Mitochondrial Dysfunction is Associated With an Immune Paralysis Phenotype in Pediatric Sepsis.

OBJECTIVE: Immune dysregulation is a defining feature of sepsis, but the role for mitochondria in the development of immunoparalysis in pediatric sepsis is not known. We sought to determine if mitochondrial dysfunction measured in peripheral blood mononuclear cells (PBMCs) is associated with immunoparalysis and systemic inflammation in children with sepsis. DESIGN: Prospective observational study. SETTING: Single-academic pediatric intensive care unit (PICU). PATIENTS: One hundred sixty-one children with sepsis/septic shock and 18 noninfected PICU controls. MEASUREMENTS AND MAIN RESULTS: Mitochondrial respiration in PBMCs, markers of immune function, and plasma cytokines were measured on days 1 to 2 (T1), 3 to 5 (T2), and 8 to 14 (T3) after sepsis recognition, and once for controls. Immunoparalysis was defined as whole-blood ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-α) ≤200 pg/mL or monocyte human leukocyte antigen-DR ≤30%. Mitochondrial respiration was lower in children with versus without immunoparalysis measured at the same timepoint. Mitochondrial respiration measured early (at T1 and T2) was also lower in those with immunoparalysis at T2 and T3, respectively. Although most patients with immunoparalysis exhibited low mitochondrial respiration, this metabolic finding was not specific to the immunoparalysis phenotype. Plasma cytokines, including IL-8, IL-10, TNF-α, and MCP-1, were highest in the subset of sepsis patients with immune paralysis or low mitochondrial respiration at T1. CONCLUSIONS: Children with sepsis had lower PBMC mitochondrial respiration when immunoparalysis was present compared with those without immunoparalysis. The subsets with immune paralysis and low mitochondrial respiration exhibited the highest levels of systemic inflammation.

Substance P-mediated chemokine production promotes monocyte migration.

The neuropeptide SP has physiologic and pathophysiologic roles in CNS and peripheral tissues and is involved in crosstalk between nervous and immune systems in various conditions, including HIV and SIV infection. Increased SP levels were demonstrated in plasma of HIV+ individuals as well as in the CNS of SIV-infected, nonhuman primates. SP increases HIV infection in macrophages through interaction with its receptor, NK1R. The SP effect on immune system is both pro- and anti-inflammatory and includes up-regulation of a number of cytokines and cell receptors. The main goal of this study was to determine whether there is interplay between monocyte exposure to SP and recruitment into sites of inflammation. We now demonstrate that exposure of either human macrophages or PBMCs to SP leads to increased production of chemokines, including MCP-1, for which expression is limited to cells of the myeloid lineage. This effect is inhibited by the NK1R antagonist, aprepitant. Exposure to conditioned medium derived from SP-treated PBMCs resulted in increased monocyte migration through semipermeable membranes and an in vitro human BBB model. Monocyte migration was blocked by anti-MCP-1 antibodies. Our results suggest that increased SP levels associated with HIV and other inflammatory conditions may contribute to increased monocyte migration into the CNS and other tissues through a MCP-1-dependent mechanism.

Decreased PD-1 Expression on CD8 Lymphocyte Subsets and Increase in CD8 Tscm Cells in Children with HIV Receiving Raltegravir.

