Secreted effectors in Toxoplasma gondii and related species: determinants of host range and pathogenesis?

Recent years have witnessed the discovery of a number of secreted proteins in Toxoplasma gondii that play important roles in host-pathogen interactions and parasite virulence, particularly in the mouse model. However, the role that these proteins play in driving the unique features of T. gondii compared to some of its nearest apicomplexan relatives (Hammondia hammondi and Neospora caninum) is unknown. These unique features include distinct dissemination characteristics in vivo and a vast host range. In this review we comprehensively survey what is known about disease outcome, the host response and host range for T. gondii, H. hammondi, and N. caninum. We then review what is presently known about recently identified secreted virulence effectors in these three genetically related, but phenotypically distinct, species. Finally we exploit the existence of genome sequences for these three organisms and discuss what is known about the presence, and functionality, of key T. gondii effectors in these three species.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide.

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca(2+) -sensitive K(+) channels. More recent studies have revealed the ability of CO to inhibit T-type Ca(2+) channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Molecular and biological characterization of first isolates of Hammondia hammondi from cats from Ethiopia.

Toxoplasma gondii oocysts are morphologically and antigenically similar to oocysts of another feline coccidian, Hammondia hammondi. The distinction between H. hammondi and T. gondii is important from an epidemiological perspective because all isolates of T. gondii are potentially pathogenic for humans and animals, whereas H. hammondi is not known to cause clinical disease in any naturally infected intermediate or definitive hosts. In the present report, H. hammondi (designated HhCatEt1 and HhCatEt2) oocysts were found microscopically in the feces of 2 of 36 feral domestic cats (Felis catus) from Addis Ababa, Ethiopia. Oocysts were orally infective to Swiss Webster and gamma interferon gene knockout mice; the inoculated mice developed tissue cysts in their muscles. Laboratory-raised cats fed mouse tissues of infected mice shed H. hammondi oocysts with a prepatent period of 5 days. The DNA extracted from sporulated oocysts reacted with H. hammondi-specific primers, and sequences were deposited in GenBank (accession nos. JX477424, and KC223619). This is the first report of isolation of H. hammondi from cats from the African continent.

Beyond the genome: recent advances in Toxoplasma gondii functional genomics.

Recent years have witnessed an explosion in the amount of genomic information available for Toxoplasma gondii and other closely related pathogens. These data, many of which have been made publicly available prior to publication, have facilitated a wide variety of functional genomics studies. In this review, we provide a brief overview of existing database tools for querying the Toxoplasma genome and associated genome-wide data and review recent publications that have been facilitated by these data. Topics covered include strain comparisons and quantitative trait loci mapping, gene expression analyses during the cell cycle as well as during parasite differentiation, and proteomics.

Hypoxic modulation of ca(2+) signaling in human venous and arterial endothelial cells.

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca(2+) homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O(2), 24 h). Basal [Ca(2+)]( i ) and store depletion-mediated Ca(2+) entry were significantly different between the two cell types, yet agonist (ATP)-mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca(2+) entry only in venous cells. Clearly, Ca(2+) signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.

Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue.

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, can cause severe disease in humans with an immature or suppressed immune system. The outcome of Toxoplasma infection is highly dependent on the strain type, as are many of its in vitro growth properties. Here we use genetic crosses between type II and III lines to show that strain-specific differences in the modulation of host cell transcription are mediated by a putative protein kinase, ROP16. Upon invasion by the parasite, this polymorphic protein is released from the apical organelles known as rhoptries and injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways and consequent downstream effects on a key host cytokine, interleukin (IL)-12. Our findings provide a new mechanism for how an intracellular eukaryotic pathogen can interact with its host and reveal important differences in how different Toxoplasma lineages have evolved to exploit this interaction.

Polymorphic secreted kinases are key virulence factors in toxoplasmosis.

The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F(1) progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.

Hypoxic regulation of Ca2+ signalling in astrocytes and endothelial cells.

