R.G.H. Downer

Lipophorins, a major class of lipoproteins of insect haemolymph

Abstract The name Lipophorin (Gk. lipos , fat; phoros , bearing) is proposed as a generic term for the class of insect haemolymph lipoproteins that serve to transport lipids between organs of absorption, storage and utilisation.

The Effects of Eicosanoid Biosynthesis Inhibitors On Prophenoloxidase Activation, Phagocytosis and Cell Spreading in Galleria mellonella.

The invertebrate immune system produces melanotic nodules in response to bacterial infections and this has previously been shown to be mediated by eicosanoids. Nodulation occurs in two phases: the first involves hemocyte degranulation and activation of the prophenoloxidase cascade; the second involves formation of a cellular capsule by attachment and spreading of hemocytes. We demonstrate that inhibitors of eicosanoid biosynthesis affect both of these phases of nodulation in Galleria mellonella. The phospholipase A(2) inhibitor, dexamethasone, as well as the cyclooxygenase inhibitor, indomethacin, significantly inhibit phagocytosis in vitro and prophenoloxidase activation in vivo. The inhibitory effects of dexamethasone were abolished by the addition of exogenous arachidonic acid. Furthermore, 5,8,11,14- eicosatetraynoic acid, dexamethasone and indomethacin inhibit hemocyte spreading in vitro. The findings support the idea that eicosanoid derivatives mediate both phases of the nodulation response and are consistent with previous studies which attribute roles for eicosanoids in other species as modulators of cell activity.

In vitro studies on the effects of octopamine on locust fat body.

Abstract The effects of octopamine on the fat body of locusts in vitro have been examined. The isolated fat body responded to octopamine by releasing lipid in a dose-dependent manner. Maximal stimulation was obtained with 5 × 10−6 M octopamine. The octopamine stimulated release of lipid was blocked by α-adrenergic receptor antagonists but unaffected by β-adrenergic receptor antagonists. Octopamine was more potent than synephrine, dopamine, or tyramine in eliciting the response. Octopamine (5 × 10−6 M) in the presence of IBMX, resulted in a six- to sevenfold elevation of cyclic AMP in the fat body; this response was also blocked by α- but not β-adrenergic receptor antagonists. Cyclic GMP levels were unaffected by octopamine. Adipokinetic hormone (AKH) in the presence of IBMX, also stimulated an elevation in cyclic AMP in the fat body, and stimulated the release of lipid. Unlike the octopamine stimulation, however, the AKH effect was not inhibited by the α-adrenergic receptor antagonists. Furthermore, AKH inhibited protein synthetic activity of fat body, while octopamine was without effect. It is concluded that a release of lipid from fat body of locusts is probably mediated by a rapid activation of an octopamine-sensitive adenylate cyclase. Furthermore, the mechanism of action and the receptors for octopamine and AKH are different.

Induction of hypertrehalosemia by excitation in Periplaneta americana.

An excitation-induced hypertrehalosemic (EXIT) response has been demostrated in the American cockroach, Periplaneta americana. The response occurs in normal insects and in insects that were ligated at the neck 24 hr prior to experimentation. Several known and putative insect neurotransmitters were tested for their ability to elicit rapidly a hypertrehalosemic state in the cockroach. Octopamine was the most effective hypertrehalosemic agent, and produced a 100% increase in haemolymph trehalose levels within 15 min of being injected into the haemocoel. Less pronounced hypertrehalosemic responses were obtained with dopamine and tyramine, but not with synephrine or 5-hydroxytryptamine. Hypertrehalosemia was observed also with normetanephrine, but not with isoproterenol. A slight inhibition of the EXIT response occurred with the β-blocking agents, propranolol and dichloroisoproterenol, but the monoamine oxidase inhibitors, iproniazid phosphate and nialamide, were without effect. It is suggested that the EXIT-response may be one of several octopamine-mediated responses to excitation in the insect.

Octopamine and 5-hydroxytryptamine enhance the phagocytic and nodule formation activities of cockroach (Periplaneta americana) haemocytes

Abstract The effects of 5-hydroxytryptamine, octopamine and dopamine on the phagocytic and nodule formation activities of cockroach haemocytes have been investigated. Octopamine and 5-hydroxytryptamine (5-HT) increase phagocytosis in vitro with a maximal effect at 10−6 and 10−8 M, respectively. Dopamine also increases phagocytosis, but only at high concentrations (10−4 M). Antagonist and agonist studies for phagocytosis show that both 5-HT- and octopamine-sensitive receptors are present in cockroach haemocytes. Survival of cockroaches exposed to a LD50 dose of Staphylococcus aureus is enhanced in the presence of 5-HT and octopamine while dopamine has no effect. Antagonist and agonist stuides of cockroach survival support the results obtained in vitro, suggesting that both octopamine- and 5-HT-sensitive receptors are found in cockroach haemocytes and these are involved in the recovery of cockroaches from bacterial infection. Examination of the haemocoel and haemocytes of treated cockroaches showed that at least two mechanisms by which 5-HT and octopamine are able to increase survival is through the elevation of phagocytosis and nodule formation.

