Ion transport across leech integument

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 - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - 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 - 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     

Apparent intracellular Mg2+ buffering in neurons of the leech Hirudo medicinalis

The apparent intracellular Mg2+ buffering, or muffling (sum of processes that damp changes in the free intracellular Mg2+ concentration, [Mg2+](i), e.g., buffering, extrusion, and sequestration), was investigated in Retzius neurons of the leech Hirudo medicinalis by iontophoretic injection of H+, OH-, or Mg2+. Simultaneously, changes in intracellular pH and the intracellular Mg2+, Na+, or K+ concentration were recorded with triple-barreled ion-selective microelectrodes. Cell volume changes were monitored measuring the tetramethylammonium (TMA) concentration in TMA-loaded neurons. Control measurements were carried out in electrolyte droplets (diameter 100-200 microm) placed on a silver wire under paraffin oil. Droplets with or without ATP, the presumed major intracellular Mg2+ buffer, were used to quantify the pH dependence of Mg2+ buffering and to determine the transport index of Mg2+ during iontophoretic injection. The observed pH dependence of [Mg2+](i) corresponded to what would be expected from Mg2+ buffering through ATP. The quantity of Mg2+ muffling, however, was considerably larger than what would be expected if ATP were the sole Mg2+ buffer. From the decrease in Mg2+ muffling in the nominal absence of extracellular Na+ it was estimated that almost 50% of the ATP-independent muffling is due to the action of Na+/Mg2+ antiport.     

Proteinase inhibitors from the medicinal leech Hirudo medicinalis

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, -chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of -chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.     

RFamide Peptides in the Leech, Hirudo medicinalis

RFamide peptides have been localized to a number of neuronsof the CNS of the leech, Hirudo medicinalis, using immunocytochemicaltechniques. The majority of this immunoreactivity appears tobe due to the peptide FMRFamide. Most of the identified RFamideimmunoreactive cells are cholinergic motor neurons, though someare interneurons. Superfused FMRFamide is active on the targetsof these identified neurons; in a few well studied cases, ithas been possible to show that FMRFamide mimics a specific physiologicalaction of an identified neuron on its target. In the leech as in other phyla where they occur, RFamide peptidesare widely distributed in neurons, and are neuromodulators withdiverse physiological effects.     

Pyroglutamate stimulates Na+ -dependent glutamate transport across the blood-brain barrier

Regulation of Na(+)-dependent glutamate transport was studied in isolated luminal and abluminal plasma membranes derived from the bovine blood-brain barrier. Abluminal membranes have Na(+)-dependent glutamate transporters while luminal membranes have facilitative transporters. This organization allows glutamate to be actively removed from brain. gamma-Glutamyl transpeptidase, the first enzyme of the gamma-glutamyl cycle (GGC), is on the luminal membrane. Pyroglutamate (oxoproline), an intracellular product of GGC, stimulated Na(+)-dependent transport of glutamate by 46%, whereas facilitative glutamate uptake in luminal membranes was inhibited. This relationship between GGC and glutamate transporters may be part of a regulatory mechanism that accelerates glutamate removal from brain.     

Na+-sensitive component of 3-O-methylglucose uptake in frog skeletal muscle

Summary A Na⁺-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na⁺ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na⁺ removed. At a 3-O-MG concentration of 2mm, the Na⁺-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na⁺-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na⁺-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na⁺-sensitive component was at a 3-O-MG concentration of about 13mm, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5mm), a potent inhibitor specific for Na⁺-coupled glucose transport, reduced the uptake in a solution containing Na⁺ to the level in Na⁺-free Ringer's solution. Glucose of concentrations higher than 20mm suppressed 3-O-MG uptake to a level slightly lower than that in Na⁺-free Ringer's solution. These observations indicate that there are Na⁺-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG.     

Antithrombotic effect of preparations from the leeches Hirudo medicinalis

Antithrombotic effect of leech salivary gland secretion was maximal after intravenous administration into rats and was slightly decreased in cases of peroral administration. Blood from leech intestinal tract and leech homogenate exhibited less distinct antithrombotic action. Effect of these preparations was maintained after peroral administration. The antithrombotic effect of the leech preparations did not depend on their antithrombic activity caused by hirudin. These leech preparations appear to elongate a period of blood plasma recalcification caused by kallikrein inhibitor as well as apparently due to their capacity to inhibit aggregation of the thrombocytes.     

