Gregory L. Orr

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.

Interaction of formamidines with octopamine-sensitive adenylate cyclase receptor in the nerve cord of Periplaneta americana L.

Chlordimeform (CDM) and demethylchloridimeform (DCDM) mimic the action of octopamine in elevating adenylate cyclase activity in intact nerve cords of the American cockroach, Periplaneta americana. At a concentration of 1 x 10(-5)M, DCDM (13.5x increase within 20 minutes) is a more potent effector of the response than CDM (3x increase within 20 minutes), but both compounds show less efficacy than octopamine (23.5x increase within 15 minutes). DCDM also mimics the stimulatory effect of octopamine on adenylate cyclase activity in nerve cord homogenates whereas CDM has no demonstrable effect on this preparation. The octopamine- and DCDM-induced responses are competitively inhibited by phentolamine (1 x 10(-6)M) and cyproheptadine (1 x 10(-6)M) but not by propranolol (1 x 10(-6)M). DCDM and CDM inhibit the octopamine-induced activation of adenylate cyclase by 33% and 44% respectively. The results are discussed in light of the proposal that DCDM serves as a partial agonist and CDM as an antagonist of the octopamine receptor.

Cyclic AMP does not mediate the action of synthetic hypertrehalosemic peptides from the corpus cardiacum of Periplaneta americana

Two synthetic peptides identical to those present in the corpus cardiacum of the American cockroach, Periplaneta americana, were tested for their effect on the production of cyclic AMP and the activation of glycogen phosphorylase in cockroach fat body. The peptides activate glycogen phosphorylase and promote trehalose production in incubated tissue when calcium is included in the incubation medium, but have no obvious effect on cyclic AMP levels. The lack of effect of the peptides on cyclic AMP production was confirmed in a fragmented membrane preparation. By contrast, an aqueous extract of corpus cardiacum activates glycogen phosphorylase, promotes trehalose production and elevates cyclic AMP levels in incubated tissue; the extract also enhances cyclic AMP production in the fragmented cell membrane preparation. Observations on the nature of cyclic AMP production in cockroach fat body indicate that the adenylate cyclase has a requirement for GTP and magnesium ions, is stimulated by fluoride and forskolin and, therefore, is similar to the adenylate cyclase complex of other eukaryotes. The results suggest that increases in intracellular calcium concentrations may mediate the expression of hypertrehalosemic effects by the synthetic peptides.

Pharmacological characterisation of the dopamine-sensitive adenylate cyclase in cockroach brain: Evidence for a distinct dopamine receptor

Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 microM and at 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 microM and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 microM respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D2-dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 microM. Other dopamine agonists (apomorphine, SKF-82526, SKF-38393) have no stimulatory effects. The octopamine-sensitive AC is inhibited by a variety of antagonists known to affect octopamine and dopamine receptors, with the following order of potency: mianserin greater than phentolamine greater than cyproheptadine greater than piflutixol greater than cis-flupentixol greater than SCH-23390 greater than (+)-butaclamol greater than SKF-83566 greater than SCH-23388 greater than sulpiride greater than spiperone greater than haloperidol. The dopamine-sensitive AC is inhibited by the same compounds with the following order of potency: piflutixol greater than cis-flupentixol greater than (+)-butaclamol greater than spiperone greater than or equal to SCH-23390 greater than cyproheptadine greater than SKF-83566 greater than SCH 23388 greater than mianserin greater than phentolamine greater than sulpiride greater than haloperidol. With the exception of mianserin, 3H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D1- and D2-dopamine receptors.

Characterisation of an octopamine-sensitive adenylate cyclase in haemocyte membrane fragments of the American cockroach Periplaneta americana L.

Abstract An octopamine-sensitive adenylate cyclase has been studied in a fragmented haemocyte-membrane preparation of the American cockroach, Periplaneta americana . The enzyme complex is similar to that of adenylate cyclases from other eukaryotic cells in having a requirement for magnesium ions and GTP and in displaying elevated prodution of cyclic AMP in the presence of sodium fluoride and forskolin. The preparation responds to GTP in a dose-dependent manner and a positive dose-dependent response to calmodulin is also reported. Octopamine and synephrine are the most potent stimulators of cyclic AMP production in the haemocyte-membrane preparation and additivity studies indicate that synephrine and another activator, tyramine, are interacting with the enzyme complex through the octopamine-receptor. The adrenergic agonists, naphazoline and clonidine increased cyclic AMP production and the formamidines, demethylchlordimeform and BTS 27271 also were effective in this regard. Hydroxymandelic acid caused a slight but statistically significant decrease in cyclic AMP production. The most potent antagonists of the octopamine-mediated response were mianserin > promethazine > phentolamine > gramine > cis- flupenthixol > dibenamine. Lineweaver-Burk plots indicate that the mianserin, promethazine and gramine-effects, as with earlier reports for phentolamine, result from competitive inhibition

Characterization of a potent agonist of the insect octopamine-receptor-coupled adenylate cyclase

Abstract The imidazoline, 2,3-xylylaminomethyl-2′-imidazoline (XAMI) was evaluated for its effects on octopamine-sensitive adenylate cyclase (OSAC) in crude membrane preparations of neural and non-neural tissues of the American cockroach, Periplaneta americana and ventral-nerve cord homogenates of the tobacco hornworm, Manduca sexta . In the cockroach nerve cord, XAMI was found to be a partial agonist with a V max 80% of p -octopamine (OA) and a K a of 30 nM. The affinity is 185 times greater than that of OA. Additivity studies suggest that at maximally stimulating concentrations, XAMI interacts primarily with the OSAC. The antagonist profile for XAMI mimics that of OA with mianserin being the best antagonist, followed by the α-adrenergic antagonist phentolamine. These antagonists were shown to act competitively at the XAMI-binding site. β-adrenergic and dopaminergic antagonists were ineffective. These data indicate that XAMI has the highest reported affinity of any OA-receptor agonist and may be a suitable ligand for studies of the OA receptor.