We investigated the effect of combination antiretroviral therapy (cART) on immune recovery, particularly on the percentages of PD-1-positive cells within the major leukocyte subsets. Cryopreserved peripheral blood mononuclear cells and plasma samples collected longitudinally from a subset of 13 children and adolescents (between 9.7 and 18.2 years old) who were enrolled in the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) P1066 were used for this study. Immunophenotyping by flow cytometry was performed to determine the effect of raltegravir-containing cART regimen on the distribution of leukocyte populations, on the expression of PD-1 on T cell subpopulations, and on the expression of well-established markers of T cell activation (CD38 and HLA-DR) on CD8 T cells. C reactive protein (CRP), lipopolysaccharide (LPS), IL-6, and soluble CD163 were assayed in plasma samples by an enzyme-linked immunosorbent assay. Plasma viral loads were decreased in all subjects (by an average of 2.9 log units). The cART regimen, including raltegravir, induced changes in CD8 T cell subsets, consistent with an effective antiretroviral outcome and improved immunologic status, including increased percentages of CD8 stem cell memory T cells (Tscm). The percentages of CD8 PD-1-positive cells decreased significantly as compared with baseline levels. Among the proinflammatory markers measured in plasma, sCD163 showed a decline that was associated with cART. cART therapy, including raltegravir, over 48 weeks in children is associated with immune restoration, consistent with effective antiretroviral therapy, namely decreased percentages of PD-1+ CD8+ T cells, an increase in CD8 Tscm cells, and decreased levels of sCD163.

Mitochondrial dysfunction in peripheral blood mononuclear cells in pediatric septic shock.

OBJECTIVES: Mitochondrial dysfunction in peripheral blood mononuclear cells has been linked to immune dysregulation and organ failure in adult sepsis, but pediatric data are limited. We hypothesized that pediatric septic shock patients exhibit mitochondrial dysfunction within peripheral blood mononuclear cells which in turn correlates with global organ injury. DESIGN: Prospective observational study. SETTING: Academic PICU. PATIENTS: Thirteen pediatric patients with septic shock and greater than or equal to two organ failures and 11 PICU controls without sepsis or organ failure. INTERVENTIONS: Ex vivo measurements of mitochondrial oxygen consumption and membrane potential (ΔΨm) were performed in intact peripheral blood mononuclear cells on day 1-2 and day 5-7 of septic illness and in controls. The Pediatric Logistic Organ Dysfunction score, inotrope score, and organ failure-free days were determined from medical records. MEASUREMENTS AND MAIN RESULTS: Spare respiratory capacity, an index of bioenergetic reserve, was lower in septic peripheral blood mononuclear cells on day 1-2 (median, 1.81; interquartile range, 0.52-2.09 pmol O2/s/10 cells) compared with controls (5.55; 2.80-7.21; p = 0.03). Spare respiratory capacity normalized by day 5-7. Patients with sepsis on day 1-2 exhibited a higher ratio of LEAK to maximal respiration than controls (17% vs < 1%; p = 0.047) with normalization by day 5-7 (1%; p = 0.008), suggesting mitochondrial uncoupling early in sepsis. However, septic peripheral blood mononuclear cells exhibited no differences in basal or adenosine triphosphate-linked oxygen consumption or ΔΨm. Oxygen consumption did not correlate with Pediatric Logistic Organ Dysfunction score, inotrope score, or organ failure-free days (all p > 0.05). Although there was a weak overall association between ΔΨm on day 1-2 and organ failure-free days (Spearman ρ = 0.56, p = 0.06), patients with sepsis with normal organ function by day 7 exhibited higher ΔΨm on day 1-2 compared with patients with organ failure for more than 7 days (p = 0.04). CONCLUSIONS: Mitochondrial dysfunction was present in peripheral blood mononuclear cells in pediatric sepsis, evidenced by decreased bioenergetic reserve and increased uncoupling. Mitochondrial membrane potential, but not respiration, was associated with duration of organ injury.

Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling.

Exocytosis is tightly regulated in many cellular processes, from neurite expansion to tumor proliferation. Rab8, a member of the Rab family of small GTPases, plays an important role in membrane trafficking from the trans-Golgi network and recycling endosomes to the plasma membrane. Rabin8 is a guanine nucleotide exchange factor (GEF) and major activator of Rab8. Investigating how Rabin8 is activated in cells is thus pivotal to the understanding of the regulation of exocytosis. Here we show that phosphorylation serves as an important mechanism for Rabin8 activation. We identified Rabin8 as a direct phospho-substrate of ERK1/2 in response to EGF signaling. At the molecular level, ERK phosphorylation relieves the autoinhibition of Rabin8, thus promoting its GEF activity. We further demonstrate that blocking ERK1/2-mediated phosphorylation of Rabin8 inhibits transferrin recycling to the plasma membrane. Together, our results suggest that ERK1/2 activate Rabin8 to regulate vesicular trafficking to the plasma membrane in response to extracellular signaling.

