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First published online June 7, 2004
Journal of Experimental Biology 207, 2507-2518 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01052

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Dopamine stimulates snail albumen gland glycoprotein secretion through the activation of a D1-like receptor

S. T. Mukai, L. Kiehn and A. S. M. Saleuddin^*

Department of Biology, Faculty of Pure and Applied Sciences, York University, Toronto, Ontario, Canada M3J 1P3

View larger version (19K): [in a new window]   Fig. 1. Effect of dopamine on the in vitro secretion of protein by the albumen gland. Basal protein secretion was quantified using a protein detection kit (line graph) or by collecting the medium around the albumen glands and analyzing secreted proteins by SDS-PAGE. Basal protein secretion was monitored for 60 min, then 10 µmol l-1 dopamine was added for an additional 20 min. The major protein secreted by the albumen gland of Helisoma duryi is the 66 kDa glycoprotein subunit of Helisoma duryi albumen gland protein (HdAGP), which appears as a heavily stained band. Addition of dopamine induced a rapid increase in the amount of HdAGP released. Removal of the stimulus at 80 min caused HdAGP secretory rate to rapidly return to basal levels. Each point on the line graph represents the mean ± S.E.M. of 6–8 samples.

View larger version (14K): [in a new window]   Fig. 2. The time course of cAMP production by the albumen gland of H. duryi after application of dopamine (DA) or the adenylate cyclase activator forskolin (FSK). The application of dopamine (10 µmol l-1) or forskolin (10 µmol l-1) significantly increased albumen gland cAMP production between 5 and 20 min. Note, in the presence of forskolin, dopamine-stimulated cAMP production was potentiated. Each point represents the mean ± S.E.M. of 4–6 samples. CON, control.

View larger version (12K): [in a new window]   Fig. 3. The effect of different concentrations of (A) dopamine and (B) forskolin on albumen gland cAMP production during a 10 min incubation. Note that dopamine caused a dose-dependent elevation in albumen gland cAMP levels between 0.1 and 10 µmol l-1. Dopamine treatment induced a statistically significant elevation of albumen gland cAMP levels at a concentration of 10 and 100 µmol l-1 when compared with untreated control glands (*P<0.001, Tukey test). Forskolin caused a dose-dependent increase in albumen gland cAMP production between 0.1 and 100 µmol l-1 (*P<0.05 and **P<0.001, Tukey test). Note that forskolin is a more potent stimulant than dopamine on albumen gland cAMP production. Bars represent the mean ± S.E.M. of 6 samples.

View larger version (11K): [in a new window]   Fig. 4. The effect of dopamine and various D1-selective agonists on albumen gland cAMP production. Albumen glands were quartered and treated with either dopamine, a D1-selective agonist or dopamine + agonist. (A) Dopamine (DA) caused a significant elevation of albumen gland cAMP levels when compared with untreated control glands (CON), whereas dihydrexidine (DH) caused a modest but statistically significant elevation of albumen gland cAMP levels. Application of dopamine and dihydrexidine together was unable to elevate cAMP levels above those of dopamine-treated glands (*P<0.05, **P<0.001, Tukey test). (B) The non-selective dopamine receptor agonist 6,7-ADTN was equally as potent as dopamine on albumen gland cAMP production (*P<0.05, Tukey test). (C) The partial D1-selective agonist SKF81297 (SKF) induced a modest elevation above basal cAMP production in the albumen gland but it was not statistically significant when compared with control glands (*P<0.001, Tukey test). All agents were used at a concentration of 10 µmol l-1. Bars represent the mean ± S.E.M. of 5–6 samples.

View larger version (11K): [in a new window]   Fig. 5. The effect of the D2-selective agonists apomorphine and bromocriptine on albumen gland cAMP production. All agents were applied to albumen glands at a concentration of 10 µmol l-1 for 10 min. (A) Apomorphine (APO) had no effect on basal cAMP production when compared with control glands (CON) but, surprisingly, it suppressed dopamine-stimulated cAMP production (*P<0.05, **P<0.001, Tukey test). (B) Bromocriptine (BR) had no effect on either basal or dopamine-stimulated cAMP production. Bars represent the mean ± S.E.M. of 5–6 samples.

View larger version (10K): [in a new window]   Fig. 6. The effect of D1-selective antagonists on dopamine-stimulated cAMP production in the albumen gland of H. duryi. All agents were used at a concentration of 10 µmol l-1 and applied to the glands for 10 min. For the dopamine plus antagonist combinations, the glands were first pre-incubated in antagonist for 20 min.Dopamine (DA) significantly increased cAMP levels in the albumen glands compared with untreated control glands (CON). (A) The D1-selective antagonist SCH23390 had no effect on basal cAMP levels but significantly inhibited dopamine-stimulated cAMP production (*P<0.001, Tukey test). (B) The D1-selective antagonist SKF83566 significantly suppressed dopamine-stimulated cAMP production and had no effect on basal cAMP levels (*P<0.001, Tukey test). (C) By contrast, flupenthixol (FLU), another D1-selective antagonist, did not cause a statistically significant suppression of dopamine-stimulated cAMP production (*P<0.05, Tukey test). All agents were used at a concentration of 10 µmol l-1. Bars represent the mean ± S.E.M. of 5–6 samples.

