Pharmacological characterization of dopamine-sensitive adenylate cyclase in the salivary glands of Locusta migratoria L.

The locust (Locusta migratoria) salivary gland adenylate cyclase was stimulated by both dopamine and serotonin (6 and 5-fold stimulation respectively). Since their effects were additive, it is concluded that these neurotransmitters are acting on two discrete receptor types.The agonist and antagonist specificity profiles were very similar to those of the D₁ receptor coupled with an adenylate cyclase in vertebrates. They were clearly different from those of the D₂ receptor of vertebrates. No evidence for octopamine sensitive adenylate cyclase was found in the salivary glands of L. migratoria.In addition to previous electrophysiological and histological studies (House and Ginsborg, 1979) these results suggest that dopamine has a neurotransmitter function in the salivary glands of locusts and that its function is mediated by cyclic AMP.     

Modulation of quail oviduct adenylate cyclase activity by estradiol and progesterone

Oviduct adenylate cyclase activity of the quail was measured by radiochemical analysis following different hormonal treatments. A single injection of estradiol benzoate (EB) to immature female quails resulted in a prereplicative surge of adenylate cyclase activity. A second surge of enzyme activity was observed during the proliferative phase induced by EB. Estradiol-17 alpha, estrone, estriol and testosterone were ineffective. Tamoxifen completely inhibits the growth-promoting effect of EB and the second surge of adenylate cyclase activity but does not inhibit the prereplicative increase of enzyme activity. This prereplicative increase of adenylate cyclase activity was also observed, even in the absence of increased plasma estradiol, when estradiol-17 beta (E2) was perfused through the hepatic portal vein. Moreover, E2 had no effect on enzyme activity when added directly to the oviduct homogenate preparation, at concentrations ranging from 10(-9) to 10(-7) M. In response to progesterone injection, oviduct adenylate cyclase activity followed a different pattern, beginning its increase after 3 h and remaining elevated up to 24 h. The activation by estradiol was independent of the presence of guanylylimidodiphosphate. Moreover, the enzyme was more sensitive to forskolin at submaximal concentration in estradiol treated birds than in control. These results demonstrate that transient activation of adenylate cyclase at the early stages of the action of estradiol does not occur through the classic nuclear receptor-gene activation pathway or a membrane receptor mediated process, but involves an indirect pathway, yet to be defined.     

Stimulatory and inhibitory effects of guanyl-5'-yl imidodiphosphate on adenylate cyclase activity of cardiac sarcolemma.

A nucleotide phosphohydrolase-resistant analog of GTP, guanyl-5′-yl imidodiphosphate [GMP-P(NH)P], caused stimulation of basal adenylate cyclase activity of cardiac sarcolemma when ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) was absent in the assay mixture, whereas the nucleotide, in the presence of EGTA, inhibited basal cyclase activity. GTP, GDP, GMP, and guanosine failed to show such an inhibition of basal enzyme activity. The degree of both stimulatory and inhibitory effects of GMP-P(NH)P depended on the concentration of magnesium ions. The apparent affinities toward magnesium ions of the metal binding site and toward MgATP^2? of the catalytic site of control and ?GMP-P(NH)P-inhibited” enzyme were similar. Isoproterenol reversed the inhibitory effect, whereas calcium ions failed to revert it. Both in the presence and absence of EGTA, GMP-P(NH)P plus isoproterenol caused a synergistic stimulation of the enzyme activity, the degree of stimulation being lower with EGTA present. Exposure of sarcolemma to GMP-P(NH)P (with and without isoproterenol and in the absence and presence of EGTA) caused an activation of adenylate cyclase, the degree of activation being higher with isoproterenol present. The activated enzyme displayed increased affinity toward Mg²⁺ at the metal binding site. When activated enzyme preparations were assayed in the presence of EGTA, reversal of the activated state was observed in the case of the GMP-P(NH)P-activated enzyme but not in the case of the GMP-P(NH)P + isoproterenol-activated enzyme.     

Cytochemical localization of adenylate cyclase in the various tissues of Locusta migratoria (migratorioides R.F.)

