Aquaporins from pathogenic protozoan parasites: structure, function and potential for chemotherapy

Infectious diseases, caused by protozoa, such as malaria, sleeping sickness, Chagas' disease or leishmaniasis, are a global threat. The increase in the number of affected individuals and the rapid spread of drug-resistant strains call for specific novel strategies to combat human pathogenic parasites. In the search for novel drug targets, transport proteins for nutrients and metabolites of the parasite-host interface are getting into focus. The present review summarizes and discusses the currently available results on protozoan aquaporins. Various genes coding for aquaporin water and solute channels have been identified in the protozoan genomes and they are probable elements of the parasite's cell membrane. Phylogenetic analysis reveals that individual aquaporin genes are of bacterial or plant origin. So far, six protozoan aquaporins have been cloned and functionally characterized. Typically, these are bifunctional channels and pass water at intermediate to high rates as well as uncharged solutes. In the present review, amino acid compositions of the individual pore entries are compared and permeability properties are attributed to specific protein features. Furthermore, possible physiological roles in osmotic protection and metabolism are discussed. Finally, the potential of protozoan aquaporins for use as a target or entry pathway for chemotherapeutic compounds is reviewed.     

A surgical model for studying biliary bile acid and cholesterol metabolism in swine.

Techniques were developed in young growing pigs to simultaneously collect and reinfuse bile. Silastic cannulae were designed and surgically implanted in the common bile duct and the duodenum. Direct sampling of the hepatic bile was achieved by bypassing the gallbladder. The techniques allowed for steady-state studies of hepatic function to be conducted in conscious swine in two different studies. Pigs, thus surgically modified, can serve as an appropriate model for physiologic, pharmacologic, and nutritional research that involves bile sampling.     

The effects of different pretreatment conditions and fixation regimes on serotonin immunoreactivity: a quantitative light microscopic study

An investigation was designed to evaluate the effects of three different fixation regimes on the retention of serotonin-like immunoreactivity in rat midbrain tissue sections. The effects of pretreatment with pargyline-HCl and l-tryptophan on the volume fraction of serotonin-like immunoreactive processes were also examined. Rat brain tissue was fixed with 4% paraformaldehyde (Pf), 4% paraformaldehyde-0.2% picric acid-0.05% glutaraldehyde (Pf-Pa-G), or 4% paraformaldehyde-0.2% glutaraldehyde (Pf-G). Tissue was subsequently processed for immunohistochemistry using a modified peroxidase-antiperoxidase technique and quantified at the light microscopic level by point counting. Fixation with Pf resulted in higher volume fraction determinations of axonal serotonin immunoreactivity than did fixation with Pf-Pa-G or Pf-G. These results provide quantitative data which indicate that even low levels of glutaraldehyde in the fixative significantly decrease serotonin immunoreactivity. Pretreatment with pargyline and tryptophan increased the amount of serotonin immunoreactivity in tissue fixed with Pf-G but not in tissue fixed with Pf. Pretreatment with pargyline and tryptophan is thus recommended when using glutaraldehyde in the fixation process to assure adequate serotonin immunoreactivity. Pretreatment in conjunction with glutaraldehyde fixation, however, appears to cause differential increases in serotonin-like immunoreactivity within brain nuclei that may compromise the interpretation of results.     

Influence of a plasma fraction of human blood, rich in high density lipoprotein, on in vitro formation of prostaglandin I2 (PGI2) and thromboxane A2 (TXA2)

The influence of a plasma fraction of human blood, rich in high density lipoprotein (HDL), was investigated on the "in vitro" formation of PGI2 and TXA2. The addition of 1 mg HDL-cholesterol per ml incubation fluid stimulated significantly the biotransformation of prostaglandin H2 into PGI2 by the microsomal fraction of pig aorta. The TXB2 formation capacity of whole clotted blood was inhibited by administration of HDL in a dose dependent manner. These results suggest that added HDL is able to enhance the ratio PGI2:TXA2. This did not depend on the preparation of HDL either by ultracentrifugation or by precipitation.     

Monoclonal antibody specific for carbodiimide-fixed glutamate: immunocytochemical localization in the rat CNS

Glutamate is widely distributed in the central nervous system (CNS) and is present in greater amounts than any other putative neurotransmitter. To study its distribution in the CNS, a monoclonal antibody was raised against gamma-L-glutamyl-L-glutamic acid (gamma-Glu-Glu) conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehydeborohydride. By use of this antibody, indirect immunoperoxidase staining was observed in CNS tissue fixed with carbodiimide to form gamma-Glu-Glu from glutamate and post-fixed with glutaraldehyde or paraformaldehyde. In contrast, immunoreactivity was quite low in tissues fixed only with glutaraldehyde. Absorption controls indicated that the staining of carbodiimide-fixed tissue could be inhibited by micromolar concentrations of gamma-Glu-Glu but not by other small molecules. Using ELISA, the antibody reacted strongly with the gamma-Glu-Glu/KLH conjugate used to immunize the mouse, but not with other small molecules conjugated to KLH. The reactivity of the antibody with the gamma-Glu-Glu/KLH conjugate on ELISA was inhibited by free gamma-Glu-Glu in micromolar concentrations, but not by similar dipeptides or amino acids. Dense immunocytochemical staining was observed in cortical pyramidal cells, cerebellar granule cells, and the cochlear nuclei. Staining with this monoclonal antibody correlated well with other methods of localizing glutamate in the CNS.     

