Inositol phospholipid hydrolysis may mediate the action of proctolin on insect visceral muscle

The formation of inositol phosphates in response to the neuropeptide proctolin was studied in locust oviducts. Glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate were identified in the locust oviducts using anion-exchange chromatography. Proctolin stimulated the release of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate during a 5-min incubation. In the presence of lithium ions the effects of proctolin were enhanced, with elevations of 98%, 42%, and 45% of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, respectively. Physiologically the effects of proctolin upon muscular contraction of locust oviducts were mimicked by the active phorbol ester, phorbol 12-myristate 13-acetate, and by the diacylglycerol analogue, 1-oleoyl-2-acetylglycerol. The inactive phorbol ester, 12-myristate 13-acetate 4-O-methyl ether, was without effect. The effects of the active phorbol ester and the diacylglycerol analogue were calcium-dependent requiring micromolar concentrations of calcium. The results indicate that the locust oviducts possess proctolin receptors that are linked to phosphatidylinositol metabolism and that inositol phospholipid hydrolysis may mediate the physiological action of proctolin.     

The effect of proctolin analogues and other peptides on locust oviduct muscle contractions

The locust oviduct bioassay system was used to assess the ability of a variety of peptides to induce oviducal contractions. Proctolin analogues were three orders of magnitude less potent than proctolin. Proctolin supra-analogue and Arg-Tyr-Leu-Ala-Thr demonstrated high activity. Perhaps the most significant finding was the discrepancy between the high binding capacity of the proctolin analogue Arg-Tyr-Ser-Pro-Thr and its relatively low myotropic activity. This observation argues for a crucial role for the leucine residue in activating the proctolin receptor. Several other myotropic peptides were tested for their effect on oviduct contractions. FMRFamide caused contractions at doses several orders of magnitude higher than proctolin. The FLRFamide leucomyosuppressin inhibited proctolin-induced contractions. In addition, myomodulin and catch relaxing peptide caused oviducal contractions at low concentrations. The enkephalins had no effect when applied alone but potentiated proctolin-induced oviduct contractions. The mechanism of the potentiation is not known. The data argue for the presence of several binding sites on the oviduct membrane.     

Characterization of proctolin binding sites on locust oviduct membranes

The binding of [³H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K_d of 400 ± 82 nM and a B_max of 23.7 ± 6.7 pmol/mg protein. The second had a K_d of 2.4 ± 0.2 μM and a B_max of 96.3 ± 16.7 pmo/mg protein.

Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [³]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.     

Comparative studies on the mode of action of proctolin and phorbol-12,13-dibutyrate in their ability to contract the locust mandibular closer muscle.

The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease-dissociated muscle cells. Both the phorbol ester, phorbol-12,13-dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1-10 nM and 10-100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1-1 nM for proctolin, and 0.1-1 pM for phorbol-12,13-dibutyrate. The effect of phorbol-12,13-dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of a protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly converging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx.     

The role of proctolin and glutamate in the excitation-contraction coupling of insect visceral muscle

Proctolin (1 X 10(-10) to 1 X 10(-9) M) had a minimal effect on the spontaneous and evoked electrical events of the hindgut of the cockroach Leucophea maderae. Spontaneous action potentials and contractile activity stopped when the hindgut was exposed to 2 mM Mn2+. Eighty per cent of the response of the hindgut to glutamate was blocked by manganese, but only 35% of the response to proctolin. Hindguts were responsive to proctolin in a calcium-free medium, but not to glutamate. Moreover, proctolin appeared to facilitate the reentry of calcium after depleted preparations were returned to normal levels of external calcium. The results offer evidence for two calcium transmembrane channels in insect visceral muscle.     

A cyclic GMP-dependent calcium-activated chloride current in smooth-muscle cells from rat mesenteric resistance arteries

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 microM BAY K8644, 0.4 microM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 microM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN- > Br- > I- > Cl- > acetate > F- > aspartate, but the conductance sequence was I- > Br- > Cl- > acetate > F- > aspartate = SCN-. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.     

Neuromodulator octopamine attenuates extrajunctional glutamate sensitivity in insect muscle

Octopamine was found to decrease extrajunctional, but not junctional glutamate responses, in mealworm neuromuscular preparations. This action of octopamine was mimicked by forskolin, 8-(4-chlorophenylthio)-adenosine 3′:5′-cyclic monophosphate (CPT-cyclic AMP), and 8-bromoguanosine 3′:5′-cyclic monophosphate (8-bromo-cyclic GMP), but not by 1,2-oleoylacetylglycerol (OAG), a protein kinase C activator. We suggest that the octopamine-induced reduction in the glutamate sensitivity of extrajunctional membranes may enable the muscle to more closely follow its neuronal input by preventing a depolarization (and hence a conductance increase) due to the discharge of unsequestered transmitter molecules at nonsynaptic sites.     