Summary The ultrastructural cytochemical procedure to demonstrate adenyl cyclase in mammalian organs was used in insects. After several modifications, an utilizable method was applied for the detection of the enzyme in the various tissues. Adenylate cyclase which can be stimulated with octopamine was localized on the membrane of the glial cells and the axolemma of certain large axons in the insect brain. Adenylate cyclase which could be activated by NaF and isoproterenol was also demonstrated in the lipid droplets of glial cells of the brain. With the simultaneous application of NaF and isoproterenol, rather strong adenylate cyclase activity could be detected on the surface of the corpora allata cells both in the cells situated on the glandular surface and the central part of the gland. In contrast in the corpus cardiacum enzyme activity was only observable on the basal lamina of the glandular surface. An appreciable amount of reaction product, indicating the presence of the enzyme, could be found on the surface of the lipid droplets in the fat body situated near the glandular tissues. In the heart muscle, reaction product referring to enzyme activation could not be demonstrated with the help of the methods applied.     

Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum

Detailed analysis of the action of prostaglandins (PGs) on the corpus luteum in primate species is very limited. In this study we examined the response of the adenylate cyclase system to PGs in homogenates prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. The conversion of [alpha 32p] ATP to [32p] cyclic AMP (cAMP) was assessed in the absence (control activity; 50 microM GTP) and presence of various concentrations of seven PGs and arachidonic acid, either alone or in combination with 250 nM hCG. Cyclic AMP production increased up to three-fold in the presence of PGD2, PGE2, PGI2 or PGF2 alpha; however PGA2, PGB2, 13, 14-dihydro-15-keto PGE2 and arachidonic acid alone did not alter cAMP levels. In dose-response studies, adenylate cyclase was 10 and 100-fold more sensitive to PGD2 (Vmax at 1 X 10(-5) M) than to PGE2 or to PGI2 and PGF2 alpha, respectively. Activity in the presence of hCG plus either PGD2, PGE2, PGI2 or PGF2 alpha did not differ from that for hCG (or the PG) alone. In contrast, addition of PGA2 or arachidonate inhibited (p less than 0.05) hCG-stimulated cAMP production by 50 and 100 percent. We conclude that the gonadotropin-sensitive adenylate cyclase of the macaque corpus luteum is also modulated by several PGs. These factors may either mimic (e.g., PGD2, PGE2, PGI2) or suppress (PGA2) gonadotropin-stimulated cAMP production and possibly cAMP-mediated events in luteal cells.     

Ultrastructural and immunocytochemical studies of neuromuscular junctions in oviduct of Locusta migratoria

The ultrastructure of nerve endings in the oviduct visceral muscles of Locusta migratoria was studied by electron microscopy and by immunogold labeling for two kinds of neuromodulators, the pentapeptide proctolin and FMRFamide-related peptides. Nerve endings contained electron-lucent round vesicles and two kinds of granules (round and avoid), and formed two types of synapses or release sites with the muscle. The morphologically distinct nerve endings were classified into three different categories based on the composition of synaptic vesicles and granules. Type-I nerve endings were dominated by electron-lucent round vesicles and contained only a few round electron-dense granules. Type-II nerve endings contained mostly electron-dense round granules and electron-lucent round vesicles. A few electron-dense ovoid granules were also present. Electron-dense ovoid granules dominated the type-III nerve endings, which usually contained less electron-lucent vesicles than either type-I or II nerve endings. Both proctolin and FMRFamide-like immunoreactivity was associated with electron-dense round granules. However, FMRFamide-like immunoreactivity was only found in the type-II nerve endings, while proctolin immunoreactivity was found within type-I nerve endings as well as in some type-II nerve endings. Immunological results therefore allow us to further divide type-II nerve endings into type-IIa (immunonegative for proctolin) and type-IIb (immunopositive for proctolin). Type-III nerve endings show no immunolabeling to either proctolin or FMRFamide.     

Spontaneous and neurally evoked contractions of visceral muscles in the oviduct of Locusta migratoria

The oviducts of Locusta migratoria are innervated by a pair of nerves which arise from, the seventh abdominal ganglion. A distinctive network of striated muscle fibres occurs in the oviducts. The lateral oviducts and common oviduct consist of an inner circular layer of muscle and an outer longitudinal layer of muscle. At the junction of the lateral and common oviduct an additional thin longitudinal layer is found adjacent to the basement epithelium. The oviducts contracted spontaneously when isolated from the central nervous system. These myogenic contractions took the form of peristaltic contractions in the lateral oviduct, and intermittent phasic-like contractions of the posterior regions of the lateral oviduct and the common oviduct. These phasic-like contractions were associated with individual complex potentials recorded extracellularly from the muscle fibres. In locusts that had been interrupted in the process of egg laying, there were large-amplitude action potentials, firing in a bursting pattern, in the oviducal nerves. These large action potentials were absent in locusts that had not been egg-laying. These action potentials were associated with both bioelectric potentials and mechanical events in the posterior region of the lateral oviduct and the common oviduct. Electrical stimulation of the oviducal nerve mimicked the effects of spontaneous action potentials, resulting in the appearance of monophasic potentials and contractions. The contractions were graded and dependent upon both frequency and duration of stimulation. It is concluded that the oviducts of Locusta are both myogenic and neurogenic. The role of these contractions in oviposition is discussed.     