Autosomal dominant hypoparathyroidism caused by germline mutation in GNA11: phenotypic and molecular characterization.

CONTEXT: Most cases of autosomal dominant hypoparathyroidism (ADH) are caused by gain-of-function mutations in CASR or dominant inhibitor mutations in GCM2 or PTH. OBJECTIVE: Our objectives were to identify the genetic basis for ADH in a multigenerational family and define the underlying disease mechanism. SUBJECTS: Here we evaluated a multigenerational family with ADH in which affected subjects had normal sequences in these genes and were shorter than unaffected family members. METHODS: We collected clinical and biochemical data from 6 of 11 affected subjects and performed whole-exome sequence analysis on DNA from two affected sisters and their affected father. Functional studies were performed after expression of wild-type and mutant Gα11 proteins in human embryonic kidney-293-CaR cells that stably express calcium-sensing receptors. RESULTS: Whole-exome-sequencing followed by Sanger sequencing revealed a heterozygous mutation, c.179G>T; p.R60L, in GNA11, which encodes the α-subunit of G11, the principal heterotrimeric G protein that couples calcium-sensing receptors to signal activation in parathyroid cells. Functional studies of Gα11 R60L showed increased accumulation of intracellular concentration of free calcium in response to extracellular concentration of free calcium with a significantly decreased EC50 compared with wild-type Gα11. By contrast, R60L was significantly less effective than the oncogenic Q209L form of Gα11 as an activator of the MAPK pathway. Compared to subjects with CASR mutations, patients with GNA11 mutations lacked hypercalciuria and had normal serum magnesium levels. CONCLUSIONS: Our findings indicate that the germline gain-of-function mutation of GNA11 is a cause of ADH and implicate a novel role for GNA11 in skeletal growth.

HIV infection of macrophages is enhanced in the presence of increased expression of CD163 induced by substance P.

Activation of NK1R by SP contributes to increased HIV-1 infection in macrophages. The scavenger receptor CD163 is expressed on cells of monocyte-macrophage origin. Our main goal was to determine if there is interplay among SP, CD163 expression, and HIV infection in macrophages. We showed that SP triggers intracellular calcium elevation and increased CD163 expression in human monocytes in a time- and concentration-dependent manner. The role of CD163 on HIV infection was examined by RT-PCR in sorted monocytes (CD163(low) and CD163(high)) and in macrophages having CD163 knocked down using siRNA. We found that the productivity of HIV infection was higher in CD163(high) cells. Additionally, in macrophages with CD163 expression knocked down, we found a significant decrease of HIV infection. Furthermore, Hb-Hp complexes, which function as an endogenous ligand for CD163, decreased HIV infection in macrophages in a dose-dependent manner. Thus, we demonstrate that SP induces higher levels of CD163 in monocytes and that high expression of CD163 is associated with increases HIV infection in macrophages. Thus, in addition to being a prognostic marker of HIV infection, the expression of CD163 on macrophages may be critical in HIV immunopathogenesis.

Analog of somatostatin vapreotide exhibits biological effects in vitro via interaction with neurokinin-1 receptor.