Acute hypoxia is well known to modulate plasmalemmal ion channels in specific tissue types, thereby modulating [Ca2+]i. Alternative mechanisms by which acute hypoxia could modulate [Ca2+]i are less well explored, particularly in non-excitable cells. Here, we describe experiments employing microfluorimetric recordings from Fura-2-loaded rat cortical astrocytes and human saphenous vein endothelial cells designed to explore any effects of hypoxia (pO2 20-30 mmHg) on [Ca2+]i. In both cell types, hypoxia evoked small rises of [Ca2+]i in the majority of cells during perfusion with a Ca(2+)-free solution, indicating hypoxia can release Ca2+ from an intracellular pool. Capacitative Ca2+ entry was observed when Ca2+ was subsequently restored to the extracellular solution. These effects were abolished by pre-treatment of cells with thapsigargin or prior application of inositol 1,4,5-trisphosphate (IP3)-generating agonists. Antioxidants fully prevented this effect of hypoxia in both cell types. Mitochondrial uncoupling significantly enhanced the effects of hypoxia in astrocytes, yet markedly suppressed the effects of hypoxia in endothelial cells. Our findings indicate that hypoxia can modulate [Ca2+]i in non-excitable cells; most importantly, it can evoke Ca2+ release from intracellular stores via a mechanism which involves reactive oxygen species. The involvement of mitochondria in this effect appears to be tissue specific.

Amyloid peptides mediate hypoxic increase of L-type Ca2+ channels in central neurones.

Prolonged hypoxia, encountered in individuals suffering from various cardiorespiratory diseases, enhances the likelihood of subsequently developing Alzheimer's disease (AD). However, the underlying mechanisms are unknown, as are the mechanisms of neurodegeneration of amyloid beta peptides (AbetaPs), although the latter involves disruption of Ca2+ homeostasis. Here, immunohistochemistry demonstrated that hypoxia increased production of AbetaPs, an effect which was prevented by inhibition of either beta or gamma secretase, the enzymes required for liberation of AbetaP from its precursor protein. Whole-cell patch clamp recordings showed that hypoxia selectively increased functional expression of L-type Ca2+ channels. This was prevented by inhibition of either beta or gamma secretase, indicating that hypoxic channel up-regulation is dependent upon AbetaP formation. Our results indicate for the first time that hypoxia promotes AbetaP formation in central neurons, and show that this leads to abnormally high selective expression of L-type Ca2+ channels whose blockade has previously been shown to be neuroprotective in AD models. These findings provide a cellular basis for understanding the increased incidence of AD following prolonged hypoxia.

Hypoxic regulation of Ca2+ signaling in cultured rat astrocytes.

Acute hypoxia modulates various cell processes, such as cell excitability, through the regulation of ion channel activity. Given the central role of Ca2+ signaling in the physiological functioning of astrocytes, we have investigated how acute hypoxia regulates such signaling, and compared results with those evoked by bradykinin (BK), an agonist whose ability to liberate Ca2+ from intracellular stores is well documented. In Ca2+-free perfusate, BK evoked rises of [Ca2+]i in all cells examined. Hypoxia produced smaller rises of [Ca2+]i in most cells, but always suppressed subsequent rises of [Ca2+]i induced by BK. Thapsigargin pre-treatment of cells prevented any rise of [Ca2+]i evoked by either BK or hypoxia. Restoration of Ca2+ to the perfusate following a period of acute hypoxia always evoked capacitative Ca2+ entry. During mitochondrial inhibition (due to exposure to carbonyl cyanide p-trifluromethoxyphenyl hydrazone (FCCP) and oligomycin), rises in [Ca2+]i (observed in Ca2+-free perfusate) evoked by hypoxia or by BK, were significantly enhanced, and hypoxia always evoked responses. Our data indicate that hypoxia triggers Ca2+ release from endoplasmic reticulum stores, efficiently buffered by mitochondria. Such liberation of Ca2+ is sufficient to trigger capacitative Ca2+ entry. These findings indicate that the local O2 level is a key determinant of astrocyte Ca2+ signaling, likely modulating Ca2+-dependent astrocyte functions in the central nervous system.

Pharmacological and autoradiographical characterization of serotonin transporter-like activity in sporocysts of the human blood fluke, Schistosoma mansoni.

The present study focuses on the role of the biogenic monoamine serotonin (5-hydroxytryptamine) in the biology of sporocyst stages of the human blood fluke, Schistosoma mansoni, and its importance during obligate development within its snail host Biomphalaria glabrata. Based on previous work demonstrating that snails infected with S. mansoni have reduced levels of 5-hydroxytryptamine, we hypothesized that sporocysts actively transport this molecule from the host milieu. Intact sporocysts isolated in vitro take up exogenous 5-hydroxytryptamine via a high-affinity mechanism (K(m)=1.4 micromol l(-1)), and this serotonin transporter-like activity is dependent upon extracellular Na(+) and Cl(-) and is highly sensitive to previously characterized serotonin transporter inhibitors. Autoradiography suggests that transported [(3)H]5-hydroxytryptamine localizes within the body of the sporocyst, and in many cases is found in apical gland cells. Moreover, serotonin transporter-like activity is absent in free-swimming miracidia, the infective stage for the snail host, and the increase in larval serotonin transporter-like activity after miracidium-to-sporocyst transformation is accompanied by a corresponding decrease in steady-state levels of transcripts for tryptophan hydroxylase, the rate-limiting enzyme in serotonin biosynthesis. Overall our data suggest that S. mansoni larvae express surface-exposed serotonin transporter-like molecules, and that the transition from free-living miracidium to parasitic mother sporocyst is characterized by an increased dependence upon exogenous 5-hydroxytryptamine.