Crustacean cardioactive peptide is a modulator of oviduct contractions in Locusta migratoria

Crustacean cardioactive peptide (CCAP) stimulates the contractions of locust oviducts. CCAP increased the basal tonus and increased the frequency and amplitude of phasic contractions, as well as the amplitude of neurally-evoked oviduct contractions in a dose-dependent manner. Oviducts from Vth instar larvae and adult locusts aged 10 days or less, were more sensitive to CCAP than oviducts from adult locusts aged 12 days or more. This may be indicative of a differential expression of number or subtypes of CCAP receptors on the oviducts at different ages, and may be related to reproductive functions or to functions of CCAP on the oviducts during ecdysis. The oviducts appear more sensitive to CCAP when compared with previously published reports of CCAP actions on the hindgut. CCAP actions on the amplitude of neurally-evoked contractions of the oviducts are similar to those of proctolin, however, the oviducts are more sensitive to CCAP. No CCAP-like immunoreactive structures were discovered in the nerves innervating the oviducts, or on the oviducts themselves, confirming the previously published suggestion (Dircksen et al., 1991) that CCAP acts as a neurohormone at the oviducts. Cells showing CCAP-like immunoreactivity were discovered in the fat body associated with the oviducts and represent a potential source of CCAP, along with CCAP released from the transverse nerve and perivisceral organs.     

Influence of age on chiasma number in male Locusta migratoria

The influence of age on chiasma formation in Locusta migratoria has been studied both in wild-type and irradiated males. Samples were obtained on the one hand from different individuals and on the other hand from the same individuals at different ages through repeated biopsies. It was observed that mean chiasmata per cell decreased in ageing animals. Chi-squared tests and correlation coefficients showed that the decrease of chiasmata with age is progressive but not continuous. Intermediate values coincide with the first mating. It seems that older individuals tend to transmit their genetic combinations unaltered, increasing the probability of transmitting gene combinations of adaptive value.     

Some pharmacological properties of neuromuscular transmission in the oviduct of the locust,Locusta migratoria

The effects of various pharmacological agents on neurally evoked contractions of the visceral muscles of the oviduct of Locusta migratoria have been examined. The pentapeptide, proctolin, at low concentrations (10^?11 M?10^?10 M), induced an increase in the amplitude of neurally evoked contractions and basal tonus, and induced the appearance and increased the frequency of myogenic contractions. Glutamate, at 10^?4 M, produced a small transient contraction which in some preparations was accompanied by a reduction in amplitude of neurally evoked contractions. Octopamine, at 10^?6 M, reduced the amplitude of neurally evoked contractions and also resulted in a relaxation of the muscles. The octopaminergic effects were inhibited by the α-aminergic antagonist phentolamine. Neurally evoked contractions were unaffected by dopamine, 5-HT or the acetylcholine receptor antagonists atropine and hexamethonium. Acetylcholine increased the amplitude of neurally evoked contractions, but only at the high concentration of 10^?3 M. The possible role of proctolin and glutamate as excitatory neuro-transmitters and the inhibitory action of octopamine is discussed.     

Comparison of the myotropic activity of position-2 modified analogues of proctolin on the hindgut of Periplaneta americana and the oviduct of Locusta migratoria

The largest series of position-2 modified proctolin analogues to have been examined to date were tested for their ability to mimic the basal contraction induced by proctolin on hindgut of the cockroach, Periplaneta americana, and oviduct of the locust, Locusta migratoria. Twelve analogues of proctolin (Arg-Tyr-Leu-Pro-Thr), differing in the substituent (H, OMe, OEt, OPr, F, Cl, Br, I, NO(2), NH(2), N(3), Me) located at the para-position of the aromatic amino acid, caused dose-dependent contractions of both tissues at concentrations quite similar to proctolin. Seven showed greater or equal potency on the hindgut but, with one exception, they were less active on the oviduct than proctolin. The rank order of potency of the analogues depends on the tissue, lending more support to the notion that insects have more than one type of proctolin receptor. No relationship was observed between myoactivity and lipophilic, steric, electron donating or electron withdrawing properties of the substituents at the para-position of the aromatic amino acid. This may be the result of more than one sub-type of proctolin receptor on the specific tissue with differing structural requirements for optimum activity.     