OBJECTIVES: Vapreotide, a synthetic analog of somatostatin, has analgesic activity most likely mediated through the blockade of neurokinin-1 receptor (NK1R), the substance P (SP)-preferring receptor. The ability of vapreotide to interfere with other biological effects of SP has yet to be investigated. METHODS: We studied the ability of vapreotide to antagonize NK1R in three different cell types: immortalized U373MG human astrocytoma cells, human monocyte-derived macrophages (MDM) and a human embryonic kidney cell line, HEK293. Both U373MG and MDM express endogenous NK1R while HEK293 cells, which normally do not express NK1R, are stably transformed to express human NK1R (HEK293-NK1R). RESULTS: Vapreotide attenuates SP-triggered intracellular calcium increases and nuclear factor-κB activation in a dose-dependent manner. Vapreotide also inhibits SP-induced interleukin-8 and monocyte chemotactic protein-1 production in HEK293-NK1R and U373MG cell lines. Vapreotide inhibits HIV-1 infection of human MDM in vitro, an effect that is reversible by SP pretreatment. CONCLUSIONS: Our findings indicate that vapreotide has NK1R antagonist activity and may have a potential application as a therapeutic intervention in HIV-1 infection.

Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

Substance P induces rapid and transient membrane blebbing in U373MG cells in a p21-activated kinase-dependent manner.

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.

Substance P inhibits natural killer cell cytotoxicity through the neurokinin-1 receptor.

SP is a potent neuroimmunomodulator that functions through ligating members of the neurokinin receptor family, one of which, NK1R, is widely expressed in immune cells. As in humans, circulating SP levels are increased in pathologic states associated with impairment of NK cell functions, such as depression and HIV infection, we hypothesized that SP has a direct, inhibitory effect upon NK cells. We have studied a clonal human NK cell line (YTS) as well as ex vivo human NK cells and have determined that truncated and full-length NK1R isoforms are expressed in and SP bound by ex vivo NK cells and the YTS NK cell line. Incubation of YTS cells with 10⁻6 M SP and ex vivo NK cells with 10⁻5 M SP inhibited cytotoxic ability by ∼20% and reduced degranulation. This inhibitory effect upon cytotoxicity was partially prevented by the NK1R antagonist CP96,345. The treatment of YTS or ex vivo NK cells with SP neither down-modulated NCR expression nor affected triggering receptor-induced NF-κB activation. Preincubation of YTS cells with SP, however, did abbreviate the typically prolonged intracellular calcium increase induced by target cell engagement and reduced triggering receptor-induced pERK. Thus, SP has the potential to regulate NK cell functions and acts downstream from neurokinin receptors to modulate NK cell activation signaling. This mechanism may contribute to impairment of NK cell function in certain disease states associated with increased circulating SP. Antagonism of this system may present an opportunity to augment NK cell function therapeutically in selected human diseases.

Neurokinin-1 receptor (NK1-R) expression in the brains of SIV-infected rhesus macaques: implications for substance P in NK1-R immune cell trafficking into the CNS.

Recent studies suggest a link between neuropsychiatric disorders and HIV/SIV infection. Most evidence indicates that monocytes/macrophages are the primary cell type infected within the CNS and that they contribute to CNS inflammation and neurological disease. Substance P (SP), a pleotropic neuropeptide implicated in inflammation, depression, and immune modulation via interaction with its cognate receptor, the neurokinin 1 receptor (NK1-R), is produced by monocyte/macrophages. While the presence of NK1-R on neurons is well known, its role on cells of the immune system such as monocyte/macrophages is just beginning to emerge. Therefore, we have examined the expression of SP and NK1-R and their relationship to SIV/HIV encephalitis (SIVE/HIVE) lesions and SIV-infected cells. These studies demonstrated intense expression of SP and NK1-R in SIVE lesions, with macrophages being the principal cell expressing NK1-R. Interestingly, all of the SIV-infected macrophages expressed NK1-R. Additionally, we examined the functional role of SP as a proinflammatory mediator of monocyte activation and chemotaxis. These studies demonstrated that treatment of monocytes with SP elicited changes in cell-surface expression for CCR5 and NK1-R in a dose-dependent manner. Moreover, pretreatment with SP enhanced both SP- and CCL5-mediated chemotaxis. All of these findings suggest that SP and NK1-R are important in SIV infection of macrophages and the development of SIVE lesions.