Forskolin-insensitive adenylate cyclase in cultured cells of Choristoneurafumiferana (Insecta)

Adenylate cyclase from a spruce budworm cell line, IBRI-Cfl, is activated by octopamine (Ka = 50 microM), guanine nucleotides and sodium fluoride but not by forskolin. In addition, forskolin does not potentiate the octopamine-sensitive response. To our knowledge, this is the first published report of a hormone-sensitive adenylate cyclase, with a functional guanine nucleotide regulatory protein, that is insensitive to forskolin both in intact cells and washed membrane preparations.

Modulation of octopamine-mediated production of cyclic AMP by phorbol-ester-sensitive protein kinase C in an insect cell line

The presence of protein kinase C (EC 2.7.1.37) in an insect cell line has been demonstrated. Phorbol 12-myristate 13-acetate (PMA), in micromolar concentrations, activated protein kinase C with a translocation of the enzyme from the cytosol to the particulate fraction. Cyclic AMP production in the presence of PMA, octopamine and a combination of both increased in a dose-dependent and time-dependent fashion. The biologically inactive 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity or on octopamine-mediated cyclic AMP production. Pretreatment of the cells with pertussis toxin had no effect on the response of cells to octopamine or PMA. However, pretreatment with cholera toxin resulted in increased cyclic AMP production which was further enhanced when both cholera toxin and PMA were used in combination. Our data indicate that the octopamine-mediated cyclic AMP production is modulated by protein kinase C.

Pharmacological Characteristics of Octopamine-Sensitive Adenylate Cyclase and N-Acetyl Octopamine Transferase in Insects

p-Octopamine (OA) occurs in the nervous systems of many invertebrates at much higher concentrations than in vertebrate tissues (Klemm, 1985). A variety of physiological effects have been ascribed to OA including modulation of the activity of excitable tissues in annelids (Tanaka and Webb, 1983), crustacea (Evans et al, 1987) and insects (Hoyle, 1975; Evans and O’Shea, 1978) and the induction of specific behavioural responses in nematodes (Horitz et al, 1982) and crustacea (Livingston et al, 1980; Bevengut and Clarac, 1982; Kravitz et al, 1981). In insects, OA has also been implicated in excitation of the firefly lantern (Carlson, 1986), stimulation of glycogenolysis in nerve cord and fat body (Robertson and Steele, 1972; Downer, 1979a,b). Stimulation of cardiac contraction (Collins and Miller, 1977), enhancement of substrate utilisation by flight muscle (Goosey and Candy, 1980), release of diacylglycerol from locust fat body (Orchard et al. 1982) and regulation of release of peptide hormones from the corpus cardiacum (Orchard et al., 1983; Downer et al., 1984).

The regulation of basal and dopamine-sensitive adenylate cyclase by salts of monovalent cations in brain of the American cockroach, Periplaneta americana L.

Abstract Production of cyclic AMP by cell-free preparations of cockroach brain is increased by dopamine and salts of monovalent cations. The effects of dopamine are GTP dependent with apparent plateaus in cyclic AMP production occurring at dopamine concentrations of between 1 and 10 μM and at 10 mM. Sodium chloride and potassium chloride elicit similar dose-dependent increases in cyclic AMP production ( K m = 15–20 mM) and choline chloride is slightly less effective ( K m = 29 mM). Sodium acetate has no effect of cyclic AMP production, sodium sulphate decreases cyclic AMP levels and sodium bromide enhances cyclic AMP production ( K m = 10 mM) in a manner similar to that of potassium chloride and sodium chloride. The cyclic AMP increase observed with sodium chloride is not dependent upon added GTP and doses of sodium chloride above 50 mM inhibit the stimulatory effects of 5′-guanylylimido-diphosphate. Forskolin enhances the effects of sodium chloride on cyclic AMP production by increasing maximal production and reducing the K m for sodium chloride. The dopamine-mediated (1.0 mM) increase in cyclic AMP production is reduced in the presence of sodium chloride, potassium chloride, choline chloride and sodium bromide with sodium chloride being most effective. Sodium acetate and sodium sulphate enhance the dopamine-mediated increase in cyclic AMP production. A fixed dose of sodium chloride (120 mM) shifts the dopamine dose-response curve to the right, thereby increasing the K m for the amine and reducing maximal stimulation. Inhibition of [ 3 H]piflutixol ([ 3 H]PIF) binding by dopamine is reduced in the presence of sodium chloride and lithium chloride but not by potassium chloride and choline chloride. The results indicate that the anionic moiety of the salt is most important in the stimulation of basal cyclic AMP production and in the inhibition of the dopamine-mediated increase in cyclic AMP. Halogens appear to be the most effective anions in mediating both effects. The ability of dopamine to inhibit [ 3 H]PIF binding is reduced in the presence of the monovalent cations Na + and Li + .