Chronic hypoxia potentiates capacitative Ca2+ entry in type-I cortical astrocytes.

Prolonged hypoxia exerts profound effects on cell function, and has been associated with increased production of amyloid beta peptides (A beta Ps) of Alzheimer's disease. Here, we have investigated the effects of chronic hypoxia (2.5% O2, 24 h) on capacitative Ca2+ entry (CCE) in primary cultures of rat type-I cortical astrocytes, and compared results with those obtained in astrocytes exposed to A beta Ps. Chronic hypoxia caused a marked enhancement of CCE that was observed after intracellular Ca2+ stores were depleted by bradykinin application or by exposure to thapsigargin (1 microM). Exposure of cells for 24 h to 1 microM A beta P(1-40) did not alter CCE. Enhancement of CCE was not attributable to cell hyperpolarization, as chronically hypoxic cells were significantly depolarized as compared with controls. Mitochondrial inhibition [by FCCP (10 microM) and oligomycin (2.5 microg/mL)] suppressed CCE in all three cell groups, but more importantly there were no significant differences in the magnitude of CCE in the three astrocyte groups under these conditions. Similarly, the antioxidants melatonin and Trolox abolished the enhancement of CCE in hypoxic cells. Our results indicate that chronic hypoxia augments CCE in cortical type-I astrocytes, a finding which is not mimicked by A beta P(1-40) and appears to be dependent on altered mitochondrial function.

Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells expressing a familial Alzheimer's disease presenilin-1 mutation.

Presenilins are involved in the proteolytic production of Alzheimer's amyloid peptides, but are also known to regulate Ca(2+) homeostasis in various cells types. In the present study, we examined intracellular Ca(2+) stores coupled to muscarinic receptors and capacitative Ca(2+) entry (CCE) in the human neuroblastoma SH-SY5Y cell line, and how these were modulated by over-expression of either wild-type presenilin 1 (PS1wt) or a mutant form of presenilin 1 (PS1 deltaE9) which predisposes to early-onset Alzheimer's disease. Ca(2+) stores discharged by application of 100 microM muscarine (in Ca(2+)-free perfusate) in PS1wt and PS1 DeltaE9 cells were significantly larger than those in control cells, as determined using Fura-2 microfluorimetry. Subsequent CCE, observed in the absence of muscarine when Ca(2+) was re-admitted to the perfusate, was unaffected in PS1wt cells, but significantly suppressed in PS1 deltaE9 cells. However, when Ca(2+) stores were fully depleted with thapsigargin, CCE was similar in all three cell groups. Western blots confirmed increased levels of PS1 in the transfected cells, but also demonstrated that the proportion of intact PS1 in the PS1 deltaE9 cells was far greater than in the other two cell groups. This study represents the first report of modulation of both Ca(2+) stores and CCE in a human, neurone-derived cell line, and indicates a distinct effect of the PS1 mutation deltaE9 over wild-type PS1.

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.

Projection of diabetes burden through 2050: impact of changing demography and disease prevalence in the U.S.

OBJECTIVE: To project the number of people with diagnosed diabetes in the U.S. through 2050, accounting for changing demography and diabetes prevalence rates. RESEARCH DESIGN AND METHODS: We combined age-, sex-, and race-specific diagnosed diabetes prevalence rates-predicted from 1980-1998 trends in prevalence data from the National Health Interview Survey-with Bureau of Census population demographic projections. Sensitivity analyses were performed by varying both prevalence rate and population projections. RESULTS: The number of Americans with diagnosed diabetes is projected to increase 165%, from 11 million in 2000 (prevalence of 4.0%) to 29 million in 2050 (prevalence of 7.2%). The largest percent increase in diagnosed diabetes will be among those aged > or =75 years (+271% in women and +437% in men). The fastest growing ethnic group with diagnosed diabetes is expected to be black males (+363% from 2000-2050), with black females (+217%), white males (+148%), and white females (+107%) following. Of the projected 18 million increase in the number of cases of diabetes in 2050, 37% are due to changes in demographic composition, 27% are due to population growth, and 36% are due to increasing prevalence rates. CONCLUSIONS: If recent trends in diabetes prevalence rates continue linearly over the next 50 years, future changes in the size and demographic characteristics of the U.S. population will lead to dramatic increases in the number of Americans with diagnosed diabetes.

