No abstract

Loading of Eimeria bovis-infected Vero cells with membrane-permeant acetoxymethyl esters (AM-esters) of ion-sensitive dyes provided us with a noninvasive method for investigation of the permeability of the parasitophorous vacuole membrane (PVM) and simultaneous measurement of Ca2+ and H+ concentrations in different compartments of the infected cells. The distribution patterns of the cleaved membrane-impermeant dyes argue against the existence of nonselective pores in the PVM. There is also no indication of a parasitophorous duct connecting the vacuolar space with extracellular media. The pH inside the parasitophorous vacuole (PV) was lower than that in the cytoplasm of the host cell or the parasite, whereas the [Ca2+] in these compartments did not differ significantly. In HT29 cells infected with E. separata for 24 h the Ca2+ response to extracellular adenosine triphosphate (ATP) was significantly reduced, indicating influences on the host cell's intracellular signaling.

Electrogenic cation transport across the caecal epithelium of the leech Hirudo medicinalis was investigated using modified Ussing chambers. Transepithelial resistance (RT) and potential difference (VT) were 61.0 +/- 3.5 omega.cm2 and -1.1 +/- 0.2 mV (n = 149), respectively, indicating that leech caecal epithelium is a "leaky" epithelium. Under control conditions short circuit current (ISC) and transepithelial Na+ transport rate (INa) averaged at 22.1 +/- 1.5 microA.cm-2 and 49.7 +/- 2.6 microA.cm-2, respectively. Mucosal application of amiloride (100 mumol.l-1) or benzamil (50 mumol.l-1) influenced neither ISC nor INa. The transport system in the apical membrane showed no pronounced cation selectivity and a linear dependence on mucosal Na+ concentration. Removal of mucosal Ca2+ increased ISC by about 50% due to an increase of transepithelial Na+ transport. Trivalent cations (La3+ and Tb3+, 1 mmol.l-1 both) added to the mucosal Ringer solution reduced INa by more than 40%. Serosal ouabain (1 mmol.l-1) almost halved ISC and INa while 0.1% (= 5.4 mmol.l-1) DNP decreased INa to 11.8 +/- 5.1% of initial values. Serosal addition of cAMP increased both ISC and INa whereas the neurotransmitters. FMRFamide, acetylcholine, GABA, L-dopa, serotonin and dopamine failed to show any effects; octopamine, glycine and L-glutamate reduced INa markedly. On the basis of these results we conclude that in leech caecal epithelium apical uptake of monovalent cations is mediated by non-selective cation conductances which are sensitive to extracellular Ca2+ but insensitive to amiloride. Basolaterally Na+ is extruded via ouabain-sensitive and -insensitive ATPases. cAMP activates Na+ transport across leech caecal epithelium, although the physiological stimulus for cAMP-production remains unknown.

An increase in intracellular cyclic AMP concentration stimulates transepithelial Na(+) transport across the skin of the leech Hirudo medicinalis, but it is unclear how cytosolic cyclic AMP levels are elevated in vivo. In search of this external stimulus, we performed Ussing chamber experiments to test several peptide hormones and neurotransmitters for their effect on Na(+) transport across leech dorsal integument. Although all the peptide hormones under investigation significantly affected ion transport across leech integument, none of them mimicked the effect of an experimental rise in intracellular cyclic AMP level. The invertebrate peptides conopressin and angiotensin II amide inhibited short-circuit-current- (I(sc)) and amiloride-sensitive Na(+) transport (I(amil)), although to slightly different degrees. The vertebrate peptide hormones 8-arginine-vasopressin and 8-lysine-vasopressin both produced an inhibition of I(amil) comparable with that caused by angiotensin II amide. However, 8-lysine-vasopressin reduced I(sc), whereas 8-arginine-vasopressin induced a moderate increase in I(sc). The neurotransmitter dopamine, which occurs in the leech central nervous system in relatively large amounts, and its precursor l-dopamine both induced large decreases in I(sc) and I(amil). However, the reactions evoked by the catecholamines showed no pronounced similarity to the effects of intracellular cyclic AMP. Two other neurotransmitters known to occur in leeches, serotonin (5-hydroxytryptamine) and gamma-n-aminobutyric acid (GABA), had no influence on transepithelial ion transport in leech skin.

Electrogenic cation transport across the caecal epithelium of the leech Hirudo medicinalis was investigated using modified Ussing chambers. Transepithelial resistance (RT) and potential difference (VT) were 61.0 +/- 3.5 omega.cm2 and -1.1 +/- 0.2 mV (n = 149), respectively, indicating that leech caecal epithelium is a "leaky" epithelium. Under control conditions short circuit current (ISC) and transepithelial Na+ transport rate (INa) averaged at 22.1 +/- 1.5 microA.cm-2 and 49.7 +/- 2.6 microA.cm-2, respectively. Mucosal application of amiloride (100 mumol.l-1) or benzamil (50 mumol.l-1) influenced neither ISC nor INa. The transport system in the apical membrane showed no pronounced cation selectivity and a linear dependence on mucosal Na+ concentration. Removal of mucosal Ca2+ increased ISC by about 50% due to an increase of transepithelial Na+ transport. Trivalent cations (La3+ and Tb3+, 1 mmol.l-1 both) added to the mucosal Ringer solution reduced INa by more than 40%. Serosal ouabain (1 mmol.l-1) almost halved ISC and INa while 0.1% (= 5.4 mmol.l-1) DNP decreased INa to 11.8 +/- 5.1% of initial values. Serosal addition of cAMP increased both ISC and INa whereas the neurotransmitters. FMRFamide, acetylcholine, GABA, L-dopa, serotonin and dopamine failed to show any effects; octopamine, glycine and L-glutamate reduced INa markedly. On the basis of these results we conclude that in leech caecal epithelium apical uptake of monovalent cations is mediated by non-selective cation conductances which are sensitive to extracellular Ca2+ but insensitive to amiloride. Basolaterally Na+ is extruded via ouabain-sensitive and -insensitive ATPases. cAMP activates Na+ transport across leech caecal epithelium, although the physiological stimulus for cAMP-production remains unknown.