Modified Plasmodium falciparum Ring-Stage Survival Assay with ML10 Kinase Inhibitor.

The ring-stage survival assay is the reference assay to measure in vitro Plasmodium falciparum artemisinin partial resistance. The main challenge of the standard protocol is to generate 0-to-3-h postinvasion ring stages (the stage least susceptible to artemisinin) from schizonts obtained by sorbitol treatment and Percoll gradient. We report here a modified protocol facilitating the production of synchronized schizonts when multiple strains are tested simultaneously, by using ML10, a protein kinase inhibitor, that reversibly blocks merozoite egress.

CDC50 Orthologues in Plasmodium falciparum Have Distinct Roles in Merozoite Egress and Trophozoite Maturation.

In model organisms, type IV ATPases (P4-ATPases) require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite Plasmodium falciparum, guanylyl cyclase alpha (GCα) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GCα is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function. We sought to investigate whether any of the three CDC50 orthologues (termed A, B, and C) encoded by P. falciparum are required for GCα function. Using gene tagging and conditional gene disruption, we demonstrate that CDC50B and CDC50C but not CDC50A are expressed in the clinically important asexual blood stages and that CDC50B is a binding partner of GCα whereas CDC50C is the binding partner of another putative P4-ATPase, phospholipid-transporting ATPase 2 (ATP2). Our findings indicate that CDC50B has no essential role for intraerythrocytic parasite maturation but modulates the rate of parasite egress by interacting with GCα for optimal cGMP synthesis. In contrast, CDC50C is essential for blood stage trophozoite maturation. Additionally, we find that the CDC50C-ATP2 complex may influence parasite endocytosis of host cell hemoglobin and consequently hemozoin formation. IMPORTANCE Malaria morbidity arises due to successive rounds of replication of Plasmodium parasites within red blood cells. Mature daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that a unique bifunctional guanylyl cyclase, GCα, initiates egress by synthesis of cGMP. GCα has an N-terminal P4-ATPase domain of unknown function. In model organisms, P4-ATPases function through interaction with a CDC50 partner protein. Here, we investigate the role of CDC50 orthologues in P. falciparum and show that GCα binds CDC50B, an interaction that regulates egress efficiency. We also find that CDC50C is essential and binds a putative P4-ATPase, ATP2, in a complex that influences endocytosis of host hemaglobin. Our results highlight the heterogenous and critical role of CDC50 proteins in P. falciparum.

Low Prior Exposure and Incidence of Hepatitis C in Human Immunodeficiency Virus-Negative Gay and Bisexual Men Taking Preexposure Prophylaxis (PrEP): Findings From the Expanded PrEP Implementation in Communities-New South Wales Prospective Implementation Study.

BACKGROUND: The use of preexposure prophylaxis (PrEP) for the prevention of human immunodeficiency virus (HIV) has raised concerns of increased sexual risk behaviors. These behaviors may be associated with increased incidence of sexually acquired hepatitis C virus (HCV) among gay and bisexual men. METHODS: The Expanded PrEP Implementation in Communities-New South Wales (EPIC-NSW) study was a cohort study of daily coformulated tenofovir disoproxil fumarate and emtricitabine for HIV prevention. We recruited 9596 people at high risk of HIV acquisition from 31 clinics across New South Wales and the Australia Capital Territory in Australia. We report prior exposure to HCV and incidence in this cohort between 2016 and 2019. RESULTS: At least 1 HCV test result was available for 8658 (90.2%) participants. These individuals had a median age of 34 years (interquartile range, 28-43), most of whom were male (8530, 98.5%), identified as gay (7944, 91.8%), and were born in Australia (51.8%). Prior exposure to HCV was detected among 81 participants at baseline (0.9%; 95% confidence interval [CI]: .71.2). Twenty of 8577 participants were diagnosed with incident infection (rate 0.2/100 person-years [95% CI: .1-.3/100 person-years]). They were significantly older (median age 41 years vs 34 years, P = .044), and more likely to report methamphetamine use at baseline (incidence rate ratio, 2.7 [95% CI: 1.00-7.2]) than those without incident infection. CONCLUSIONS: In this population of PrEP users, HCV prior exposure and incidence were low. With high levels of HCV and HIV testing and treatment, the dual goals of HIV and HCV elimination could be achieved in this population. Clinical Trials Registration: number NCT02870790.