The action of iontophoretically applied L-glutamate on an insect visceral muscle

1) lontophoretic application of L-glutamate was employed to study the distribution of glutamate receptors in the superior longitudinal (SL) muscles of the locust (Locusta migratoria) hindgut, in which spontaneous activity was inhibited using normal saline containing 5 mM MgCl_2. 2) Junctional glutamate potentials with a rise time of 50–100 ms (peak) and a decay time of 250–400 ms were recorded at localized sites using ejection pulses in the range 5–10 nC. Most active sites were found in interfiber clefts and were spaced at about 250–300 μm intervals. 3) Desensitization of glutamate receptors occurred using ejection frequencies > 0.2 Hz. Desensitization could be irreversibly blocked using the lectin concanavalin A. 4) Depolarizing (D-) and biphasic depolarizing/hyperpofarizing (DH -) extrajunctional glutamate potentials were observed using ejection pulses > 15 nC. 5) δ-Philanthotoxin (δ-PTX) at concentrations > 0.3 Uml^?1 inhibited junctional glutamate potentials in a dose-dependent manner, 50% inhibition was achieved using 0.45 Uml^?1 δ-PTX. 6) Subthreshold concentrations of proctolin (up to 5 × 10^?10M) had no visible effect on glutamate potentials, suggesting that proctolin possibly does not act by modulating glutamate activity. 7) It is proposed that glutamate plays a transmitter role in SL muscles, while the role of proctolin is still unclear.     

Modulatory effects of proctolin on a crab ventilatory muscle

Proctolin enhances nerve-evoked, phasic contractions of a selected respiratory muscle of the shore crab, Carcinus maenas, but has no effect on muscle tonus. Proctolin also increases the work and power output of this muscle. These effects are functionally appropriate in view of previous reports that proctolin stimulates the ventilatory rhythm. They also suggest that proctolin exerts coordinated modulatory control at the central and peripheral levels of the gill ventilatory system. In contrast, serotonin, dopamine and octopamine have no effect on this muscle.     

Central effects of the peptides,SchistoFLRFamide and proctolin,on locust oviduct contraction

We have developed a semi-intact preparation-consisting of an isolated oviduct with abdominal ganglia VII and VIII intact and attached-with which to characterize the effects on oviduct contraction, of peptides that are bath applied to CNS tissues. The work presented here offers a qualitative analysis of the central effects of SchistoFLRFamide and proctolin upon action potentials recorded from the oviducal nerves and upon oviduct contraction. In the process of this, we hope to demonstrate that a previously characterized putative CNS SchistoFLRFamide receptor [Peptides 23 (2002) 765] is a functional receptor.SchistoFLRFamide (10(-6)M), bath applied to abdominal ganglion VII, caused an increase in action potential frequencies recorded from the oviducal nerves, as well as an increase in the frequency of phasic contractions of the oviduct. Although the function of this response is not known, these results further support the possibility that the putative CNS SchistoFLRFamide receptors are functional receptors.Proctolin (10(-6)M), bath applied to abdominal ganglion VIII, altered the rhythmic bursting of action potentials recorded from the oviducal nerve and changed the appearance and cycle duration of neurogenic oviduct contractions.     

Myogenic contractions in locust muscle induced by proctolin and by wasp, Philanthus triangulum, venom

Apparently myogenic slow contractions of the extensor tibiae of Locusta migratoria can be induced by proctolin in a concentration of 10^?10 to 10^?9 mole per liter perfusion fluid. Proctolin in a concentration of 10^?8 mole/l causes a prolonged contraction interrupted by rhythmical relaxations. Higher concentrations of proctolin cause a powerful but irreversible contraction.In some preparations in which proctolin is ineffective in a concentration of 10^?9 mole/l, a short stimulation of nerve 3b can initiate a series of rhythmic contractions. If, however, nerve 3b is stimulated at the peak of such a contraction a rapid relaxation is induced.Administration of the venom of Philanthus triangulum in a concentration which blocks the excitatory and inhibitory neuromuscular transmission, induces similar myogenic contractions. Stimulation of nerve 3b at the peak of such a contraction again causes a relaxation. Similar myogenic contractions can also be induced by administration of a homogenate of a locust.     