Trehalose production in isolated fat body of the american cockroach, Periplaneta americana

Abstract 1. The trehalogenic capacity of incubating fat body tissue may be expressed in terms of the trehalogenic index (Trehalose produced during incubation/Trehalose produced + Glycogen remaining after incubation). 2. Pieces of fat body from the same individual have comparable trehalogenic indexes. 3. The trehalogenic index is relatively independent of pH flux in the incubating medium. 4. The trehalogenic index is elevated by calcium, but not by magnesium, whereas potassium ions tend to depress the trehalogenic index. 5. Aqueous extracts of corpora cardiaca, 10 −4 M octopamine and 10 −4 M dopamine increase the trehalogenic index, whereas 10 −4 M tyramine causes the trehalogenic index to decrease.

Octopamine, a modulator of the haemocytic nodulation response of non-immune Galleria mellonella larvae

Octopamine (1) injected into Galleria mellonella induced changes in the haemograms of the larvae identical to those in insects infected with Bacillus cereus, (2) was elevated during bacterial infection in vivo, and (3) accelerated the removal of microorganisms from the haemolymph. The modulation of these non-self responses by octopamine was independent of prophenoloxidase activation. Octopamine in vitro bound to the surface of the insect pathogens B. cereus and Xenorhabdus nematophilus. Injecting the octopamine-coated bacteria accelerated their removal from haemolymph over a 30 min post-injection period. It is proposed that octopamine is a bifunctional molecule acting as both a modulator of haemocyte activity and mediating bacterial attachment to haemocytes.

Characterization of tyramine and octopamine receptors in the insect (Locusta migratoria migratorioides) brain

The kinetic and pharmacological properties of [3H]tyramine and [3H]octopamine binding to membrane preparations of locust (Locusta migratoria migratorioides) brain were studied to characterize the tyramine and octopamine receptors. [3H]Tyramine and [3H]octopamine bind specifically and reversibly to the locust brain membrane with equilibrium achieved after 20 min. The dissociation of [3H]tyramine is monophasic while that of the [3H]octopamine shows a biphasic tendency. Scatchard analysis of the saturation curves reveals a single high affinity binding site for each of tyramine and octopamine. The mean (+/- S.E.M.) values of Kd and Bmax are 6.11 +/- 0.71 nM and 21.45 +/- 3.0 fmol/mg tissue for tyramine and 5.65 +/- 0.91 nM and 15.0 +/- 2.4 fmol/mg tissue for octopamine, respectively. Pharmacological analysis of the binding suggests the presence of both tyramine and octopamine receptors in the locust brain. alpha-Adrenergic agonists and antagonists have a high affinity to the octopamine but not the tyramine receptor whereas dopaminergic drugs have a higher affinity to the tyramine receptor than the octopamine receptor. No highly effective inhibitors of tyramine binding were identified. The serotonergic blockers, mianserin, LSD, BOL are effective blockers for both tyramine and octopamine receptors, whereas the serotonergic antagonist gramine is more active against the octopamine than the serotonin receptor. The results suggest that a G-protein binding mechanism is involved in the expression of both the tyramine and octopamine effects.

The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach

Abstract Incubation of corpora cardiaca from adult male Periplaneta americana in the presence of octopamine results in elevated tissue levels of cyclic AMP. The octopamine-induced elevation of cyclic AMP is partially blocked by phentolamine, gramine and cyproheptadine but not by propranolol. Dopamine and 5-hydroxytryptamine also increase cyclic AMP levels in the corpus cardiacum and additivity studies indicate that separate octopamine- and dopamine-binding sites are present within the tissue. Cyclic AMP levels in the corpus cardiacum also increase in response to electrical stimulation of nervi corporis cardiaci II (NCC II) and the electrically induced effect is eliminated in the presence of phentolamine. A factor, which causes elevated haemolymph trehalose levels when injected into adult cockroaches, is released from corpora cardiaca incubated in the presence of octopamine. The active factor is denatured by incubation in the presence of pronase. The hypertrehalosemic factor is also released when corpora cardiaca are incubated in the presence of dibutyryl cyclic AMP or 40 mM potassium chloride; however dopamine and 5-hydroxytryptamine fail to effect a marked release of the hypertrehalosemic factor. The results are discussed in light of the proposal that the release of hypertrehalosemic hormone from corpora cardiaca is regulated by octopaminergic neurones contained within NCC II.

Pharmacology of aminergic receptors mediating an elevation in cyclic AMP and release of hormone from locust neurosecretory cells.

Synaptic activation of the neurosecretory cells within the glandular lobe of the corpus cardiacum of locusts results in the release of bioassayable adipokinetic hormones and an elevation in cAMP. The effectiveness of several putative aminergic neurotransmitters in mimicking these actions of the natural transmitter, and the effects of aminergic antagonists, have been studied. The receptors mediating the release of adipokinetic hormones exhibited a specificity for the monophenolic amines octopamine and synephrine at 10(-7) M. These two amines were also the most effective, at 5 X 10(-6) M, in elevating cAMP levels. Octopamine induced a dose-dependent elevation in cAMP with half-maximal stimulation occurring at 5 X 10(-6) M. 5-Hydroxytryptamine (5-HT) was also capable of elevating cAMP levels, but, unlike the octopamine response, or the response to the natural transmitter, the response to 5-HT was not blocked by the antagonist phentolamine. Gramine was also an effective antagonist of the octopamine-induced response. The results are consistent with the hypothesis that octopamine is the natural transmitter within this neurosecretory system.