Plasmodium falciparum Guanylyl Cyclase-Alpha and the Activity of Its Appended P4-ATPase Domain Are Essential for cGMP Synthesis and Blood-Stage Egress.

Guanylyl cyclases (GCs) synthesize cyclic GMP (cGMP) and, together with cyclic nucleotide phosphodiesterases, are responsible for regulating levels of this intracellular messenger which mediates myriad functions across eukaryotes. In malaria parasites (Plasmodium spp), as well as their apicomplexan and ciliate relatives, GCs are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here, we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood-stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesize cGMP or mobilize calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that P4-ATPase activity is upstream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress.IMPORTANCE The clinical manifestations of malaria arise due to successive rounds of replication of Plasmodium parasites within red blood cells. Once mature, daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that the activation of cyclic GMP (cGMP) signaling is critical for initiating egress. Here, we demonstrate that GCα, a unique bifunctional enzyme, is the sole enzyme responsible for cGMP production during the asexual blood stages of Plasmodium falciparum and is required for the cellular events leading up to merozoite egress. We further demonstrate that in addition to the GC domain, the appended ATPase-like domain of GCα is also involved in cGMP production. Our results highlight the critical role of GCα in cGMP signaling required for orchestrating malaria parasite egress.

cAMP signalling and its role in host cell invasion by malaria parasites.

Cyclic adenosine monophosphate (cAMP) is an important signalling molecule across evolution, but until recently there was little information on its role in malaria parasites. Advances in gene editing - in particular conditional genetic approaches and mass spectrometry have paved the way for characterisation of the key components of the cAMP signalling pathway in malaria parasites. This has revealed that cAMP signalling plays a critical role in invasion of host red blood cells by Plasmodium falciparum merozoites through regulating the phosphorylation of key parasite proteins by the cAMP-dependent protein kinase (PKA). These insights will help us to investigate parasite cAMP signalling as a target for novel antimalarial drugs.

Antimalarial activity of primaquine operates via a two-step biochemical relay.

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

High-throughput screening of the Plasmodium falciparum cGMP-dependent protein kinase identified a thiazole scaffold which kills erythrocytic and sexual stage parasites.

Antimalarial drug resistance compels the quest for new compounds that target alternative pathways to current drugs. The Plasmodium cyclic GMP-dependent protein kinase (PKG) has essential functions in all of the major life cycle developmental stages. An imidazopyridine PKG inhibitor scaffold was previously shown to clear P. falciparum infection in a rodent model in vivo and blocked transmission to mosquitoes providing proof of concept for this target. To find new classes of PKG inhibitors to serve as alternative chemical starting points, we performed a high-throughput screen of the GSK Full Diversity Collection using recombinant P. falciparum PKG. We developed a robust enzymatic assay in a 1536-well plate format. Promising compounds were then tested for activity against P. falciparum asexual blood stage growth, selectivity and cytotoxicity. By using a scoring system we selected the 66 most promising PKG inhibitors (comprising nine clusters and seven singletons). Among these, thiazoles were the most potent scaffold with mid-nanomolar activity on P. falciparum blood stage and gamete development. Using Kinobeads profiling we identified additional P. falciparum protein kinases targeted by the thiazoles that mediate a faster speed of the kill than PKG-selective compounds. This scaffold represents a promising starting point to develop a new antimalarial.

Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery.

Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACß) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.

Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion.

Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase ß (PDEß) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEß-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEß plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.

Subclinical Infection of Macaques and Baboons with A Baboon Simarterivirus.

Simarteriviruses (Arteriviridae: Simarterivirinae) are commonly found at high titers in the blood of African monkeys but do not cause overt disease in these hosts. In contrast, simarteriviruses cause severe disease in Asian macaques upon accidental or experimental transmission. Here, we sought to better understand the host-dependent drivers of simarterivirus pathogenesis by infecting olive baboons (n = 4) and rhesus monkeys (n = 4) with the simarterivirus Southwest baboon virus 1 (SWBV-1). Surprisingly, none of the animals in our study showed signs of disease following SWBV-1 inoculation. Three animals (two rhesus monkeys and one olive baboon) became infected and sustained high levels of SWBV-1 viremia for the duration of the study. The course of SWBV-1 infection was highly predictable: plasma viremia peaked between 1 × 107 and 1 × 108 vRNA copies/mL at 3⁻10 days post-inoculation, which was followed by a relative nadir and then establishment of a stable set-point between 1 × 106 and 1 × 107 vRNA copies/mL for the remainder of the study (56 days). We characterized cellular and antibody responses to SWBV-1 infection in these animals, demonstrating that macaques and baboons mount similar responses to SWBV-1 infection, yet these responses are ineffective at clearing SWBV-1 infection. SWBV-1 sequencing revealed the accumulation of non-synonymous mutations in a region of the genome that corresponds to an immunodominant epitope in the simarterivirus major envelope glycoprotein GP5, which likely contribute to viral persistence by enabling escape from host antibodies.

