Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine.

Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX1 and OX2, Gq-coupled GPCRs. We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+]i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP3) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine-orexin A interaction in nucleus accumbens neurons.

1-Chromonyl-5-Imidazolylpentadienone Demonstrates Anti-Cancer Action against TNBC and Exhibits Synergism with Paclitaxel.

Curcumin has been well studied for its anti-oxidant, anti-inflammatory, and anti-cancer action. Its potential as a therapy is limited due to its low bioavailability and rapid metabolism. To overcome these challenges, investigators are developing curcumin analogs, nanoparticle formulations, and combining curcumin with other compounds or dietary components. In the present study, we used a 1-chromonyl-5-imidazolylpentadienone named KY-20-22 that contains both the pharmacophore of curcumin and 1,4 benzopyrone (chromone) moiety typical for flavonoids, and also included specific moieties to enhance the bioavailability. When we tested the in vitro effect of KY-20-22 in triple-negative breast cancer (TNBC) cell lines, we found that it decreased the cell survival and colony formation of MDA-MB-231 and MDA-MB-468 cells. An increase in mitochondrial reactive oxygen species was also observed in TNBC cells exposed to KY-20-22. Furthermore, KY-20-22 decreased epithelial-mesenchymal formation (EMT) as evidenced by the modulation of the EMT markers E-cadherin and N-cadherin. Based on the fact that KY-20-22 regulates interleukin-6, a cytokine involved in chemotherapy resistance, we combined it with paclitaxel and found that it synergistically induced anti-proliferative action in TNBC cells. The results from this study suggested that 1-chromonyl-5-imidazolylpentadienone KY-20-22 exhibited anti-cancer action in MDA-MB-231 and MDA-MB-468 cells. Future studies are required to evaluate the anti-cancer ability and bioavailability of KY-20-22 in the TNBC animal model.

Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV.

Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.

Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease.

Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.

Modulation of Calcium Entry by the Endo-lysosomal System.

Endo-lysosomes are acidic organelles that besides the role in macromolecules degradation, act as intracellular Ca(2+) stores. Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+)-mobilizing second messenger, produced in response to agonist stimulation, activates Ca(2+)-releasing channels on endo-lysosomes and modulates a variety of cellular functions. NAADP-evoked signals are amplified by Ca(2+) release from endoplasmic reticulum, via the recruitment of inositol 1,4,5-trisphosphate and/or ryanodine receptors through a Ca(2+)-induced Ca(2+)- release (CICR) mechanism. The endo-lysosomal Ca(2+) channels activated by NAADP were recently identified as the two-pore channels (TPCs). In addition to TPCs, endo-lysosomes express another distinct family of Ca(2+)- permeable channels, namely the transient receptor potential mucolipin (TRPML) channels, functionally distinct from TPCs. TPCs belong to the voltage-gated channels, resembling voltage-gated Na(+) and Ca(2+) channels. TPCs have important roles in vesicular fusion and trafficking, in triggering a global Ca(2+) signal and in modulation of the membrane excitability. Depletion of acidic Ca(2+) stores has been shown to activate store-operated Ca(2+) entry in human platelets and mouse pancreatic ß-cells. In human platelets, Ca(2+) influx in response to acidic stores depletion is facilitated by the tubulin-cytoskeleton and occurs through non-selective cation channels and transient receptor potential canonical (TRPC) channels. Emerging evidence indicates that activation of intracellular receptors, situated on endo-lysosomes, elicits canonical and non-canonical signaling mechanisms that involve CICR and activation of non-selective cation channels in plasma membrane. The ability of endo-lysosomal Ca(2+) stores to modulate the Ca(2+) release from other organelles and the Ca(2+) entry increases the diversity and complexity of cellular signaling mechanisms.

The Lysophosphatidylinositol Receptor GPR55 Modulates Pain Perception in the Periaqueductal Gray.

