion was investigated with fluorescence measurements. [Ca2+]i of cells loaded with Fura 2 was determined by photon counting and imaging at wavelengths of 340 and 380 nm. After application of NHD, [Ca2+]i increased within 3 min from a value of 93 +/- 36 nM to 153 +/- 51 nM, corresponding to an enhancement to 164 +/- 35%. In Ca(2+)-free solution, [Ca2+]i was lowered (52 +/- 6 nM) and NHD no longer affected the intracellular calcium level. The presence of 0.1 mM Cd2+, in normal saline, prevented the NHD-induced increase of [Ca2+]i. The results were explained by postulating a Ca2+ resting current in these cells which is augmented by NHD.