Blockade of chloride channels reveals relaxations of rat small mesenteric arteries to raised potassium.

1. Raised extracellular K(+) relaxes some arteries, and has been proposed as Endothelium-Derived Hyperpolarizing Factor (EDHF). However, relaxation of rat small mesenteric arteries to K(+) is highly variable. We have investigated the mechanism of K(+)-induced dilatation and relaxation of pressurized arteries and arteries mounted for measurement of isometric force. 2. Raising [K(+)](o) from 5.88 - 10.58 mM did not dilate or relax pressurized or isometric arteries. Relaxation to raised [K(+)](o) was revealed in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB); this effect of NPPB was concentration-dependent (IC(50): 1.16 microM). 3. Relaxations to raised [K(+)](o) in the presence of NPPB, were abolished by 30 microM Ba(2+) or endothelial-denudation. Acetycholine (10 microM) relaxed endothelium-intact arteries in presence of raised [K(+)](o) NPPB and Ba(2+). 4. Relaxations to raised [K(+)](o) were revealed in hyperosmotic superfusate (+60 mM sucrose). These relaxations were abolished by 30 microM Ba(2+). In the presence of raised [K(+)](o), 60 mM sucrose and 30 microM Ba(2+), 10 microM acetycholine still relaxed all arteries. 5. Fifty microM 18 alpha-glycyrrhetinic acid (18 alpha-GA), a gap junction inhibitor, depressed relaxations to both 10 microM acetylcholine and raised [K(+)](o), in the presence of 10 microM NPPB. 6. In summary, blockade of a volume-sensitive Cl(-) conductance in small rat mesenteric arteries, using NPPB or hyperosmotic superfusion, reveals a endothelium-dependent, Ba(2+) sensitive dilatation or relaxation of rat mesenteric arteries to raised [K(+)](o). We conclude that inwardly rectifying potassium channels on the endothelium underlie relaxations to raised [K(+)](o) in rat small mesenteric arteries.

Receptor-ligand interactions and cellular signalling at the host-parasite interface.

Although the effects of trematode infection on snail host physiology or host responses on parasite development have been well described in the literature, very little is known regarding the underlying mechanisms and specific molecules responsible for mediating those effects. It is presumed that many host-parasite interactions are communicated through receptor-mediated events, in particular those involving haemocytic immune responses to invading parasites, larval motility and migration through host tissues, and larval acquisition of host molecules either as nutrients or critical developmental factors. The intent of this chapter is to review current knowledge of molecules (both receptors and their ligands or counter-receptors) involved in molecular communication at the interface between larval trematodes, especially the mother or primary sporocyst stage, and host cells/tissues in intimate proximity to developing larvae. Information to date suggests that the molecular exchange at this interface is a highly complex and dynamic process, and appears to be regulated in specific cases. Topics discussed will focus on snail cell receptor interactions with the sporocyst tegument and its secretions, host cell-cell and cell-substrate adhesion receptors and their related signal transduction pathways, and sporocyst tegumental surface receptors and ligands involved in the binding of soluble host molecules.

Potassium does not mimic EDHF in rat mesenteric arteries.

1. K(+) has been proposed to be EDHF in small arteries. We compared ACh-stimulated, EDHF-mediated dilatation/relaxation with raised [K(+)](o) in rat mesenteric arteries. 2. In pressurized arteries, ACh (10 microM) dilated all arteries. Raising [K(+)](o) from 5.88 to 10. 58 mM only dilated 30% of arteries. Ba(2+) (30 microM) did not affect dilatation to ACh, but abolished 40% of dilatations to raised [K(+)](o). 3. If [K(+)](o) was lowered to 1.18 mM, restoring [K(+)](o) to 5.88 mM produced dilatation which was depressed by Ba(2+) or ouabain (1 mM). Combined application of Ba(2+) and ouabain abolished dilatation. In 1.18 mM K(+), dilatation to ACh was depressed by ouabain, but not by Ba(2+). Combined application of Ba(2+) and ouabain depressed dilatation further. Gap junction inhibitors (Gap-27; 300 microM and 18-alpha-glycyrrhetinic acid; 100 microM) also depressed dilatation to ACh. 4. In arteries mounted isometrically, ACh (1 microM) relaxed endothelium intact (+E), but not endothelium denuded (-E) arteries. Raising [K(+)](o) from 5.9 - 10.9 mM failed to relax all arteries. When [K(+)](o) was lowered to 1 mM, raising [K(+)](o) to 6 mM produced relaxation. In -E arteries, relaxation was unaffected by Ba(2+) but abolished by ouabain. In +E arteries, Ba(2+) depressed and ouabain abolished relaxation. In +E arteries, with 1 mM K(+), ACh relaxation was depressed by ouabain but not Ba(2+). The combined application of Ba(2+) and ouabain further depressed relaxation. 5. In summary, both EDHF and raised [K(+)](o) dilate/relax rat mesenteric arteries, though sensitivities to barium and ouabain differ. K(+) may be a relaxing factor in this tissue, but its characteristics differ from EDHF. Gap junction inhibitors depress EDHF, implying an important role for myo-endothelial gap junctions.