Effect of azadirachtin A on neuroendocrine activity in Locusta migratoria

Summary The neurosecretory activity of azadirachtin A-treated Locusta migratoria that failed to mature was compared with that of rapidly maturing control females by means of histological techniques. High resolution drymount autoradiography was used to localize [22,23-³H₂]dihydroazadirachtin A in the brain and corpus cardiacum (CC). The neurosecretory system of the locusts treated with this insecticide was accompanied by an unusually high accumulation of paraldehyde fuchsin (PAF)-stainable neurosecretory material (NSM) in the brain fibers and in the storage lobes of the CC. The control females never had such an intense accumulation of NSM. The accumulation of NSM, resulting from its poor release, may influence its synthesis which is controlled by feed-back regulation. [22,23-³H₂]Dihydroazadirachtin A is localized mainly at the peripheral blood-brain barrier and does not penetrate into the brain effectively, whereas it completely covers the CC. It is concluded that azadirachtin A affects the endocrine activity of the brain in an indirect way.     

Octopamine- and dopamine-sensitive adenylate cyclase in the brain ofLocusta migratoria during its development

Octopamine- and dopamine-sensitive adenylate cyclases were studied in the brain of Locusta migratoria during its metamorphosis. In the adult brain the effects of octopamine and dopamine on adenylate cyclase were additive, suggesting the presence of separate populations of adenylate cyclase-linked receptors for octopamine and dopamine. There are no separate receptors for noradrenaline. Octopamine stimulates adenylate cyclase in both adult and larval brain; however, in adult brain octopamine is more potent than in larval brain. Dopamine stimulates adenylate cyclase activity only in adult brain. The sensitivity of adenylate cyclase to octopamine changes during the development of the animal. Phentolamine and cyproheptadine are potent antagonists of octopamine-stimulated adenylate cyclase, while propranolol has a weak effect. No cytosol factor which would modulate either basal or octopamine-stimulated adenylate cyclase was found. The effect of GTP and octopamine on adenylate cyclase was synergistic in adult brain but not in larval brain, while the effect of GppNHp and octopamine was synergistic in both adult and larval brains.     

Divergent effects of phenothiazines on Leydig tumor cell steroidogenesis and adenylate cyclase activity

The dose and temporal (1-24 h) effects of two phenothiazines, chlorpromazine and trifluoperazine, on steroidogenesis and adenylate cyclase activity of gonadotropin-responsive Leydig tumor cells (M5480A) in primary culture were examined. At low doses (e.g. 0.1-1 microM) these antipsychotic drugs were slightly inhibitory (trifluoperazine) or without effect (chlorpromazine), while at 25 microM each drug was weakly stimulatory to basal testosterone production. Trifluoperazine was, in general, inhibitory to HCG-stimulated testosterone production, but chlorpromazine exhibited paradoxical effects. At 5 and 10 microM this neuroleptic agent increased HCG-stimulated steroidogenesis, while at 25 microM testosterone production was inhibited. In a particulate fraction prepared from the tumor the activity of adenylate cyclase was stimulated 3.4-fold in the presence of 10 microM 5'-guanylimidodiphosphate and 5-fold in the presence of HCG plus the non-hydrolyzable GTP analogue. Between doses of 1-100 microM neither drug altered the basal activity of adenylate cyclase. Trifluoperazine at doses of 1-100 microM inhibited 5'-guanylimidodiphosphate-stimulated adenylate cyclase activity both with and without added gonadotropin. At doses of 1-10 microM chlorpromazine had no effect on adenylate cyclase activity, but it stimulated activity in the dose range of 20-100 microM. Interestingly, in the presence of 5'-guanylimidodiphosphate this drug did not alter the stimulated enzymic activity achieved with a maximal dose of HCG. Therefore, these phenothiazines exhibit quite divergent dose-dependent effects and their actions must occur at multiple loci. Also, it seems unlikely that the effects of these agents on steroidogenesis and adenylate cyclase activity can be reconciled solely in terms of calmodulin-mediated processes.     

Effects of intestinal insulin-like peptide on glucose catabolism in male adult Locusta migratoria

An insulin-like peptide (ILP) extracted from midgut of 25-day-old male adult Locusta migratoria can modify the relative activity of the two main pathways of glucose catabolism. The effect of ILP on the activity of the pentose cycle and the glycolytic-citric acid cycle in Locusta migratoria was evaluated by a radiorespirometric method by means of [1-¹⁴C] glucose and [6-¹⁴C]glucose as substrates. The time course of the ILP effect was determined. The insulin-like peptide increases the relative activity of the pentose cycle. This effect is rapid and of short duration. Injected mammalian insulin has a similar effect.     