Emerging evidence indicates the involvement of GPR55 and its proposed endogenous ligand, lysophosphatidylinositol (LPI), in nociception, yet their role in central pain processing has not been explored. Using Ca(2+) imaging, we show here that LPI elicits concentration-dependent and GPR55-mediated increases in intracellular Ca(2+) levels in dissociated rat periaqueductal gray (PAG) neurons, which express GPR55 mRNA. This effect is mediated by Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptors and by Ca(2+) entry via P/Q-type of voltage-gated Ca(2+) channels. Moreover, LPI depolarizes PAG neurons and upon intra-PAG microinjection, reduces nociceptive threshold in the hot-plate test. Both these effects are dependent on GPR55 activation, because they are abolished by pretreatment with ML-193 [N-(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)-phenyl)-6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carboxamide], a selective GPR55 antagonist. Thus, we provide the first pharmacological evidence that GPR55 activation at central levels is pronociceptive, suggesting that interfering with GPR55 signaling in the PAG may promote analgesia.

G protein-coupled estrogen receptor-mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells.

G protein-coupled estrogen receptor (GPER) is a relatively recently identified non-nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca(2+) concentration, [Ca(2+)](i), nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G-1, a selective GPER agonist, or extracellular administration of 17-ß-estradiol and tamoxifen, increased [Ca(2+)](i) in RBMVEC. The effect of G-1 on [Ca(2+)](i) was abolished in Ca(2+) -free saline or in the presence of a L-type Ca(2+) channel blocker. G-1 increased nitric oxide production in RBMVEC; the effect was prevented by NG-nitro-l-arginine methyl ester. G-1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate-conductance Ca(2+) -activated K(+) channels, apamin, and charibdotoxin. GPER-mediated responses were sensitive to G-36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G-1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER-mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature. Activation of the G protein-coupled estrogen receptor (GPER) in rat brain microvascular endothelial cells (RBMVEC) increases [Ca(2+)](i) by promoting Ca(2+) influx. The increase in [Ca(2+)](i) leads to membrane hyperpolarization, nitric oxide (NO) production, and to cytoskeletal changes and increased cell stiffness. Our results unravel the mechanisms underlying GPER-mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

Direct evidence of intracrine angiotensin II signaling in neurons.

The existence of a local renin-angiotensin system (RAS) in neurons was first postulated 40 years ago. Further studies indicated intraneuronal generation of ANG II. However, the function and signaling mechanisms of intraneuronal ANG II remained elusive. Since ANG II type 1 receptor (AT1R) is the major type of receptor mediating the effects of ANG II, we used intracellular microinjection and concurrent Ca(2+) and voltage imaging to examine the functionality of intracellular AT1R in neurons. We show that intracellular administration of ANG II produces a dose-dependent elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) in hypothalamic neurons that is sensitive to AT1R antagonism. Endolysosomal, but not Golgi apparatus, disruption prevents the effect of microinjected ANG II on [Ca(2+)]i. Additionally, the ANG II-induced Ca(2+) response is dependent on microautophagy and sensitive to inhibition of PLC or antagonism of inositol 1,4,5-trisphosphate receptors. Furthermore, intracellular application of ANG II produces AT1R-mediated depolarization of hypothalamic neurons, which is dependent on [Ca(2+)]i increase and on cation influx via transient receptor potential canonical channels. In summary, we provide evidence that intracellular ANG II activates endolysosomal AT1Rs in hypothalamic neurons. Our results point to the functionality of a novel intraneuronal angiotensinergic pathway, extending the current understanding of intracrine ANG II signaling.

Differential activation of intracellular versus plasmalemmal CB2 cannabinoid receptors.