Schistosoma mansoni: effects of serotonin and serotonin receptor antagonists on motility and length of primary sporocysts in vitro.

The effects of serotonin (5-hydroxytryptamine; 5-HT) on in vitro transformed primary sporocysts of Schistosoma mansoni were investigated. Serotonin treatment significantly increased parasite motility (percentage of motile sporocysts) and length at concentrations as low as 1 microM. These effects were mimicked by the 5-HT agonist tryptamine, albeit with 10- to 100-fold less potency. The effects of 10 microM 5-HT on sporocyst motility were observed within 15 min posttreatment and on parasite length by 6 h posttreatment, and both effects were stable for up to 48 h. Receptor antagonists with varying affinities for defined vertebrate neurotransmitter receptor subtypes were examined for their effects on parasite behavior in the absence and presence of 10 microM 5-HT. In the absence of 5-HT, only methiothepin significantly inhibited normal parasite growth after 48 h of incubation. In the presence of 10 microM 5-HT, the serotonin receptor antagonists mianserin, ketanserin (both at 100 microM), and methiothepin (at 10 microM) significantly inhibited 5-HT-induced lengthening of primary sporocysts, while 3-tropanyl-indole-3-carboxylate and chlorpromazine had no significant effect. The effects of these same drugs on parasite motility were also examined. In the absence of 5-HT, 10 microM chlorpromazine increased parasite motility, while the other antagonists had no effect. When sporocysts were treated with 10 microM 5-HT for 2 h in the continued presence of antagonist, 100 microM mianserin, ketanserin, 3-tropanyl-indole-3-carboxylate, and 10 microM methiothepin inhibited 5-HT induced increases in parasite motility, while 10 microM chlorpromazine had no effect. These results show that primary sporocysts of S. mansoni exhibit behavioral responses to serotonin much like adult stages of this parasite. Furthermore, these responses appear to be mediated via receptors with pharmacological similarities to those previously described in adult worms.

Evidence against potassium as an endothelium-derived hyperpolarizing factor in rat mesenteric small arteries.

1. Endothelium-derived hyperpolarizing factor (EDHF) has recently been identified as potassium released from endothelial cells into the myo-endothelial space. The present study was designed to test this hypothesis. 2. In rat small mesenteric arteries, mounted in a wire myograph, relaxation to acetylcholine or potassium was not significantly changed following incubation with oxadiazolo-quinoxalin-1-one (ODQ, 4 microM) and indomethacin (10 microM, n = 9). 3. Maximal relaxations to acetylcholine occurred in all arteries, were maintained and were significantly greater (P < 0.01, n = 9) than the transient relaxations to potassium, which only occurred in 30-40% of vessels. 4. Removal of the vascular endothelium abolished relaxant responses both to potassium and acetylcholine (P < 0.005, n = 9). 5. Compared with responses in 5.5 mM potassium PSS, relaxation responses to added potassium in arteries maintained in 1.5 mM potassium PSS were more marked and were not dependent on the presence of an intact endothelium (n = 8). 6. Incubation with BaCl2 (50 microM) significantly inhibited the maximal relaxant response to potassium in the presence of an intact endothelium in 5.5 mM potassium PSS (P < 0.05, n = 4), but had no effect on relaxation of de-endothelialized preparations in 1.5 mM potassium PSS (n = 5). 7. Treatment with ouabain (0.1 mM) abolished the relaxant response to potassium in 1.5 mM potassium PSS (P < 0.001, n = 9), but only partly inhibited the maximal relaxant response to acetylcholine in 5.5 mM potassium PSS (P < 0.01, n = 5). 8. These data show that at physiological concentrations of potassium an intact endothelium is necessary for potassium-induced relaxation in rat mesenteric arteries. Furthermore, the response to potassium is clearly different to that from acetylcholine, indicating that potassium does not mimic EDHF released by acetylcholine in these arteries.