Early effects of Epac depend on the fine-tuning of the sarcoplasmic reticulum Ca2+ handling in cardiomyocytes.

In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including contractility, relaxation and automatism. cAMP cascade leads to the activation of the classic protein kinase A but also to the stimulation of the recently discovered exchange protein directly activated by cAMP (Epac). The role of Epac in the regulation of intracellular Ca2+ homeostasis and contractility in cardiac myocytes is still matter of debate. In this study we showed that the selective Epac activator, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (8-CPT), produced a positive inotropic effect when adult rat cardiac myocytes were stabilized at low [Ca2+]o (0.5mM), no changes at 1mM [Ca2+]o and a negative inotropic effect when [Ca2+]o was increased to 1.8mM. These effects were associated to parallel variations in sarcoplasmic reticulum (SR) Ca2+ content. At all [Ca2+]o studied, 8-CPT induced an increase in Ca2+ spark frequency and enhanced CaMKII autophosphorylation and the CaMKII-dependent phosphorylation of SR proteins: phospholamban (PLN, at Thr17 site) and ryanodine receptor (RyR2, at Ser2814 site). We used transgenic mice lacking PLN CaMKII phosphorylation site (PLN-DM) and knock-in mice with an inactivated CaMKII site S2814 on RyR2 (RyR2-S2814A) to investigate the involvement of these processes in the effects of Epac stimulation. In PLN-DM mice, 8-CPT failed to induce the positive inotropic effect at low [Ca2+]o and RyR2-S2814A mice showed no propensity to arrhythmic events when compared to wild type mice myocytes. We conclude that stimulation of Epac proteins could have either beneficial or deleterious effects depending on the steady-state Ca2+ levels at which the myocyte is functioning, favoring the prevailing mechanism of SR Ca2+ handling (uptake vs. leak) in the different situations.

Receptor crosstalk protein, calcyon, regulates affinity state of dopamine D1 receptors.

The recently cloned protein, calcyon, potentiates crosstalk between G(s)-coupled dopamine D1 receptors and heterologous G(q/11)-coupled receptors allowing dopamine D1 receptors to stimulate intracellular Ca(2+) release, in addition to cAMP production. This crosstalk also requires the participating G(q/11)-coupled receptors to be primed by their agonists. We examined the ability of calcyon and priming to regulate the affinity of dopamine D1 receptors for its ligands. Receptor binding assays were performed on HEK293 cell membrane preparations expressing dopamine D1 receptors either alone or in combination with calcyon. Co-expression of dopamine D1 receptor and calcyon affected neither the affinity of this receptor for antagonists nor the affinity of agonist binding to this receptor high and low-affinity states. However, the presence of calcyon dramatically decreased the proportion of the high-affinity dopamine D1 receptor agonist binding sites. This decrease was reversed by carbachol, which primes the receptor crosstalk by stimulating endogenous G(q/11)-coupled muscarinic receptors. Our findings suggest that calcyon regulates the ability of dopamine D1 receptors to achieve the high-affinity state for agonists, in a manner that depends on priming of receptor crosstalk.

Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein.

The synergistic response of cells to the stimulation of multiple receptors has been ascribed to receptor cross talk; however, the specific molecules that mediate the resultant signal amplification have not been defined. Here a 24-kilodalton single transmembrane protein, designated calcyon, we functionally characterize that interacts with the D1 dopamine receptor. Calcyon localizes to dendritic spines of D1 receptor-expressing pyramidal cells in prefrontal cortex. These studies delineate a mechanism of Gq- and Gs-coupled heterotrimeric GTP-binding protein-coupled receptor cross talk by which D1 receptors can shift effector coupling to stimulate robust intracellular calcium (Ca2+i) release as a result of interaction with calcyon. The role of calcyon in potentiating Ca2+-dependent signaling should provide insight into the D1 receptor-modulated cognitive functions of prefrontal cortex.

Alpha-melanotropin hormone inhibits the binding of [3H]SCH 23390 to the dopamine D1 receptor in vitro.

