Ion transport across leech integument |
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Authors: | W -M Weber B Dannenmaier W Clauss |
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Institution: | (1) Institut für Tierphysiologie, Justus-Liebig-Universität Giessen, Wartweg 95, W-6300 Giessen, Germany |
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Abstract: | The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm-2) with an initial short-circuit current of 29.0±2.9 A·cm-2. Removal of Na+ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l-1) added to the basolateral solution, depressed the short-circuit current completely. The Na+ current saturated at a concentration of 90 mmol Na+·l-1 in the apical solution (K
M=11.2±1.8 mmol·l-1). Amiloride (100 mol·l-1) on the apical side inhibited ca. 40% of the Na+ current and indicated the presence of Na+ channels. The dependence of Na+ current on the amiloride concentration followed Michaclis-Menten kinetics (K
i=2.9±0.4 mol·l-1). The amiloride analogue benzamil had a higher affinity to the Na+ channel (K
i=0.7±0.2 mol·l-1). Thus, Na+ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na+·l-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na+ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l-1) and isobutylmethylxanthine (1 mmol·l-1) nearly doubled short-circuit current and increased amiloride-sensitive Na+ currents by 50%. By current fluctuation analysis we estimated single Na+ channel current (2.7±0.9 pA) and Na+ channel density (3.6±0.6 channels·m-2) under control conditions. After cyclic adenosine monophosphate stimulation Na+ channel density increased to 5.4±1.1 channels·m-2, whereas single Na+ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na+ channel, and show the similarities and differences to vertebrate Na+ channels. Whereas the channel properties are different from the classical vertebrate Na+ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na+ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na+ channels.Abbreviations
slope of the background noise component
- ADH
antidiuretic hormone
- cAMP
cyclic adenosine monophosphate
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f
frequency
-
f
c
coner frequency of the Lorentzian noise component
- Hepes
N-hydroxyethylpiperazine-N-ethanesulphonic acid
- BMX
isobutyl-methylxanthine
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i
Na
single Na+ channel current
-
I
Na
max, maximal inhibitable Na+ current
-
I
SC
short circuit current
-
K
i
half maximal blocker concentration
-
K
M
Michaelis constandard error of the mean
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S
(f)
power density of the Lorentzian noise component
-
S
0
plateau value of the Lorentzian noise component
- TMA
tetramethylammonium
- Trizma
TRIS-hydroxymethyl-amino-methane
-
V
max
maximal reaction velocity
-
V
T
transepithelial potential
-
K
half maximal blocker concentration |
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Keywords: | Na+ channels Amiloride Benzamil Noise analysis Leech Hirudo medicinalis |
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