Role of cAMP in activation of ischemically sensitive abdominal visceral afferents

A number of metabolites produced during abdominal ischemia can stimulate and/or sensitize visceral afferents. The precise mechanisms whereby these metabolites act are uncertain. Other studies have shown that the adenylate cyclase-cAMP system may be involved in the activation of sensory neurons. Therefore, we hypothesized that cAMP contributes to the activation of ischemically sensitive abdominal visceral afferents. Single-unit activity of abdominal visceral C fibers was recorded from the right thoracic sympathetic chain in anesthetized cats before and during 7 min of abdominal ischemia. Forty-six percent of ischemically sensitive C fibers responded to intra-arterial injection of 8-bromo-cAMP (0.35-1. 0 mg/kg), an analog of cAMP, with responses during ischemia increasing from 0.50 +/- 0.06 to 0.84 +/- 0.08 impulses/s (P < 0.05, n = 11 C fibers). Conversely, an inhibitor of adenylate cyclase, 2', 5'-dideoxyadenosine (DDA; 0.1 mg/kg iv), attenuated ischemia-induced increase in activity of afferents from 0.66 +/- 0.10 to 0.34 +/- 0. 09 impulses/s (P < 0.05; n = 8). Furthermore, whereas exogenous PGE(2) (3-4 microg/kg ia) augmented the ischemia-induced increase in activity of afferents (P < 0.05, n = 10), treatment with DDA (0.1 mg/kg iv) substantially reduced the increase in discharge activity of afferents during ischemia, which was augmented by PGE(2) (1.45 +/- 0.24 vs. 0.70 +/- 0.09 impulses/s, -DDA vs. +DDA; P < 0.05) in six fibers. A time control group (n = 4), however, demonstrated similar increases in the activity of afferents with repeated administration of PGE(2). These data suggest that cAMP contributes to the activation of abdominal visceral afferents during ischemia, particularly to the action of PGs on activation and/or sensitization of these endings.     

Dopamine-stimulated adenylate cyclase in human term placenta

The effect of dopamine on adenylate cyclase activity was investigated in slices of human term placentas. Dopamine elicited a dose-dependent stimulation of cAMP formation with a ED50 value of about 1 X 10(-6)M dopamine and an increase of 110% over the control with 1 X 10(-4)M dopamine. (-)-Epinephrine and (-)-norepinephrine also increased placental cAMP formation. Apomorphine displayed a slight but non-significant stimulatory effect while bromocriptine was not effective. SCH 23390, a selective antagonist of dopamine D1 receptors caused a dose-dependent decrease of the dopamine activation. In contrast, the dopamine increase of cAMP was unaffected by beta- and alpha-adrenergic blocking drugs and by the D2 selective antagonist, (-)-sulpiride. These data indicate that dopamine stimulates cAMP formation in human term placenta through a specific mechanism via D1 dopaminergic receptors positively coupled to adenylate cyclase.     

Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

Extracellular Acidic Sulfur-Containing Amino Acids and γ-Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin-Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.     

Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.     

Role of ATP-sensitive K+ channels in electrophysiological alterations during myocardial ischemia: a study using Kir6.2-null mice

The role of cardiac ATP-sensitive K(+) (K(ATP)) channels in ischemia-induced electrophysiological alterations has not been thoroughly established. Using mice with homozygous knockout (KO) of Kir6.2 (a pore-forming subunit of cardiac K(ATP) channel) gene, we investigated the potential contribution of K(ATP) channels to electrophysiological alterations and extracellular K(+) accumulation during myocardial ischemia. Coronary-perfused mouse left ventricular muscles were stimulated at 5 Hz and subjected to no-flow ischemia. Transmembrane potential and extracellular K(+) concentration ([K(+)](o)) were measured by using conventional and K(+)-selective microelectrodes, respectively. In wild-type (WT) hearts, action potential duration (APD) at 90% repolarization (APD(90)) was significantly decreased by 70.1 +/- 5.2% after 10 min of ischemia (n = 6, P < 0.05). Such ischemia-induced shortening of APD(90) did not occur in Kir6.2-deficient (Kir6.2 KO) hearts. Resting membrane potential in WT and Kir6.2 KO hearts similarly decreased by 16.8 +/- 5.6 (n = 7, P < 0.05) and 15.0 +/- 1.7 (n = 6, P < 0.05) mV, respectively. The [K(+)](o) in WT hearts increased within the first 5 min of ischemia by 6.9 +/- 2.5 mM (n = 6, P < 0.05) and then reached a plateau. However, the extracellular K(+) accumulation similarly occurred in Kir6.2 KO hearts and the degree of [K(+)](o) increase was comparable to that in WT hearts (by 7.0 +/- 1.7 mM, n = 6, P < 0.05). In Kir6.2 KO hearts, time-dependent slowing of conduction was more pronounced compared with WT hearts. In conclusion, the present study using Kir6.2 KO hearts provides evidence that the activation of K(ATP) channels contributes to the shortening of APD, whereas it is not the primary cause of extracellular K(+) accumulation during early myocardial ischemia.     

