5-HT stimulation of heart rate inDrosophiladoes not act through cAMP as revealed by pharmacogenetics

Abstract

The fruit fly, Drosophila melanogaster, is a good experimental organism to study the underlying mechanism of heart rate (HR) regulation. It is already known that many neuromodulators (serotonin, dopamine, octopamine, acetylcholine) change the HR in Drosophila melanogaster larvae. In this study, we investigated the role of cAMP-PKA signaling pathway in HR regulation and 5-HT positive chronotropic action. In order to obtain insight into the 5-HT mechanism of action in larvae cardiomyocytes, genetic and pharmacological approaches were used. We used transgenic flies that expressed the hM4Di receptor [designer receptors exclusively activated by designer drugs (DREADDs)] as one tool. Our previous results showed that activation of hM4Di receptors (modified muscarinic acetylcholine receptors) decreases or arrests the heart from beating. In this study, it was hypothesized that the positive chronotropic effect of serotonin [5-hydroxytryptamine (5-HT)] are mediated by serotonin receptors coupled to the adenylyl cyclase pathway and downstream cAMP and PKA activity. Activation of hM4Di by clozapine-N-oxide (CNO) was predicted to block the effects of serotonin by inhibiting adenylyl cyclase activity through Gαi pathway activation. Interestingly, we found here that manipulation of adenylyl cyclase activity and cAMP levels had no significant effect on HR. The ability of hM4Di receptor activation to slow or stop the heart is therefore likely mediated by activation of GIRK channels to produce hyperpolarization of cardiomyocytes, and not through inhibition of adenylyl cyclase.

Pharmacological identification of cholinergic receptor subtypes on Drosophila melanogaster larval heart

“…As stated, M2 mAChR receptor subtype is present in mammalian cardiac tissue and was shown to attenuate adenylate cyclase activity, thereby reducing the intracellular levels of cAMP through G i (Felder, 1995). Our analysis suggest that the mAChRs present in cardiac tissue at the larval stage act through a stimulatory cascade that is not regulated by adenylate cyclase, as it has been shown that HR stimulation by 5-HT does not act through cAMP (Majeed et al 2013). In a recent study by Ren et al (2015), the group showed that A-type mAChRs couple to the G q / 11 signaling pathway, whereas B-type mAChR couple to the G i / 0 pathway.…”

“…As previously reported, mechanical disturbance plays a role in altering HR in a semi-intact, open preparation (Majeed et al 2013). In addition, the HR generally slows down over time.…”

“…In insects, neuromodulators travel in the hemolymph and affect non-neuronal tissues in addition to acting as the primary mediator of communication between cells of the nervous system . A number of neuromodulators that are prominent in larvae, including dopamine (Neve et al 2004;Titlow et al 2013), serotonin (Dasari and Cooper 2006;Majeed et al 2013) and octopamine (Johnson et al 1997), have also shown to exhibit modulatory effects on the heart. It has previously been shown that ACh at concentrations between 1 mM and 1 M, decreases heart rate (HR) in Drosophila at the larval, pupal, and adult stages with no significant changes at concentrations lower than 1 mM (Zornik et al 1999); however, these studies were performed in the intact, whole animal with injections into the hemolymph.…”

Model Organisms in G Protein–Coupled Receptor Research

“…DREADD knock-in animal models, including Drosophila and mice, are currently being used by the GPCR research community for engaging celltype selective G protein signaling. For example, the selective expression of DREADDs in the Drosophila heart clarified the signal pathway of the 5-HT receptor in the control of heartbeat (Becnel et al, 2013;Majeed et al, 2013). DREADD/cloxipinenitric oxide pharmacology does not provide spatial-temporal control; however, there is no need for complex fiber/light-emitting diode systems or expensive microscopy, and this approach has therefore gained a wide user base.…”

The neurotransmitters serotonin and glutamate accelerate the heart rate of the mosquito Anopheles gambiae

“…For example, serotonin increases the heart rate of Periplaneta americana, Rhodnius prolixus, and Manduca sexta in a concentration-dependent manner, with the confirmed bioactive concentrations ranging from 1 X10 -9 M to 1 X10 -5 M (Chiang et al, 1992;Collins and Miller, 1977;Platt and Reynolds, 1986;Prier et al, 1994). In D. melanogaster, several studies have shown that serotonin is cardioacceleratory, with the confirmed bioactive concentrations ranging between 1 X 10 -7 M and 1 X 10 -5 M (Johnson et al, 1997;Johnson et al, 2002;Majeed et al, 2013;Nichols, 2006;Zornik et al, 1999). However, another study in this same insect found that serotonin can be both cardioacceleratory and cardiodeceleratory.…”