Electrical characteristics of sensory neurons of Hirudo medicinalis

During intracellular polarization of identified sensory neurons of the leech by square pulses of hyperpolarizing current electrical parameters of the cell membranes were determined: input resistance of the neuron R_n, time constant of the membrane , the ratio between conductance of the cell processes and conductance of the soma , the resistance of the soma membrane r_s, the input resistance of the axon r _a , capacitance of the membrane C_s, and resistivity of the soma membrane R_s. The results obtained by the study of various types of neurons were subjected to statistical analysis and compared with each other. Significant differences for neurons of N- and T-types were found only between the values of , C_s, and R_s (P<0.01). These parameters also had the lowest coefficients of variation. The surface area of the soma of the neurons, calculated from the capacitance of the membrane (the specific capacitance of the membrane was taken as 1 µF/cm²) was 7–10 times (N-neurons) or 4–6 times (T-neurons) greater than the surface area of a sphere of the same diameter. The resistivity of the soma membrane R_s was 35.00 k·cm² for cells of the N-type and 19.50 k·cm² for T-neurons. The reasons for the relative stability of this parameter compared with the input resistance of the cell (coefficient of variation 22–7 and 53–31% respectively) are discussed. The possible effects of electrical characteristics on the properties of repeated discharges in neurons of different types also are discussed.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol.7, No.3, pp.295–301, May–June, 1975.     

Intracellular Na+ activity as a function of Na+ transport rate across a tight epithelium.

Liquid Na+ resin microelectrodes were used to measure intracellular Na+ activities (alpha iNa+) in the mammalian tight epithelium, rabbit urinary bladder. alpha iNa+ averaged 7 +/- 1 mM and was independent of Na+ transport rate over the range of 2 to 8 muA/muF. (1 mF congruent to 1 cm2 apical membrane area). After Na+ loading the cells the Na+ pump in the basolateral membrane was measurably electrogenic. A method for shielding the Na+-sensitive microelectrodes is described which increases the response time and eliminates an electrical shunting artifact.     

Swelling-activated Gd3+-sensitive Cation Current and Cell Volume Regulation in Rabbit Ventricular Myocytes

The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6–0.9× normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (I_Cir,swell) that was reversibly blocked by Gd³⁺, but I_Cir,swell was not detected in isotonic or hypertonic media. This current was not related to I_K1 because it was insensitive to Ba²⁺. The P_K/P_Na ratio for I_Cir,swell was 5.9 ± 0.3, implying that inward current is largely Na⁺ under physiological conditions. Increasing bath K⁺ increased g_Cir,swell but decreased rectification. Gd³⁺ block was fitted with a K _0.5 of 1.7 ± 0.3 μM and Hill coefficient, n, of 1.7 ± 0.4. Exposure to Gd³⁺ also reduced hypotonic swelling by up to ∼30%, and block of current preceded the volume change by ∼1 min. Gd³⁺-induced cell shrinkage was proportional to I_Cir,swell when I_Cir,swell was varied by graded swelling or Gd³⁺ concentration and was voltage dependent, reflecting the voltage dependence of I_Cir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that I_Cir,swell was sufficient to explain the majority of the volume change at –80 mV. In addition, swelling activated an outwardly rectifying Cl⁻ current, I_Cl,swell. This current was absent after Cl⁻ replacement, reversed at E_Cl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of I_Cl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, I_Cir,swell is expected to cause diastolic depolarization. Activation of I_Cir,swell also is likely to affect contraction and other physiological processes in myocytes.     

A novel method to quantify H+-ATPase-dependent Na+ transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na(+) is excluded from the cytosol to the apoplast or the vacuole by Na(+)/H(+) antiporters. The secondary active transport of Na(+) to apoplast against its electrochemical gradient is driven by plasma membrane H(+)-ATPases that hydrolyze ATP and pump H(+) across the plasma membrane. Current methods to determine Na(+) flux rely either on the use of Na-isotopes ((22)Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H(+) gradient due to H(+)-ATPase in the presence or absence of Na(+) by pH-sensitive probes. To date, there are no methods that can directly quantify H(+)-ATPase-dependent Na(+) transport in plasma membrane vesicles. We developed a method to measure bidirectional H(+)-ATPase-dependent Na(+) transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H(+)-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na(+) sealed for the study of H(+)-ATPase-dependent Na(+) transport. Our data implicate that Na(+) movement across vesicle membranes is highly dependent on H(+)-ATPase activity requiring ATP and Mg(2+) and displays optimum rates of 2.50 microM Na(+) mg(-1) membrane protein min(-1) at pH 6.5 and 25 degrees C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H(+)-ATPase-dependent Na(+) transport. The results demonstrate that the Na(+) content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H(+)-ATPase-dependent Na(+) transport with membrane vesicles.