Progesterone effects on vaginal cytokines in women with a history of preterm birth.

OBJECTIVE: To determine the effect of intramuscular progesterone on the vaginal immune response of pregnant women with a history of prior preterm birth. METHODS: A prospective, cohort study of women at 11-16 weeks gestation, ≥18 years of age, and carrying a singleton pregnancy was conducted from June 2016 to August 2017 after IRB approval. Women in the progesterone arm had a history of preterm birth and received weekly intramuscular 17-hydroxyprogesterone caproate. Controls comprised of women with healthy, uncomplicated pregnancies. Excluded were women with vaginitis, diabetes mellitus, hypertension, or other chronic diseases affecting the immune response. A vaginal wash was performed at enrollment, at 26-28 weeks, and at 35-36 weeks gestation. Samples underwent semi-quantitative detection of human inflammatory markers. Immunofluorescence pixel density data was analyzed and a P value <0.05 was considered significant. RESULTS: There were 39 women included, 10 with a prior preterm birth and 29 controls. The baseline demographics and pregnancy outcomes for both groups were similar in age, parity, race, BMI, gestational age at delivery, mode of delivery, and birth weight. Enrollment cytokines in women with a prior preterm birth, including IL-1 alpha (39.2±25.1% versus 26.1±13.2%; P = 0.04), IL-1 beta (47.9±26.4% versus 24.9±17%; P<0.01), IL-2 (16.7±9.3% versus 11.3±6.3%; P = 0.03), and IL-13 (16.9±12.4% versus 8.2±7.4%; P = 0.01) were significantly elevated compared to controls. In the third trimester the cytokine densities for IL-1 alpha (26.0±18.2% versus 22.3±12.0%; P = 0.49), IL-1 beta (31.8±15.9% versus 33.1±16.8%; P = 0.84), IL-2 (10.0±8.4% versus 10.9±5.9%; P = 0.71), and IL-13 (9.1±5.9% versus 10.0±6.5%; P = 0.71) were all statistically similar between the progesterone arm and controls, respectively. CONCLUSION: There is an increased cytokine presence in vaginal washings of women at risk for preterm birth which appears to be modified following the administration of 17- hydroxyprogesterone caproate to levels similar to healthy controls.

Control of endothelial cell tube formation by Notch ligand intracellular domain interactions with activator protein 1 (AP-1).

Notch signaling is a ubiquitous signal transduction pathway found in most if not all metazoan cell types characterized to date. It is indispensable for cell differentiation as well as tissue growth, tissue remodeling, and apoptosis. Although the canonical Notch signaling pathway is well characterized, accumulating evidence points to the existence of multiple, less well-defined layers of regulation. In this study, we investigated the function of the intracellular domain (ICD) of the Notch ligand Delta-like 4 (DLL4). We provide evidence that the DLL4 ICD is required for normal DLL4 subcellular localization. We further show that it is cleaved and interacts with the JUN proto-oncogene, which forms part of the activator protein 1 (AP-1) transcription factor complex. Mechanistically, the DLL4 ICD inhibited JUN binding to DNA and thereby controlled the expression of JUN target genes, including DLL4 Our work further demonstrated that JUN strongly stimulates endothelial cell tube formation and that DLL4 constrains this process. These results raise the possibility that Notch/DLL4 signaling is bidirectional and suggest that the DLL4 ICD could represent a point of cross-talk between Notch and receptor tyrosine kinase (RTK) signaling.

Cyclic nucleotide signalling in malaria parasites.