The therapeutic and psychoactive properties of cannabinoids have long been recognized. The type 2 receptor for cannabinoids (CB2) has emerged as an important therapeutic target in several pathologies, as it mediates beneficial effects of cannabinoids while having little if any psychotropic activity. Difficulties associated with the development of CB2-based therapeutic agents have been related to its intricate pharmacology, including the species specificity and functional selectivity of the CB2-initiated responses. We postulated that a plasmalemmal or subcellular location of the receptor may contribute to the differential signaling pathways initiated by its activation. To differentiate between these two, we used extracellular and intracellular administration of CB2 ligands and concurrent calcium imaging in CB2-expressing U2OS cells. We found that extracellular administration of anandamide was ineffective, whereas 2-arachidonoyl glycerol (2-AG) and WIN55,212-2 triggered delayed, CB2-dependent Ca(2+) responses that were Gq protein-mediated. When microinjected, all agonists elicited fast, transient, and dose-dependent elevations in intracellular Ca(2+) concentration upon activation of Gq-coupled CB2 receptors. The CB2 dependency was confirmed by the sensitivity to AM630, a selective CB2 antagonist, and by the unresponsiveness of untransfected U2OS cells to 2-AG, anandamide, or WIN55,212-2. Moreover, we provide functional and morphological evidence that CB2 receptors are localized at the endolysosomes, while their activation releases Ca(2+) from inositol 1,4,5-trisphosphate-sensitive- and acidic-like Ca(2+) stores. Our results support the functionality of intracellular CB2 receptors and their ability to couple to Gq and elicit Ca(2+) signaling. These findings add further complexity to CB2 receptor pharmacology and argue for careful consideration of receptor localization in the development of CB2-based therapeutic agents.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger in muscarinic receptor-induced contraction of guinea pig trachea.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is increasingly being demonstrated to be involved in calcium signaling in many cell types and species. Although it has been shown to play a role in smooth muscle cell contraction in several tissues, nothing is known about its possible role in tracheal smooth muscle, a muscle type that is clinically relevant to asthma. To determine whether NAADP functions as a second messenger in tracheal smooth muscle contraction, we used the criteria set out by Sutherland for a molecule to be designated a second messenger. We report that NAADP satisfies all five criteria as follows. First, the NAADP antagonist Ned-19 inhibited contractions in tracheal rings and calcium increases in isolated smooth muscle cells induced by the muscarinic agonist carbachol. Second, NAADP increased cytosolic calcium in isolated cells when microinjected and was blocked by Ned-19. Third, tracheal homogenates could synthesize NAADP by base exchange from exogenous NADP and nicotinic acid and metabolize exogenous NAADP to nicotinic acid adenine dinucleotide by a 2'-phosphatase. Fourth, carbachol induced a rapid and transient increase in endogenous NAADP levels. Fifth, tracheal homogenates contained NAADP-binding sites of high affinity. Taken together, these data demonstrate that NAADP functions as a second messenger in tracheal smooth muscle, and therefore, steps in the NAADP signaling pathway might provide possible new drug targets.

Differential activation of cultured neonatal cardiomyocytes by plasmalemmal versus intracellular G protein-coupled receptor 55.

The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca(2+) signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca(2+) entry via L-type Ca(2+) channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca(2+) release. The latter signal is further amplified by Ca(2+)-induced Ca(2+) release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca(2+) release from acidic-like Ca(2+) stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca(2+)-induced Ca(2+) release via ryanodine receptors. Extracellularly applied LPI produces Ca(2+)-independent membrane depolarization, whereas the Ca(2+) signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders.

HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats.

The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1-86) produced a concentration-dependent increase in cytosolic Ca(2+) in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca(2+) elevation: 1) lysosomal Ca(2+) mobilization, 2) Ca(2+) release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca(2+) influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca(2+) entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.

Aldosterone increases cardiac vagal tone via G protein-coupled oestrogen receptor activation.