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca(2+) permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASIC1a-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca(2+) elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.     

Dopamine mimics the cardioprotective effect of ischemic preconditioning via activation of alpha1-adrenoceptors in the isolated rat heart

The aim of the present study was to clarify whether pharmacological preconditioning with dopamine protects the heart against ischemia and whether this effect is mediated through dopaminergic receptors (D1 and D2) or alpha1-adrenoceptors. Isolated perfused rat hearts were either non-preconditioned, preconditioned with 5 min ischemia, or treated for 5 min with dopamine (1, 5 or 10 microM) before being subjected to 45 min of sustained ischemia followed by 60 min reperfusion. Postischemic functional recovery and infarct size were used as indices of the effects of ischemia. Treatment with the lower concentration of dopamine (1 microM), did not provide any protection to the ischemic myocardium. On the other hand, treatment with 5 microM dopamine resulted in significantly improved functional recovery, whereas administration of dopamine (10 microM) resulted in significantly improved functional recovery as well as reduction of infarct size. Pretreatment with the mixed D1/D2 dopaminergic receptor antagonist haloperidol or the beta-adrenoceptor selective antagonist propranolol did not attenuate the protective effect of pharmacological preconditioning with 10 microM dopamine with respect to both functional recovery and infarct size reduction. On the other hand, the cardioprotective effect of dopamine was blocked when the alpha1-adrenoceptor selective antagonist, prazosin, was administered. In conclusion, pharmacological preconditioning with dopamine protects the myocardium against ischemia and this effect seems to be mediated through activation of alpha1-adrenoceptors.     

An islet activating protein-sensitive G protein is involved in dopamine inhibition of angiotensin and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells

In primary culture of anterior pituitary cells, dopamine inhibited the angiotensin (AII)-stimulated inositol phosphate production by 28 +/- 2.5% (n = 14), with an EC50 of 660 +/- 228 nM (n = 8). This effect was blocked by (+)-butaclamol, a specific dopamine receptor antagonist. RU 24926, a D2 specific agonist, but not SKF 38393, a specific D1 agonist, inhibited AII-stimulated inositol phosphate production, suggesting that this dopamine effect is mediated through a dopamine receptor of the D2 subtype. Dopamine also partially inhibited (25%) inositol phosphate production stimulated by thyrotropin-releasing hormone (TRH). Our results suggest that the dopamine-mediated inhibition of hormonally stimulated inositol phosphate production is probably not mediated through the known inhibitory effects of dopamine on cAMP and Ca2+ intracellular concentrations. Although unknown, the mechanism by which dopamine inhibited the AII and TRH-stimulated inositol phosphate production implicates a GTP binding protein sensitive to the islet activating protein (IAP) since dopamine effects were blocked by this toxin. The alpha subunit of the GTP binding protein involved could be one of the three ADP-ribosylated proteins found in anterior pituitary cells in primary cultures, the alpha o (39 kDa), the alpha i (41 kDa), and an alpha subunit of 40 kDa. Indeed, we show here that this 40-kDa IAP substrate, already described in a few tissues, is present in anterior pituitary cells. The negative coupling between dopamine receptors and the AII or TRH inositol phosphate production systems, could be implicated in the dopamine inhibition of the AII- and TRH-stimulated prolactin release since such an inhibition is blocked by IAP. Our results suggest that the negative regulation of inositol phosphate production is one of the mechanisms by which dopamine controls hormonally stimulated prolactin release.     