Physiological and biochemical effects of Olea europaea leaf extracts from four phenological growth stages on the oogenesis of female locust Locusta migratoria

The effects of olive leaf extract (OLE) are studied on several reproductive variables and the ovarian biochemical composition of Locusta migratoria (Orthoptera: Acrididae) adult females. The methanolic extracts are prepared from the leaves sampled during four phenological growth stages of olive tree: cluster formation (Cf), swelling inflorescence buds (Sib), full flowering (Ff) and endocarp hardening (Eh). When applied to adult females during the pre‐ovipositional phase, the treatment elicites a significant adverse effect on their reproductive potential. Indeed, OLE significantly reduces both fecundity and fertility and affects oocyte growth during the first gonadotrophic cycle, as indicated by measurements of ovarian weight, length of terminal oocytes and ovarian index. Furthermore, OLE is examined with respect to ovarian biochemical components. Biochemical analyses reveal a significant reduction of ovarian contents of proteins, lipids and carbohydrates, suggesting a disruption in the incorporation of the haemolymph metabolites in the oocytes and an interference of OLE with the vitellogenesis process. The antigonadotrophic effect is confirmed by a histological study of the ovaries, which clearly shows a delay in ovarian development and in yolk accumulation in the basal oocytes of treated females. The most effect is noted with the extract prepared from the leaves collected at the swelling inflorescence buds for all measured parameters, which appears to be related to its high content of polyphenols.     

Differential effects of Ca2+-calmodulin on adenylate cyclase activity cyclase activity in mouse and rat pancreatic islets.

The subcellular localization of the beta-galactoside-binding protein, or lectin, from rat lung was investigated by the specific binding of anti-lectin immunoglobulin G to subcellular fractions. We used both adult and immature (12-day-old) rats; the immature rat lungs have an 8-10-fold greater concentration than adult rat lungs [Powell & Whitney (1980) Biochem. J. 188, 1-8]. In both groups of animals we observed greater specific binding of anti-lectin immunoglobulin G to intracellular membrane (mitochondrial and microsomal fractions) than to plasma membranes. Pre-incubation of membrane fractions with lactose resulted in a marked diminution of anti-lectin immunoglobulin G binding. In the adult rat lung most (approx. 80%) of the lectin activity was membrane-associated. In the immature rat lung only approx. 30% of the lectin activity was membrane associated and most of the beta-galactoside-binding protein appeared to be a soluble cytoplasmic component. The rat lung beta-galactoside-binding protein appeared to have a broad but predominantly intracellular location, being associated with membranes through one of its galactoside-binding sites.     

Calmodulin regulation of adenylate cyclase activity

Calmodulin-dependent stimulation of adenylate cyclase was initially thought to be a unique feature of neural tissues. In recent years evidence to the contrary has accumulated, calmodulin-dependent stimulation of adenylate cyclase now being demonstrated in a wide range of structurally unrelated tissues and species. Demonstration of the existence of calmodulin-dependent adenylate cyclase has in nearly all instances required the removal of endogenous calmodulin. It is not yet clear whether calmodulin-dependent and calmodulin-independent forms of the enzyme exist and whether some tissues (such as heart) lack a calmodulin-dependent adenylate cyclase. The presence of calmodulin appears largely responsible for the ability of the adenylate cyclase enzyme to be stimulated by submicromolar concentrations of calcium; it may not be relevant to the inhibition of the enzyme which occurs at higher concentrations of calcium. The physical relationship of calmodulin to the plasma membrane bound enzyme (or to the soluble forms of the enzyme) is not known nor is the mechanism of adenylate cyclase activation by calmodulin clear; current data suggest some involvement with both the N and C units of the enzyme. Finally, it is possible that in vivo calcium contributes to the duration of the hormone stimulated cyclic AMP signal. Thus current in vitro data suggest that optimal hormonal activation of calmodulin-dependent adenylate cyclase occurs at very low intracellular calcium concentrations, comparable to those found in the resting cell; conversely the enzyme is inhibited as intracellular calcium increases, following for example agonist stimulation of the cell. These higher calcium concentrations would then activate calmodulin-dependent phosphodiesterase. Such differential effects of calcium on adenylate cyclase and phosphodiesterase would ultimately restrict the duration of the hormone-induced cyclic AMP signal.