In addition to acting on mineralocorticoid receptors, aldosterone has been recently shown to activate the G protein-coupled oestrogen receptor (GPER) in vascular cells. In light of the newly identified role for GPER in vagal cardiac control, we examined whether or not aldosterone activates GPER in rat nucleus ambiguus. Aldosterone produced a dose-dependent increase in cytosolic Ca(2+) concentration in retrogradely labelled cardiac vagal neurons of nucleus ambiguus; the response was abolished by pretreatment with the GPER antagonist G-36, but was not affected by the mineralocorticoid receptor antagonists, spironolactone and eplerenone. In Ca(2+)-free saline, the response to aldosterone was insensitive to blockade of the Ca(2+) release from lysosomes, while it was reduced by blocking the Ca(2+) release via ryanodine receptors and abolished by blocking the IP3 receptors. Aldosterone induced Ca(2+) influx via P/Q-type Ca(2+) channels, but not via L-type and N-type Ca(2+) channels. Aldosterone induced depolarization of cardiac vagal neurons of nucleus ambiguus that was sensitive to antagonism of GPER but not of mineralocorticoid receptor. in vivo studies, using telemetric measurement of heart rate, indicate that microinjection of aldosterone into the nucleus ambiguus produced a dose-dependent bradycardia in conscious, freely moving rats. Aldosterone-induced bradycardia was blocked by the GPER antagonist, but not by the mineralocorticoid receptor antagonists. In summary, we report for the first time that aldosterone decreases heart rate by activating GPER in cardiac vagal neurons of nucleus ambiguus.

Intracellular endothelin type B receptor-driven Ca2+ signal elicits nitric oxide production in endothelial cells.

Endothelin-1 exerts its actions via activation of ET(A) and ET(B) G(q/11) protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET(B) receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca(2+) signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET(B)-transfected cells and endothelial cells; this response is sensitive to ET(B) but not ET(A) receptor blockade. In endothelial cells, the endothelin-1-induced Ca(2+) response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET(B)-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET(B) receptors and increase cytosolic Ca(2+) and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET(B) to induce nitric oxide formation, thus modulating endothelial function.

Nesfatin-1 activates cardiac vagal neurons of nucleus ambiguus and elicits bradycardia in conscious rats.

Nesfatin-1, a peptide whose receptor is yet to be identified, has been involved in the modulation of feeding, stress, and metabolic responses. More recently, increasing evidence supports a modulatory role for nesfatin-1 in autonomic and cardiovascular activity. This study was undertaken to test if the expression of nesfatin-1 in the nucleus ambiguus, a key site for parasympathetic cardiac control, may be correlated with a functional role. As we have previously demonstrated that nesfatin-1 elicits Ca²âº signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca²âº in cardiac pre-ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin-1 increases cytosolic Ca²âº concentration via a Gi/o-coupled mechanism. The nesfatin-1-induced Ca²âº rise is critically dependent on Ca²âº influx via P/Q-type voltage-activated Ca²âº channels. Repeated administration of nesfatin-1 leads to tachyphylaxis. Furthermore, nesfatin-1 produces a dose-dependent depolarization of cardiac vagal neurons via a Gi/o-coupled mechanism. In vivo studies, using telemetric and tail-cuff monitoring of heart rate and blood pressure, indicate that microinjection of nesfatin-1 into the nucleus ambiguus produces bradycardia not accompanied by a change in blood pressure in conscious rats. Taken together, our results identify for the first time that nesfatin-1 decreases heart rate by activating cardiac vagal neurons of nucleus ambiguus. Our results indicate that nesfatin-1, one of the most potent feeding peptides, increases cytosolic Ca²âº by promoting Ca²âº influx via P/Q channels and depolarizes nucleus ambiguus neurons; both effects are Gi/o-mediated. In vivo studies indicate that microinjection of nesfatin-1 into nucleus ambiguus produces bradycardia in conscious rats. This is the first report that nesfatin-1 increases the parasympathetic cardiac tone.

Bradycardic effects mediated by activation of G protein-coupled estrogen receptor in rat nucleus ambiguus.