Distinct axonemal processes underlie spontaneous and stimulated airway ciliary activity

Cilia are small organelles protruding from the cell surface that beat synchronously, producing biological transport. Despite intense research for over a century, the mechanisms underlying ciliary beating are still not well understood. Even the nature of the cytosolic molecules required for spontaneous and stimulated beating is debatable. In an effort to resolve fundamental questions related to cilia beating, we developed a method that integrates the whole-cell mode of the patch-clamp technique with ciliary beat frequency measurements on a single cell. This method enables to control the composition of the intracellular solution while the cilia remain intact, thus providing a unique tool to simultaneously investigate the biochemical and physiological mechanism of ciliary beating. Thus far, we investigated whether the spontaneous and stimulated states of cilia beating are controlled by the same intracellular molecular mechanisms. It was found that: (a) MgATP was sufficient to support spontaneous beating. (b) Ca(2+) alone or Ca(2+)-calmodulin at concentrations as high as 1 microM could not alter ciliary beating. (c) In the absence of Ca(2+), cyclic nucleotides produced a moderate rise in ciliary beating while in the presence of Ca(2+) robust enhancement was observed. These results suggest that the axonemal machinery can function in at least two different modes.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon

Summary Using patch-clamp techniques, we have studied Ca²⁺-activated K⁺ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately –45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K⁺ over Na⁺. Channel activity correlated directly with bath Ca²⁺ concentration in the excised patches. Channel currents were blocked by 5mm TEA⁺ and 1mm Ba²⁺. In cell-attached patches, after addition of the Ca²⁺ ionophore A23187, which increases intracellular Ca²⁺, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca²⁺. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca²⁺ and cAMP.     

Mechanisms of action of pH-induced effects on vascular smooth muscle

It is clear that pH has many effects on vascular smooth muscle and the overall action of pH on force will depend on the type of vascular smooth muscle in question and the combined effects on all the potential modulatory mechanisms. The major effects of pH on force appear to be mediated via modulation of [Ca]_i rather than changes in the sensitivity of the contractile machinery to Ca²⁺. There are still numerous gaps in our understanding of the actions of pH and as more data become available, we will be able to better understand the major mechanisms involved. (Mol Cell Biochem 263: 163–172, 2004)     

Human bone cell cultures: a new model for studying the mechanism of action of calcitonin

An investigation on cell cultures obtained from temporal human bone fragments showed that they provide a suitable model for studying the mechanism involved in calcitonin action on bone cells. Furthermore they demonstrated: a transitory increase in 45Ca uptake that returned to control values ten minutes after the hormone was added; a relation between 45Ca uptake and increased cAMP concentrations when these were measured at the same time intervals; a reproduction of the salmon calcitonin (sCT) effect after incubation of the cultures with either db-cAMP or db-cGMP and inhibition of 45Ca uptake and parallel decrease in cAMP levels with propanol. These results suggest that in human bone cell cultures, sCT acts as a temporary promoter of 45Ca uptake, probably by activating an adenylate-cyclase system through a beta-receptor.     

The action of philanthotoxin-343 and photolabile analogues on locust (Schistocerca gregaria) muscle

The effects of philanthotoxin-343 (PhTX-343; tyrosyl-butanoyl-spermine) and photolabile analogues of this synthetic toxin on locust (Schistocerca gregaria) skeletal muscle have been investigated using whole muscle preparations (twitch contractions), single muscle fibres (excitatory postsynaptic currents (EPSCs)) and muscle membrane patches containing single quisqualate-sensitive glutamate receptors (qGluR). Analogues containing an azido group attached to either the butanoyl side-chain of PhTX-343 or as a substitute for the hydroxyl moiety of the tyrosyl residue were about 6 fold more potent antagonists than PhTX-343; those with an azido group located at the distal end of the toxin molecule were generally 2–3 fold less potent than PhTX-343. When these compounds were tested in subdued light, they were reversible antagonists of the muscle twitch, EPSC and qGluR. When a muscle was irradiated with U.V. during application of photolabile toxin combined with either neural stimulation of the muscle orl-glutamate application, antagonism of the twitch, EPSC and qGluR was complete and irreversible.     

Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance

Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4DeltaC) from which its C-terminal, with the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full-length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T-DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.     

Thermostable extracellular cyclic nucleotide phosphodiesterase of the Physarum polycephalum plasmodium

Cyclic nucleotide phosphodiesterase secreted by the Physarum polycephalum plasmodium was partially purified by ion-exchange chromatography on DEAE cellulose, ultrafiltration, and HPLC. The data obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing showed that the active enzyme in solution exists as a monomer of about 90 kDa with pI 3.6–4.0. The K _m values were 0.9 and 7.7 mM for cAMP and cGMP, respectively, whereas the maximal rates of hydrolysis of these nucleotides were virtually equal and reached several millimoles of hydrolyzed cyclic nucleotide per hour per milligram of enzyme. The partially purified enzyme was highly stable. It was not inactivated by heating at 100°C for 30 min. The enzyme remained active in the presence of 1% sodium dodecyl sulfate; however, it was completely inactivated under these conditions in the presence of β-mercaptoethanol.