We have previously demonstrated that the simultaneous presence of alpha-melanocyte stimulating hormone (alpha-MSH) and dopamine resulted in a reduction in cyclic AMP (cAMP) levels in slices containing caudate putamen and accumbens nuclei as compared to those treated only with dopamine or alpha-MSH. This study was carried out to explore if the interaction between alpha-MSH and dopamine could be explained on the basis of a direct interaction between alpha-MSH and the dopamine D1 receptor. Saturation curves for [n-methyl-3 H](R)-(+)-8 chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 H-3-benzazepin-7-o] hemimaleate ([3H]SCH 23390) binding in the presence of increasing concentrations of alpha-MSH were performed. Nonlinear regression in the presence of alpha-MSH revealed an increased dissociation constant (Kd). The binding capacity (Bmax) was not affected by the peptide. These data suggest an apparent competitive interaction between alpha-MSH and [3H]SCH 23390 in striatal membranes on the dopamine D1 receptor; (Ki = 1.2 X 10(-7) M). The present data show that alpha-MSH could interact with the dopamine D1 receptor modulating allosterically the affinity of [3H]SCH 23390 for the receptor or by causing a change in the lipid environment of the dopamine receptor, resulting in an inhibition of the ligand binding to it.

Interaction of alpha-MSH and substance P with interfaces containing gangliosides.

In the present work we studied the interaction of alpha-MSH and substance P neuropeptides with gangliosides using lipid monolayers, fluorescence spectroscopy, and differential scanning calorimetry techniques. The positively charged weak amphiphilic neuropeptides did not show surface activity in the range of concentrations tested (0.1-0.3 muM), but they were preferentially able to penetrate monolayers formed by acidic lipids, showing the best interaction with the more complex gangliosides. The general order of interaction found for both peptides is GTIh > GDIa = GMI > DLPA > sulphatide. Neither neuropeptide interacted with phosphatidylcholine monolayers above 10 mN.m-1. The binding of alpha-MSH to GMI micelles followed by changes in the fluorescence of its tryptophan residue takes place with an increase in the hydrophobic environment of the neuropeptide. An apparent dissociation constant of 13 muM was estimated for this process. Similar result was found with GMI:DMPC vesicles (1:10 molar ratio). The thermotropic profile of GMI micelles is modified in the presence of the neuropeptides. The calorimetric enthalpy of GMI transition increased 21% and 37% in the presence of alpha-MSH and substance P, respectively. Both neuropeptides induced the same increment in the transition temperature Tm from 19 to 20.5 degrees C. The basic physicochemical studies herein indicated that both positively charged neuropeptides, alpha-MSH and substance P, interact with interfaces containing gangliosides in a mainly electrostatic form, whereas the hydrophobic interaction seems to play a secondary role.

Synergic action of gangliosides on alpha-MSH-induced cyclic AMP levels in rat brain slices.

Gangliosides are particularly enriched in neuron cell membranes and they were postulated to be involved in the modulation of membrane-mediated transduction of information. In this study we explore the possibility that the increase in cAMP tissular levels induced by alpha-MSH may be modulated by the action of exogenously added gangliosides. We measured the level of cAMP in both tissues and medium in response to the alpha-MSH in slices previously incubated with total bovine brain gangliosides (TBG). When slices were exposed to TBG, the effect of alpha-MSH on inducing an increase in the content of cAMP was practically twice compared to the changes induced by alpha-MSH or TBG alone. We conclude that the presence of gangliosides may facilitate the alpha-MSH interaction with its receptor, increasing the cAMP levels in slices containing the CP and Acc nuclei.

Effects of alpha-MSH and cholinergic agents on cGMP and IP3 levels in rat brain slices.

On one hand, it has been demonstrated that the exposure of rat brain slices containing caudate putamen and accumbens nuclei to alpha-MSH brings about an increase in cAMP. This increase is affected when dopamine is present in the incubation medium. On the other hand, an interaction of melanotropinergic-like peptides with acetylcholinergic drugs has been showed to be similar to the one observed with dopamine. In this study we have intended to measure cGMP or IP3 in response to alpha-MSH, and also to study the interaction with cholinergic drugs by measuring the second messengers recently mentioned. cGMP and IP3 have been measured in tissues and medium in their response to the effect of alpha-MSH alone or in the presence of the peptide plus pilocarpine (selective muscarinic agonist) or atropine (selective muscarinic antagonist). None of them modified the cGMP levels when compared with the control group. The exposure of rat brain slices containing CP and Acc nuclei to alpha-MSH resulted in an increase in IP3 levels. Pilocarpine by itself brought about an increase of IP3 only when the highest doses was used. Atropine did not modify the IP3 content. However, when slices were exposured to both alpha-MSH and pilocarpine, IP3 content was similar to control values. The blockage of the muscarinic receptor with atropine blocked the IP3 increase induced by alpha-MSH as well. Therefore, we assume that alpha-MSH does not induce changes in cGMP but it does change the IP3 levels, probably acting at the muscarinic receptor level.

alpha-MSH changes cyclic AMP levels in rat brain slices by an interaction with the D1 dopamine receptor.

The exposure of rat brain slices containing caudate putamen and accumbens nuclei to alpha-MSH or dopamine (DA) results in an increase in cyclic AMP (cAMP) levels. When tissues are compared with those containing both alpha-MSH and DA, a reduction in the cyclic nucleotide is observable. This study was carried out to determine whether variations in tissular cAMP levels induced by alpha-MSH might be explained by an interaction between the peptide and some dopaminergic receptors. Therefore, we measured cAMP in tissues and medium in response to alpha-MSH in the presence of haloperidol, the selective D1 (SCH 23390) or D2 (sulpiride) antagonists, or the selective D1 (SKF 38393) or D2 (bromocriptine) agonists. Haloperidol by itself induced no changes either in the cAMP content or in the cAMP efflux to the medium. When slices were exposed to alpha-MSH and haloperidol, the latter blocked the alpha-MSH effect of inducing an increase in the content of cAMP. None of the specific antagonists (at the administered doses) induced changes in the content of cAMP when compared with the control group. The presence of SCH 23390 in the incubation medium together with alpha-MSH yielded a reduction in cAMP levels compared with those incubated with alpha-MSH. A slight stimulatory effect on cAMP formation was observed when the dopaminergic agonists (SKF 38393 10 microM) were used. We conclude that alpha-MSH interacts with the D1 dopamine receptor, changing the cAMP levels in striatum and accumbens nuclei.

Independent and simultaneous effects of alpha-MSH and dopamine on cyclic AMP levels in rat brain slices.

This study was carried out to explore whether the postulated interaction between alpha-MSH and dopamine (DA) could be explained on the basis of variations in tissular cyclic AMP (cAMP) levels. We used slices containing caudate putamen and accumbens nuclei incubated in the presence of alpha-MSH, DA, or both simultaneously. Exposure of slices to alpha-MSH or DA resulted in an increase in cAMP levels. The simultaneous presence of alpha-MSH and DA resulted in a reduction in the accumulation of the cyclic nucleotide in the tissues as compared with those treated only with DA or alpha-MSH. The effect of alpha-MSH and DA on cAMP efflux was also tested. Incubation of slices with DA led to a marked increase in cAMP efflux; this efflux could be prevented if alpha-MSH was present in the medium. The results suggest that the inhibition by alpha-MSH of DA-induced striatal cAMP accumulation and efflux are a consequence of an interaction between the peptide and the neurotransmitter; DA and alpha-MSH transducing mechanisms could be biochemically linked.

Alpha MSH-induced excessive grooming behavior involves a GABAergic mechanism.

It has been shown that MSH administered in the ventral tegmental area (VTA) elicits excessive grooming behavior (EGB) by stimulating an acetylcholinergic pathway. The present work was performed in order to evaluate the possible participation of the GABAergic system in this behavior. VTA injection of GABA antagonist bicuculline stimulated the EGB (55.5 +/- 2.4). In contrast, this effect disappeared if the animals were pretreated with atropine (33.1 +/- 1.5). When bicuculline was injected before a 200 ng/microliters dose of MSH, the EGB increased (87.6 +/- 4.4) in comparison to MSH-treated rats (46.5 +/- 3.2). Our results suggest that GABA, ACh, and MSH interact in the VTA in the induction of EGB; an increase in MSH levels appears to stimulate cholinergic neurons. GABAergic fibers probably modulate the cholinergic discharge at the presynaptic level.