The G protein-coupled estrogen receptor (GPER) has been identified in several brain regions, including cholinergic neurons of the nucleus ambiguus, which are critical for parasympathetic cardiac regulation. Using calcium imaging and electrophysiological techniques, microinjection into the nucleus ambiguus and blood pressure measurement, we examined the in vitro and in vivo effects of GPER activation in nucleus ambiguus neurons. A GPER selective agonist, G-1, produced a sustained increase in cytosolic Ca(2+) concentration in a concentration-dependent manner in retrogradely labelled cardiac vagal neurons of nucleus ambiguus. The increase in cytosolic Ca(2+) produced by G-1 was abolished by pretreatment with G36, a GPER antagonist. G-1 depolarized cultured cardiac vagal neurons of the nucleus ambiguus. The excitatory effect of G-1 was also identified by whole-cell visual patch-clamp recordings in nucleus ambiguus neurons, in medullary slices. To validate the physiological relevance of our in vitro studies, we carried out in vivo experiments. Microinjection of G-1 into the nucleus ambiguus elicited a decrease in heart rate; the effect was blocked by prior microinjection of G36. Systemic injection of G-1, in addition to a previously reported decrease in blood pressure, also reduced the heart rate. The G-1-induced bradycardia was prevented by systemic injection of atropine, a muscarinic antagonist, or by bilateral microinjection of G36 into the nucleus ambiguus. Our results indicate that GPER-mediated bradycardia occurs via activation of cardiac parasympathetic neurons of the nucleus ambiguus and support the involvement of the GPER in the modulation of cardiac vagal tone.

Intracellular cannabinoid type 1 (CB1) receptors are activated by anandamide.

Recent studies have demonstrated that the majority of endogenous cannabinoid type 1 (CB(1)) receptors do not reach the cell surface but are instead associated with endosomal and lysosomal compartments. Using calcium imaging and intracellular microinjection in CB(1) receptor-transfected HEK293 cells and NG108-15 neuroblastoma × glioma cells, we provide evidence that anandamide acting on CB(1) receptors increases intracellular calcium concentration when administered intracellularly but not extracellularly. The calcium-mobilizing effect of intracellular anandamide was dose-dependent and abolished by pretreatment with SR141716A, a CB(1) receptor antagonist. The anandamide-induced calcium increase was reduced by blocking nicotinic acid-adenine dinucleotide phosphate- or inositol 1,4,5-trisphosphate-dependent calcium release and abolished when both lysosomal and endoplasmic reticulum calcium release pathways were blocked. Taken together, our results indicate that, in CB(1) receptor-transfected HEK293 cells, intracellular CB(1) receptors are functional; they are located in acid-filled calcium stores (endolysosomes). Activation of intracellular CB(1) receptors releases calcium from endoplasmic reticulum and lysosomal calcium stores. In addition, our results support a novel role for nicotinic acid-adenine dinucleotide phosphate in cannabinoid-induced calcium signaling.

Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons.

Urocortin 3 (also known as stresscopin) is an endogenous ligand for the corticotropin-releasing factor receptor 2 (CRF(2)). Despite predominant G(s) coupling of CRF(2), promiscuous coupling with other G proteins has been also associated with the activation of this receptor. As urocortin 3 has been involved in central cardiovascular regulation at hypothalamic and medullary sites, we examined its cellular effects on cardiac vagal neurons of nucleus ambiguus, a key area for the autonomic control of heart rate. Urocortin 3 (1 nM-1000 nM) induced a concentration-dependent increase in cytosolic Ca(2+) concentration that was blocked by the CRF(2) antagonist K41498. In the case of two consecutive treatments with urocortin 3, the second urocortin 3-induced Ca(2+) response was reduced, indicating receptor desensitization. The effect of urocortin 3 was abolished by pre-treatment with pertussis toxin and by inhibition of phospolipase C with U-73122. Urocortin 3 activated Ca(2+) influx via voltage-gated P/Q-type channels as well as Ca(2+) release from endoplasmic reticulum. Urocortin 3 promoted Ca(2+) release via inositol 1,4,5 trisphosphate receptors, but not ryanodine receptors. Our results indicate a novel Ca(2+) -mobilizing effect of urocortin 3 in vagal pre-ganglionic neurons of nucleus ambiguus, providing a cellular mechanism for a previously reported role for this peptide in parasympathetic cardiac regulation.

Intracellular angiotensin II activates rat myometrium.

Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.