Neurokinin 1 receptor isoforms and the control of innate immunity.

Substance P is the prototype tachykinin peptide and triggers a variety of biological effects in both the nervous and immune system. Two naturally occurring variants of the neurokinin 1 receptor (NK1R) mediate the effects of SP: a 'classic' full-length receptor and a truncated (tail-less) form that lacks 96 amino acid residues at the C-terminus. Most research has focused on the full length receptor and the truncated NK1R has not been extensively explored. Recent data demonstrate that truncated NK1R has important functional roles, including modulation of responses triggered by cytokines, chemotaxis of macrophages and regulation of HIV replication. Targeting the truncated NK1R with pharmacologic agents might result in novel therapeutic approaches in diseases which affect the immune system, including HIV disease.

Mechanism of activation and functional role of protein kinase Ceta in human platelets.

The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

Neurokinin-1 receptor expression and function in human macrophages and brain: perspective on the role in HIV neuropathogenesis.

Substance P (SP) is upregulated in HIV infection in adult men and women, as determined by increased plasma levels. There is a reciprocal and bidirectional relationship between substance P and HIV in HIV-infected monocyte-derived macrophages and cell lines (e.g., THP-1). Substance P up-regulates HIV and HIV up-regulates SP protein expression. Neurokinin-1 receptor (NK1R) antagonists inhibit HIV infectivity through downregulation of the chemokine receptor, CCR5, and downregulation of HIV LTR. Neurokinin-1 receptor is expressed in full-length and truncated forms. The full-length NK1R is capable of signaling, whereas the truncated NK1R primes the chemokine receptor CCR5. Both full-length and truncated NK1R are expressed in several brain regions in human autopsy brains. SP-NK1R interactions have regulatory roles in inflammation and infection. The differential expression of truncated and full-length NK1R has important biological consequences. These include receptor-receptor interaction (e.g., NK1R-CCR5); changes in expression during cell differentiation (e.g., THP-1 cells); and differences in regional tissue distribution (e.g., differences in different brain regions). NK1R-SP receptor pathways are important cell regulatory pathways.

Neurokinin-1 receptor antagonist (aprepitant) inhibits drug-resistant HIV-1 infection of macrophages in vitro.

BACKGROUND: Despite the success of antiretroviral therapy in controlling HIV replication, treatment failure may ultimately occur in more than 50% of the individuals on antiretroviral therapy. Cellular targets offer an attractive alternative, as it may be more difficult for HIV to develop resistance to alternative cellular inhibitory pathways. We have previously shown that CP-96,345, a neurokinin-1 receptor (NK-1R) antagonist, inhibits HIV-1 infection of macrophages in vitro by downregulating CCR5 expression (Lai JP, Ho WZ, Zhan GX, Yi Y, Collman RG, Douglas SD 2001). We have now investigated the effects of a Food and Drug Administration (FDA)-approved NK-1R antagonist, aprepitant (Emend), on HIV infection of macrophages in an in vitro system. Aprepitant is in clinical use for the prevention of nausea and vomiting associated with cancer chemotherapy or following surgical procedures. METHODS: Monocytes isolated from healthy donors were cultured for 7 days and then treated with or without aprepitant (10(-6) M) for 2 h, followed by HIV infection with drug-resistant strains for 2 h. Untreated and HIV-infected macrophages were used as controls. Culture supernatants were harvested for p24 enzyme-linked immunosorbent assay (ELISA) or HIV reverse transcriptase (RT) activity at different time points after infection. R5X4 tropic and AZT-resistant strains (R5X4 tropic: A012 and A018) and RT inhibitor-resistant HIV strains (R5 tropic: TC60 and TC49) were used for infection. RESULTS: Aprepitant suppressed HIV Bal infection of macrophages. Treatment with aprepitant (10(-6) M) inhibited infection of macrophages with the AZT-resistant viruses (A018, A012) by 0.7 log(10). Aprepitant also suppressed infection of macrophages with RT inhibitor-resistant virus (TC 49 and TC 60) by 0.5 log(10). Furthermore, aprepitant significantly enhanced the anti-HIV activity of antiretrovirals (AZT, Efavirenz, and Indinavir) in HIV Bal-infected macrophages, and aprepitant inhibited CCR5 expression on macrophages, ranging from 50.5 to 29.6%. Donor heterogeneity was observed in antiviral activity and CCR5 receptor expression. CONCLUSION: Aprepitant is active against HIV drug-resistant isolates and enhances the anti-HIV activity of the antiretrovirals. Aprepitant downregulates CCR5 expression on macrophages. NK-1R antagonists merit further investigation as potential HIV therapeutic and immunomodulatory agents.