View larger version (16K): [in a new window]   Fig. 7. The effect of various D2-selective antagonists on dopamine-stimulated cAMP production in isolated albumen glands of H. duryi. The D2-selective inhibitors chlorpromazine (A), eticlopride (B) and haloperidol (C) and the mixed D1/D2 antagonist butaclamol (D) affected neither basal cAMP levels nor dopamine-stimulated cAMP production in the albumen glands. Abbreviations for A: CON, control; DA, dopamine (*P<0.001, Tukey test); CLR, chlorpromazine; DA+CLR, dopamine + chlorpromazine (*P<0.001, Tukey test). Abbreviations for B: CON, control; DA, dopamine (*P<0.01, Tukey test); ETIC, eticlopride; DA+ETIC, dopamine + eticlopride (*P<0.01). Abbreviations for C: CON, control; DA, dopamine (*P<0.05, Tukey test); HAL, haloperidol; DA+HAL, dopamine + haloperidol (*P<0.05, Tukey test). Abbreviations for D: CON, control; DA, dopamine (*P<0.001, Tukey test); BUT, butaclamol; DA+BUT, dopamine + butaclamol (*P<0.001, Tukey test). All agents were used at a concentration of 10 µmol l-1. Bars represent the mean ± S.E.M. of 5–6 samples.

View larger version (14K): [in a new window]   Fig. 8. The effect of the D1-selective agonists SKF81297 and dihydrexidine on the albumen gland glycoprotein secretion. (A) The partial D1 agonist SKF81297 (50 µmol l-1) induced a modest but statistically significant increase in glycoprotein secretion compared with control (CON) glands in saline. However, it was less than half the potency of dopamine (DA) at 50 µmol l-1 concentration. Bars represent the mean ± S.E.M. of 16–18 samples (*P<0.05; *P<0.001, Tukey test). (B) Dihydrexidine (DH) caused a dose-dependent increase in albumen gland glycoprotein secretion between 1 and 100 µmol l-1. Note that the shape and magnitude of the dose–response curve for dihydrexidine are similar to those of dopamine. Each point represents the mean ± S.E.M. of 18 samples (*P<0.05; *P<0.001, Tukey test).

View larger version (8K): [in a new window]   Fig. 9. The effect of the D2-selective agonists apomorphine and bromocriptine on glycoprotein secretion by the albumen gland of H. duryi. The application of apomorphine (10 µmol l-1) and bromocriptine (10 µmol l-1) had no significant effect on albumen gland protein secretion compared with control glands incubated in saline. Positive control glands incubated in 10 µmol l-1 dopamine showed a fivefold increase in protein secretion compared with saline controls. Abbreviations: CON, control; DA, dopamine; APO, apomorphine; BR, bromocriptine. Bars represent the mean ± S.E.M. of 5–8 samples (*P<0.001, Tukey test).

View larger version (11K): [in a new window]   Fig. 10. The effect of the D1-selective antagonists SKF83566 and flupenthixol on dopamine-stimulated glycoprotein secretion by the albumen glands of H. duryi. The application of dopamine (50 µmol l-1) stimulated glycoprotein secretion more than fourfold compared with control glands in saline. (A) In the presence of SKF83566 (50 µmol l-1), dopamine-stimulated glycoprotein secretion was significantly suppressed. Abbreviations: CON, control; DA, dopamine; DA+SKF, dopamine + SKF83566. Bars represent the mean ± S.E.M. of 15–20 samples (*P<0.05, Tukey test). (B) The D1-selective antagonist flupenthixol also caused a significant inhibition of dopamine-stimulated protein secretion in isolated albumen glands. Abbreviations: CON, control; DA, dopamine; DA+FLU, dopamine + flupenthixol. Neither antagonist had an effect on basal glycoprotein secretion. Bars represent the mean ± S.E.M. of 15–20 samples (*P<0.05, Tukey test).

View larger version (11K): [in a new window]   Fig. 11. The effect of protein kinase A (PKA) antagonists on dopamine-induced glycoprotein secretion by the albumen glands of H. duryi. Albumen glands were preincubated with either 1 mmol l-1 Rp-cAMP (A) or 10 µmol l-1 H-89 (B) for 60 min and then dopamine (10 µmol l-1) + Rp-cAMP or dopamine + H-89 were added to the albumen glands for an additional 20 min. Neither PKA antagonist had an affect on basal protein secretion nor did they suppress dopamine-stimulated protein secretion by the albumen glands. Abbreviations: CON, control; DA, dopamine; DA+Rp, dopamine + Rp-cAMP; H-89+DA, dopamine + H-89. Bars represent the mean ± S.E.M. of 6 samples (*P<0.05, Tukey test).