Extracellular cAMP inhibits D1 dopamine receptor expression in CAD catecholaminergic cells via A2a adenosine receptors

The expression of D1 dopamine (DA) receptor gene is regulated during development, aging, and pathophysiology. The extracellular factors and signaling mechanisms that modulate the expression of D1 DA receptor have not been well characterized. Here, we present novel evidence that endogenous D1 DA receptor expression is inhibited by extracellular cAMP in the Cath.A Derived (CAD) catecholaminergic neuronal cell line. CAD cells express the multi-drug resistance protein 5 transporters and secrete cAMP. Addition of exogenous cAMP decreases D1 receptor mRNA and protein greater than fourfold in 24 h. The cAMP-induced decrease of D1 receptor mRNA levels is blocked by cGMP and by 1,3-dipropyl-8-(p-sulfo-phenyl)xanthine, an inhibitor of ecto-phosphodiestrase. Extracellular AMP, a metabolite of cAMP, also independently decreased D1 receptor mRNA levels. Inhibitors of ecto-nucleotidases, alpha,beta-methyleneadenosine 5'-di-phosphate and GMP, completely blocked the decrease of D1 receptor mRNA by extracellular cAMP, but only partially blocked the decrease induced by extracellular AMP. Levamisole, an inhibitor of tissue non-specific alkaline phosphatase, completely blocked the AMP-induced decrease of D1 receptor mRNA. The extracellular cAMP, AMP, and adenosine (ADO)-induced decrease in D1 receptor mRNA expression are mediated by A2a ADO receptor subtype. The results suggest a novel molecular mechanism linking activation of A2a ADO receptors with inhibition of D1 DA receptor expression.     

Vagal stimulation suppresses ischemia-induced myocardial interstitial norepinephrine release

Although electrical vagal stimulation exerts beneficial effects on the ischemic heart such as an antiarrhythmic effect, whether it modulates norepinephrine (NE) and acetylcholine (ACh) releases in the ischemic myocardium remains unknown. To clarify the neural modulation in the ischemic region during vagal stimulation, we examined ischemia-induced NE and ACh releases in anesthetized and vagotomized cats. In a control group (VX, n = 8), occlusion of the left anterior descending coronary artery increased myocardial interstitial NE level from 0.46+/-0.09 to 83.2+/-17.6 nM at 30-45 min of ischemia (mean+/-SE). Vagal stimulation at 5 Hz (VS, n = 8) decreased heart rate by approximately 80 beats/min during the ischemic period and suppressed the NE release to 24.4+/-10.6 nM (P < 0.05 from the VX group). Fixed-rate ventricular pacing (VSP, n=8) abolished this vagally mediated suppression of ischemia-induced NE release. The vagal stimulation augmented ischemia-induced ACh release at 0-15 min of ischemia (VX: 11.1+/-2.1 vs. VS: 20.7+/-3.9 nM, P < 0.05). In the VSP group, the ACh release was not augmented. In conclusion, vagal stimulation suppressed the ischemia-induced NE release and augmented the initial increase in the ACh level. These modulations of NE and ACh levels in the ischemic myocardium may contribute to the beneficial effects of vagal stimulation on the heart during acute myocardial ischemia.     

Dopamine- and cAMP-regulated Phosphoprotein of 32-kDa (DARPP-32)-dependent Activation of Extracellular Signal-regulated Kinase (ERK) and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Experimental Parkinsonism

Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia.     

D1-D2 dopamine receptor interaction within the nucleus accumbens mediates long-loop negative feedback to the ventral tegmental area (VTA)

The objective of the present study was to examine the effects of perfusion of dopamine (DA) D1- and D2-like receptor agonists in the nucleus accumbens (ACB) on the long-loop negative feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats employing ipsilateral dual probe in vivo microdialysis. Perfusion of the ACB for 60 min with the D1-like receptor agonist SKF 38393 (SKF, 1-100 microM) dose-dependently reduced the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA were not changed. Similarly, application of the D2-like receptor agonist quinpirole (Quin, 1-100 microM) through the microdialysis probe in the ACB reduced the extracellular levels of DA in the ACB in a concentration-dependent manner, whereas extracellular levels of DA in the VTA were not altered. Co-application of SKF (100 microM) and Quin (100 microM) produced concomitant reductions in the extracellular levels of DA in the ACB and VTA. The reduction in extracellular levels of DA in the ACB and VTA produced by co-infusion of SKF and Quin was reversed in the presence of either 100 microM SCH 23390 (D1-like antagonist) or 100 microM sulpiride (D2-like antagonist). Overall, the results suggest that (a) activation of dopamine D1- or D2-like receptors can independently regulate local terminal DA release in the ACB, whereas stimulation of both subtypes is required for activation of the negative feedback pathway to the VTA.     

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.     

Role of calcium and cAMP in heterologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in an osteoblast cell line.

To understand further the mechanism of action of parathyroid hormone (PTH) in the stimulation of the number of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding sites in UMR 106-01 cells we studied the role of cAMP and calcium. In addition to PTH other agents known to act via the cAMP signal pathway, prostaglandin E2, forskolin and dibutyryl cAMP, caused an increase in 1,25(OH)2D3 binding. Addition of the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl)adenine resulted in a marked decrease of PTH-stimulated cAMP production but this was not followed by a reduction of 1,25(OH)2D3 receptor up-regulation by PTH. Increasing the intracellular calcium concentration by Bay K 8644 and A23817 independent of an activation of the cAMP signal pathway did not result in an increased 1,25(OH)2D3 binding. The calcium channel blockers nitrendipine and verapamil and chelating extracellular calcium with EGTA all reduced cAMP-mediated stimulation of 1,25(OH)2D3 binding. This reduction was not due to a reduce cAMP production as verapamil even potentiated PTH- and forskolin-stimulated cAMP production in a dose-dependent manner. The present study provides evidence for an interrelated action of calcium and cAMP in the heterologous up-regulation of the 1,25(OH)2D3 receptor. The current data show an interaction between the cAMP and calcium signal pathway at (1) the level of cAMP generation/degradation, and (2) a level located distal in the cascade leading to 1,25(OH)2D3 receptor up-regulation.     

Functional characterization of the novel antipsychotic iloperidone at human D2, D3, alpha 2C, 5-HT6, and 5-HT1A receptors

Iloperidone has demonstrated an interesting monoamine receptor profile in radioligand binding studies, with nanomolar affinity for certain noradrenaline, dopamine, and serotonin receptors. In this study, the agonist/antagonist activity of iloperidone was determined in cell lines expressing recombinant human D(2A), D(3), alpha(2C), 5-HT(1A), or 5-HT(6) receptors. With the exception of 5-HT(6) receptors, these receptors are negatively coupled to cyclase. Thus, after stimulation with forskolin, the agonists dopamine (at D(2A) and D(3)), noradrenaline (at alpha(2C)), or 8-OH-DPAT (at 5-HT(1A)) induced a reduction in cAMP accumulation. Conversely, activation of the 5-HT(6) receptor by 5-HT led to an increase in cAMP accumulation. Iloperidone alone was devoid of significant agonist activity but inhibited the agonist response in all 5 cell lines in a surmountable and concentration-dependent fashion. Iloperidone was most potent at D(3) receptors (pK(B) 8.59 +/- 0.20; n = 6), followed by alpha(2C) (pK(B) 7.83 +/- 0.06; n = 15), 5-HT(1A) (pK(B) 7.69 +/- 0.18; n = 10), D(2A) (pK(B) 7.53 +/- 0.04; n = 11) and 5-HT(6) (pK(B) 7.11 +/- 0.08; n = 11) receptors.     

D2‐like dopamine receptors mediate regulation of pupal diapause in Chinese oak silkmoth Antheraea pernyi

The present study investigated the pharmacological properties of dopamine receptors that functioned in the termination of pupal diapause in the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). Dopamine receptors are classified according to their structure and function into two subfamilies as D1‐ and D2‐like receptors. D1‐like receptors activate, whereas D2‐like receptors inhibit, adenylate cyclase. We examined the effects of agonists and antagonists selective for D1‐ and D2‐like receptors on the diapause state. As A. pernyi is a long‐day species, pupal diapause is maintained during short days and can be terminated by exposure to a long‐day photoperiod. The D2‐like receptor‐selective agonist quinpirole delayed the timing of adult emergence under long days, and the D2‐receptor‐selective antagonist sulpiride terminated pupal diapause even under a short‐day photoperiod. The D1‐like receptor‐selective agonist and antagonist, SKF‐38393 and SCH‐23390, respectively, caused no significant effects on diapause pupae. These results suggest that not D1‐ but D2‐like receptors mediated diapause regulation in A. pernyi. This dopamine pathway appeared to block the termination of pupal diapause. Furthermore, the actions of the cAMP analog 8‐CPT‐cAMP and dopamine receptor antagonists upon diapause pupae were similar, which supports the notion that D2‐like receptors involved in diapause of this insect prevent adenylate cyclase from producing cAMP like vertebrate D2‐like receptors. Taken together, our findings suggest that dopamine blocked diapause termination through D2‐like receptors that inhibited adenylate cyclase in A. pernyi. During short days under which diapause was maintained in pupae, the dopaminergic mechanism might be stimulated to suppress cAMP levels in cells regulating diapause.     