Messenger-specific role for nicotinic acid adenine dinucleotide phosphate in neuronal differentiation.

Cells possess several Ca2+-mobilizing messengers, which couple stimulation at the cell surface by a multitude of extracellular cues to the regulation of intracellular Ca2+-sensitive targets. Recent studies suggest that agonists differentially select from this molecular palette to generate their characteristic Ca2+ signals but it is still unclear whether different messengers mediate different functions or whether they act in a redundant fashion. In this study, we compared the effects of nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing messenger, with that of the prototypical messenger inositol trisphosphate on cytosolic Ca2+ levels and differentiation status of PC12 cells. We demonstrate that liposomal delivery of NAADP mediated release of Ca2+ from acidic Ca2+ stores and that this stimulus was sufficient to drive differentiation of the cells to a neuronal-like phenotype. In sharp contrast, cell fate was unaffected by more transient Ca2+ signals generated by inositol trisphosphate-evoked release of endoplasmic reticulum Ca2+ stores. Our data establish for the first time (i) the presence of novel NAADP-sensitive Ca2+ stores in PC12 cells, (ii) a role for NAADP in differentiation, and (iii) that Ca2+-dependent function can be messenger-specific. Thus, differential recruitment of intracellular Ca2+-mobilizing messengers and their target Ca2+ stores may represent a robust means of maintaining stimulus fidelity in the control of Ca2+-dependent cell function.

Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling.

The P2Y(1) ADP receptor activates G(q) and causes increases in intracellular Ca(2+) concentration through stimulation of PLC. In this study, we investigated the role of the amino acid residues in the COOH terminus of the human P2Y(1) receptor in G(q) activation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y(1) receptor (P2Y(1)-WT cells), P2Y(1)-DeltaR340-L373, or P2Y(1)-DeltaD356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y(1)-DeltaT330-L373, a mutant lacking the entire COOH terminus, completely lost their response to 2-MeSADP. Similar data were obtained by using these cell lines and measuring Ca(2+) mobilization upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the COOH terminus of the human P2Y(1) receptor are essential for G(q) coupling. Radioligand binding demonstrated that both the P2Y(1)-WT and P2Y(1)-DeltaT330-L373-expressing cells have almost equal binding of [(3)H]MRS2279, a P2Y(1) receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y(12) receptor with the P2Y(1) COOH terminus failed to elicit G(q) functional responses, indicating that the P2Y(1) COOH terminus is essential but not sufficient for G(q) activation. Finally, cells expressing a double-mutant P2Y(1) receptor (R333A/R334A) in the conserved BBXXB region of the COOH terminus of the G(q)-activating P2Y receptors completely lost their functional ability to activate G(q). We conclude that the two arginine residues (R333R334) in the COOH terminus of the human P2Y(1) receptor are essential for G(q) coupling.

Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor.

Nucleotides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y2 receptors. However, P2Y2 receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca2+ increases in isolated human neutrophils with an EC50 of 1 microM but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y2 receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils.