Insect Biochemistry and Molecular Biology 30 2000 755–766 www.elsevier.comlocateibmb
Pharmacological characterization of dopamine receptors in the corpus allatum of Manduca sexta larvae
Noelle A. Granger
a,
, Richard Ebersohl
a
, Thomas C. Sparks
b
a
Department of Cell Biology and Anatomy, Campus Box 7090, Taylor Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
b
DowAgro Sciences, 3221 Zionsville Road, Indianapolis, IN, USA Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
Dopamine receptors previously identified in corpora allata CA of Manduca sexta last instars on the basis of dopamine effects on JH juvenile hormoneJH acid biosynthesis and cyclic AMP cAMP accumulation, were characterized pharmacologically. For
this study, a broad spectrum of agonists or antagonists of D1, D2, D3 or D4 dopamine receptors, together with the dopamine metabolite N-acetyl-dopamine, other neurotransmitters and their agonistsantagonists, were tested for their effects on gland activity
and cAMP production. The lack of effect of other neurotransmitters supports the specificity of the effect of dopamine and the dopamine specificity of the receptors. Only the D2 receptor antagonist spiperone had a potent effect on JH biosynthesis and cAMP
formation by CA taken on day 0 of the last stadium, when dopamine stimulates both activities and thus appears to be acting via a D1-like receptor. Several other D2 receptor antagonists, and D1, D2D1 and D4,3D2 receptor antagonists were less effective.
Thus, the D1-like receptor of the Manduca CA appears to be distinct pharmacologically from vertebrate D1 receptors. By contrast, a number of D2 agonistsantagonists had a significant effect on JH acid biosynthesis and cAMP production by the CA from day
6 of the last stadium, when dopamine inhibits both activities and thus appears to be acting via a D2-like receptor. Certain D1-specific agonistsantagonists were equally effective. The Manduca D2-like receptor therefore bears some pharmacological resemblance to
vertebrate D2 receptors. N-acetyl dopamine acted as a dopamine agonist with day 6 CA, the first identified function for an N- acetylated biogenic amine in insects. Dopamine was found to have the same differential affect on the formation of cAMP in
homogenates of day 0 and day 6 brains as it did with CA, and in the same concentration range. Dopamine receptor agonistsantagonists affecting cAMP formation by day 0 and day 6 CA homogenates had similar effects with brain homogenates.
By contrast, dopamine only stimulated cAMP formation by homogenates of day 0 and day 6 abdominal or ventral nerve cord. These results suggest that D1- and D2-like dopamine receptors of Manduca are regionally as well as temporally localized.
2000
Elsevier Science Ltd. All rights reserved.
Keywords: Juvenile hormone biosynthesis; Cylic AMP cAMP; Neurotransmitter; Biogenic amines; Receptor agonist; Receptor antagonist
1. Introduction
There has been considerable interest in the molecular mechanisms
by which
juvenile hormone
JH synthesisrelease is controlled, since interruption of these
processes would affect the many individual roles JH plays in regulating development and reproduction.
Emphasis has been placed largely on understanding the
Corresponding author. Tel.: +
1-919-966-3288; fax: +
1-919-966- 1856.
E-mail address: noellemed.unc.edu N.A. Granger.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 4 7 - 3
role of neuropeptides in the control of JH synthesis, particularly the biology of allatostatins Stay et al. 1994,
1996; Stay, 2000; see Gilbert et al., 2000, for a review. Neurotransmitters have been investigated to a consider-
ably lesser extent, but there is strong evidence that in a number of species, neurotransmitters can also regulate
CA activity Lafont-Cazal and Baehr, 1988; Thompson et al., 1990; Pastor et al., 1991; Rachinsky, 1994; Kaatz
et al., 1994; Woodring and Hoffmann, 1994. In a pre- vious study, it was shown that dopamine stimulated both
JH biosynthesis and cAMP formation by the corpora allata CA during the first 2 days of the last larval stad-
ium of Manduca sexta, while it inhibited both activities
756 N.A. Granger et al. Insect Biochemistry and Molecular Biology 30 2000 755–766
by CA on days 3–6 Granger et al., 1996. According to the traditional classification of dopamine receptors
Creese et al., 1983; Gingrich and Caron, 1993, binding of ligand to a D1 receptor increases adenylyl cyclase
activity, while binding to a D2 receptor either has no effect or inhibits the activity of this enzyme. Thus these
results suggested the existence of D1- and D2-like dopa- mine receptors in the CA. A preliminary investigation
of the effects of a small number of vertebrate D1 and D2 receptor agonistsantagonists on JH biosynthesis and
cAMP formation indicated that the D1-like receptor was pharmacologically distinct from vertebrate D1 receptors,
while the D2-like receptor was more similar pharmaco- logically to vertebrate D2 receptors.
Our knowledge of neurotransmitter receptors in insects is limited, but growing for reviews see Roeder,
1994; Osborne, 1996. D1 and D2 dopamine receptors belong to a large superfamily of G protein-coupled
receptors, and the genes or cDNAs of more than 60 are known
O’Dowd, 1993,
including octopamine,
octopaminetyramine, and serotonin receptors for Droso- phila Arakawa et al., 1990; Witz et al., 1990; Saudou
et al., 1992; Robb et al., 1994; Vanden Broeck et al., 1995; Von Nickisch-Rosenegk et al., 1996. Molecular
cloning has revealed five pharmacologically distinct ver- tebrate dopamine receptor subtypes. Two of these cloned
receptor subtypes D1A and D1D5B exhibit the func- tional and pharmacological properties of the classical D1
receptor sub-family, while the other three D2S, D2L, D3, and D4 are D2-like Gingrich and Caron, 1993;
Strader et al., 1995. Several dopamine receptors have been sequenced in Drosophila Gotzes et al., 1994;
Sugamori et al., 1995; Feng et al., 1996; Han et al., 1996. While these receptors demonstrate relatively little
sequence homology to cloned vertebrate D1-like recep- tors, they are nevertheless linked to increases in adenylyl
cyclase activity when expressed in cell lines or Xenopus oocytes. The DopR99B receptor Feng et al., 1996;
Reale et al., 1997 is also coupled directly to the gener- ation of an intracellular Ca
2 +
signal. Both cloned Drosophila: Gotzes et al., 1994; Suga-
mori et al., 1995; Feng et al., 1996; Reale et al., 1997 and native Periplaneta americana: Orr et al., 1987;
Downer, 1990; Apis mellifera: Kokay and Mercer, 1996, 1997 have been examined for their ability to bind com-
pounds which act as agonists and antagonists of dopam- ine receptors in vertebrates. The results of these studies
strongly suggest that the D1-like and D2-like sub-famil- ies of dopamine receptors in insects are substantially dis-
tinct with different and variable subtype groups, parti- cularly between different orders of insects.
The objective of the present study was to characterize pharmacologically the dopamine receptors in the CA of
Manduca last instars, by examining the effects of a broad range
of vertebrate
D1 and
D2 receptor
agonistsantagonists on JHJH acid biosynthesis and cAMP formation. This has enabled a comparison of
these receptors to those of vertebrates, as well as to known insect dopamine receptors. Also examined were
the effects of other neurotransmitters, to confirm the specificity of the dopamine effects, and the effects of
dopamine
and certain
dopamine receptor
agonistsantagonists on cAMP formation in the Manduca nervous system, specifically the brain and ventral
nerve cord.
2. Materials and methods