Effect of HDL and LDL from pre and post menopausal women on prostacyclin synthesis

Earlier we found LDL and HDL to differentially affect the synthesis of PGI2 from PGH2 by pig aorta microsomes and that this depended on the gender of the lipoprotein donors. Here we report there are also differences in the effects of LDL and HDL from pre- and postmenopausal women. The influence of the lipoproteins from postmenopausal women on the PGI2 formation is similar to the action of lipoproteins from men. We suggest that endogenous PGI2 formation is regulated by the sex related composition of the lipoproteins.     

Acid evoked thermal hyperalgesia involves peripheral P2Y1 receptor mediated TRPV1 phosphorylation in a rodent model of thrombus induced ischemic pain

Background

We previously developed a thrombus-induced ischemic pain (TIIP) animal model, which was characterized by chronic bilateral mechanical allodynia without thermal hyperalgesia (TH). On the other hand we had shown that intraplantar injection of acidic saline facilitated ATP-induced pain, which did result in the induction of TH in normal rats. Because acidic pH and increased ATP are closely associated with ischemic conditions, this study is designed to: (1) examine whether acidic saline injection into the hind paw causes the development of TH in TIIP, but not control, animals; and (2) determine which peripheral mechanisms are involved in the development of this TH.

Results

Repeated intraplantar injection of pH 4.0 saline, but not pH 5.5 and 7.0 saline, for 3 days following TIIP surgery resulted in the development of TH. After pH 4.0 saline injections, protein levels of hypoxia inducible factor-1α (HIF-1α) and carbonic anhydrase II (CA II) were elevated in the plantar muscle indicating that acidic stimulation intensified ischemic insults with decreased tissue acidity. At the same time point, there were no changes in the expression of TRPV1 in hind paw skin, whereas a significant increase in TRPV1 phosphorylation (pTRPV1) was shown in acidic saline (pH 4.0) injected TIIP (AS-TIIP) animals. Moreover, intraplantar injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1 antagonist) effectively alleviated the established TH. In order to investigate which proton- or ATP-sensing receptors contributed to the development of TH, amiloride (an ASICs blocker), AMG9810, TNP-ATP (a P2Xs antagonist) or MRS2179 (a P2Y1 antagonist) were pre-injected before the pH 4.0 saline. Only MRS2179 significantly prevented the induction of TH, and the increased pTRPV1 ratio was also blocked in MRS2179 injected animals.

Conclusion

Collectively these data show that maintenance of an acidic environment in the ischemic hind paw of TIIP rats results in the phosphorylation of TRPV1 receptors via a PKC-dependent pathway, which leads to the development of TH mimicking what occurs in chronic ischemic patients with severe acidosis. More importantly, peripheral P2Y1 receptors play a pivotal role in this process, suggesting a novel peripheral mechanism underlying the development of TH in these patients.     

Concerted action of two cation filters in the aquaporin water channel

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water-specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H⁺ ≫K⁺ >Rb⁺ >Na⁺ >Cs⁺ >Li⁺. Contrary to theoretical models, we found that electrostatic repulsion at the central asn-pro-ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali-leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali-tight but solute-unselective NPA region as a feature of primordial channels and the proton-tight and solute-selective ar/R constriction variants as later adaptations within the AQP superfamily.     

N(delta)-Methylated L-arginine derivatives and their effects on the nitric oxide generating system

So far N(delta)-methyl-l-arginine (MA) is only detected in yeast cells. Assuming that MA also exists in mammalians we examined possible physiological effects of N(delta)-methylated l-arginine derivatives on the nitric oxide generating system, that is, nitric oxide synthase (NOS), arginase and dimethylarginine dimethylaminohydrolase (DDAH). N(delta)-methyl-l-citrulline (MC) turned out to be a weak non-specific inhibitor of nitric oxide synthases. Moreover, MA is hydroxylated by all human NOS isoforms to N(omega)-hydroxy-N(delta)-methyl-l-arginine (NHAM) but not converted further. This hydroxylated intermediate, however, was detected to be a potent inhibitor of bovine liver arginase with a K(i) of 17.1+/-2.2 microM.     

A single aquaporin gene encodes a water/glycerol/urea facilitator in Toxoplasma gondii with similarity to plant tonoplast intrinsic proteins

We describe a single aquaporin gene in Toxoplasma gondii which, surprisingly, has only 28% sequence similarity to the aquaglyceroporin of another apicomplexan parasite, Plasmodium falciparum. Sequence comparisons showed 47% similarity to water-specific plant aquaporins and the conservation of typical pore-forming residues. We established that the Toxoplasma aquaporin protein is a bifunctional membrane pore with intermediate water and high glycerol permeability. Furthermore, we identified hydroxyurea, an antineoplastic agent with inhibitory effects on parasite proliferation, as a permeant of this channel.