The cyclic nucleotides 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP) are intracellular messengers found in most animal cell types. They usually mediate an extracellular stimulus to drive a change in cell function through activation of their respective cyclic nucleotide-dependent protein kinases, PKA and PKG. The enzymatic components of the malaria parasite cyclic nucleotide signalling pathways have been identified, and the genetic and biochemical studies of these enzymes carried out to date are reviewed herein. What has become very clear is that cyclic nucleotides play vital roles in controlling every stage of the complex malaria parasite life cycle. Our understanding of the involvement of cyclic nucleotide signalling in orchestrating the complex biology of malaria parasites is still in its infancy, but the recent advances in our genetic tools and the increasing interest in signalling will deliver more rapid progress in the coming years.

Functional analyses of a human vascular tumor FOS variant identify a novel degradation mechanism and a link to tumorigenesis.

Epithelioid hemangioma is a locally aggressive vascular neoplasm, found in bones and soft tissue, whose cause is currently unknown, but may involve oncogene activation. FOS is one of the earliest viral oncogenes to be characterized, and normal cellular FOS forms part of the activator protein 1 (AP-1) transcription factor complex, which plays a pivotal role in cell growth, differentiation, and survival as well as the DNA damage response. Despite this, a causal link between aberrant FOS function and naturally occurring tumors has not yet been established. Here, we describe a thorough molecular and biochemical analysis of a mutant FOS protein we identified in these vascular tumors. The mutant protein lacks a highly conserved helix consisting of the C-terminal four amino acids of FOS, which we show is indispensable for fast, ubiquitin-independent FOS degradation via the 20S proteasome. Our work reveals that FOS stimulates endothelial sprouting and that perturbation of normal FOS degradation could account for the abnormal vessel growth typical of epithelioid hemangioma. To the best of our knowledge, this is the first functional characterization of mutant FOS proteins found in tumors.

Creation and Execution of a Novel Anesthesia Perioperative Care Service at a Veterans Affairs Hospital.

Physician-led perioperative surgical home models are developing as a method for improving the American health care system. These models are novel, team-based approaches that help to provide continuity of care throughout the perioperative period. Another avenue for improving care for surgical patients is the use of enhanced recovery after surgery pathways. These are well-described methods that have shown to improve perioperative outcomes. An established perioperative surgical home model can help implementation, efficiency, and adherence to enhanced recovery after surgery pathways. For these reasons, the Tennessee Valley Healthcare System, Nashville Veterans Affairs Medical Center created an Anesthesiology Perioperative Care Service that provides comprehensive care to surgical patients from their preoperative period through the continuum of their hospital course and postdischarge follow-up. In this brief report, we describe the development, implementation, and preliminary outcomes of the service.

Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating.

While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive.

Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity.

Regulation of System xc(-) by Pharmacological Manipulation of Cellular Thiols.

The cystine/glutamate exchanger (system xc (-)) mediates the transport of cystine into the cell in exchange for glutamate. By releasing glutamate, system xc (-) can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and may protect cells against oxidative stress. We tested two different compounds that deplete primary cortical cultures containing both neurons and astrocytes of intracellular GSH, L-buthionine-sulfoximine (L-BSO), and diethyl maleate (DEM). Both compounds caused significant concentration and time dependent decreases in intracellular GSH levels. However; DEM caused an increase in radiolabeled cystine uptake through system xc (-), while unexpectedly BSO caused a decrease in uptake. The compounds caused similar low levels of neurotoxicity, while only BSO caused an increase in oxidative stress. The mechanism of GSH depletion by these two compounds is different, DEM directly conjugates to GSH, while BSO inhibits γ-glutamylcysteine synthetase, a key enzyme in GSH synthesis. As would be expected from these mechanisms of action, DEM caused a decrease in intracellular cysteine, while BSO increased cysteine levels. The results suggest that negative feedback by intracellular cysteine is an important regulator of system xc (-) in this culture system.

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

Augmented cystine-glutamate exchange by pituitary adenylate cyclase-activating polypeptide signaling via the VPAC1 receptor.

In the central nervous system, cystine import in exchange for glutamate through system xc- is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc- activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine-glutamate exchange via system xc-. In this study, 24-h treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc- function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc- inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc- activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine-glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc-. Furthermore, the potentiation of system xc- activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc- activity. Synapse 68:604-612, 2014. © 2014 Wiley Periodicals, Inc.