Regulatory effects of D2 receptors in the ventral tegmental area on the mesocorticolimbic dopaminergic pathway

To investigate the regulatory effects of somatodendritic D2 receptors on the terminal's extracellular dopamine (DA) concentration, a D2 antagonist (eticlopride) was infused directly into the ventral tegmental area via a microdialysis probe in chloral hydrate-anesthetized rats. Extracellular DA changes in both the nucleus accumbens (N ACC) and the medial prefrontal cortex (mPFC) were monitored. Infusion of 10.0 fM eticlopride had no effect on DA in the mPFC (110.2 +/- 10.0% of baseline) but significantly increased DA in the N ACC (150.1 +/- 11.7%). Infusion of a higher dose of eticlopride (100.0 or 1,000.0 fM) significantly augmented the DA in the mPFC (121.1 +/- 7.6 and 180.7 +/- 25.8%, respectively) but surprisingly had no effect on DA in the N ACC (111.5 +/- 7.3 and 104.1 +/- 8.7%, respectively). To further investigate whether the bluntness of DA increase in the N ACC was due to DA receptor activation in the mPFC, eticlopride or SCH23390 was infused into the mPFC prior to and during intrategmental eticlopride infusion, and the change of DA in the N ACC was simultaneously monitored. During intra-mPFC 1.0 nM eticlopride infusion but not during 10.0 nM SCH23390 administration (95.5 +/- 6.1%), intrategmental 1,000.0 fM eticlopride infusion could further elevate DA in the N ACC (130.0 +/- 4.6%). Our results indicated that (1) the mesolimbic and the mesocortical pathways were under tonic inhibition by somatodendritic D2 receptors; (2) the DA concentration in the N ACC first increased and then returned to baseline while the intrategmental infusion dose of eticlopride increased; and (3) the bluntness of DA increase in the N ACC resulted from the D2 receptor activation in the mPFC.     

Effect of the Platelet-Activating Factor Antagonist BN 50739 and Its Diluents on Mitochondrial Respiration and Membrane Lipids During and Following Cerebral Ischemia

Abstract: Recent evidence suggests that platelet-activating factor plays a role in ischemia-induced neural injury. The Pulsinelli-Brierley four-vessel occlusion model was used to study the effect of a synthetic platelet-activating factor antagonist, BN 50739, and its solvents, either dimethyl sulfoxide or hydroxypropyl-β-cyclodextrin, on cerebral ischemia-reperfusion. Rats were subjected to either 30 min of ischemia or 30 min of ischemia followed by 60 min of recirculation. Changes in the brain mitochondrial free fatty acid pool size, fatty acyl composition of phospholipids, and respiratory function were monitored. When the BN 50739 (2 mg of BN 50739/kg of body weight i.v.) was administered at the onset of recirculation, it significantly reversed the ischemia-induced accumulation of mitochondrial free fatty acids and loss of polyunsaturated fatty acyl chains from phosphatidylcholine and phosphatidylethanolamine while simultaneously improving mitochondrial respiration. Dimethyl sulfoxide alone decreased the mitochondrial level of malonyldialdehyde and total free fatty acid pool size, but there was no improvement in mitochondrial respiration. Hydroxypropyl-β-cyclodextrin was reported to be pharmacologically inactive and capable of dissolving BN 50739. However, hydroxypropyl-β-cyclodextrin alone also caused a significant increase in content of cerebral mitochondrial membrane free fatty acids and hydrolysis of phosphatidylcholine in normoxic control animals. The overall effect of BN 50739 on mitochondrial structure and energy metabolism supports the hypothesis that platelet-activating factor may play a key role in ischemia-induced cerebral injury.