Haemodynamic and tubular renal dysfunction in rats with sustained arterial calcinosis

1. In humans, two of the principal characteristics of vascular ageing are arterial wall calcification and decreased arterial distensibility, which induce organ damage. To amplify arterial calcium accumulation in laboratory animals, it is necessary to use an overdose of vitamin D(3). 2. The aim of the present study was to assess the impact of arterial calcium overload on renal function. 3. Adult male Wistar rats were randomly divided into two groups: control and treated rats. Treated rats were injected 10 days before the experiment with a single dose of vitamin D(3) (300 000 IU/kg, i.m.). 4. Treated rats showed a decrease in renal blood flow and glomerular filtration rate. Tubular parameters were not modified under basal conditions. In contrast, a statistically significant increase in the fractional excretion of Na, K, Ca and H(2)O were observed in treated rats after the acute increment of sodium distal delivery, suggesting that the reabsorptive capacity of the thick ascending limb may be altered in treated rats. 5. Thus, Na(+)/K(+)-ATPase activity was evaluated in homogenates from renal cortex and medulla. Rats with arterial calcinosis presented a diminished activity of Na(+)/K(+)-ATPase in medulla homogenates. 6. An increment in the abundance of the Na-K-2Cl cotransporter (NKCC2) was observed in renal medulla homogenates from treated rats. It is suggested that this may compensate for the inefficiency of Na(+)/K(+)-ATPase under basal conditions but, in the presence of acute distal sodium overload, the increment in NKCC2 abundance may not be sufficient to compensate for the decrease in Na(+)/K(+)-ATPase activity. 7. In summary, in our experimental model of arterial calcinosis, renal function is impaired, presenting a vascular compromise and altered function of the medullar thick ascending limb that becomes evident in the presence of acute high distal sodium delivery.     

pH-regulatory mechanisms in in vitro perfused rectal gland tubules of Squalus acanthias

 Isolated in vitro perfused rectal gland tubules (RGT) were preincubated with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and pH-regulatory mechanisms were studied. A reduction of bath Cl^– concentration from 269 to 6 mmol/l increased the fluorescence ratio 488/436 [corresponding to cytosolic pH (pH_i)] slightly but significantly (n=10). Depolarization by Ba²⁺ (1 mmol/l) or a bath solution containing 30 mmol/l K⁺ (n=4–6) increased the fluorescence ratio (pH_i). These data suggest that HCO₃ ^– uptake and/or H⁺ extrusion is dependent on Cl^– and/or voltage. A reduction of bath Na⁺ from 278 to 5 mmol/l reduced the ratio significantly (n=3). Addition of trimethylamine (Trima⁺, 20 mmol/l) alkalinized cytosolic pH (n=7). Similarly, addition of NH₄ ⁺ (20 mmol/l) led to an initial alkalinization and a strong acidification when NH₄ ⁺ was removed (n=59). The initial pH_i-recovery rates after NH₄ ⁺ removal were quantified and the responsible H⁺ extrusion and/or HCO₃ ^– import systems were examined. The recovery was almost completely abolished when the extracellular Na⁺ concentration was reduced to 5 mmol/l. In the presence of normal Na⁺, recovery was slower in the absence as compared to the presence of HCO₃ ^– (n=5). It was inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) (0.5 mmol/l, n=11) in the presence of HCO₃ ^– and in the absence of HCO₃ ^– by the Na⁺/H⁺-exchange blocker HOE694 (0.5 mmol/l, n=6). These data suggest that acid extrusion probably occurs by basolateral Na⁺-2HCO₃ ^–/Cl^– exchange in the presence of HCO₃ ^– and by basolateral Na⁺/H⁺ exchange in the absence of HCO₃ ^–. Luminal perfusion with a solution containing a low Cl^– concentration (6 mmol/l) increased the fluorescence ratio (pH_i) (n=5). The ratio (pH_i) was further increased and pH recovery further delayed by basolateral addition of Trima⁺ (20 mmol/l, n=3). These data suggest that the HCO₃ ^–/Cl^– exchanger is present in the luminal membrane. Luminal HCO₃ ^–/Cl^– exchange and basolateral Na⁺-2HCO₃ ^–/Cl^– exchange may work in tandem to secrete HCO₃ ^– and exchange it for luminal Cl^–. Received: 7 January 1998 / Received after revision and accepted: 5 March 1998     

Regulation of the Na+2Cl–K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias

 Previously it has been shown that the Na⁺2Cl^–K⁺ cotransporter accepts NH₄ ⁺ at its K⁺ binding site. This property can be used to estimate its transport rates by adding NH₄ ⁺ to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na⁺2Cl^–K⁺ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH₄ ⁺ to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH₃ uptake. This was followed by an acidification, corresponding to NH₄ ⁺ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl^– solution (6 mmol/l, low Cl^–) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH₄ ⁺ uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba²⁺ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl^– had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl^– (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH₄ ⁺ uptake rate was increased furosemide (500 μmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na⁺2Cl^–K⁺ cotransporter probably independently: (1) lowering of cytosolic Cl^– concentration; (2) cell shrinkage; and (3) activation by cAMP. Received: 3 March 1998 / Received after revision: 22 April 1998 / Accepted: 27 April 1998     

The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR

 The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl^–conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation ΔF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (V _m), the membrane conductance (G _m) and the membrane capacitance (C _m). C _m was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 μmol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on V _m and G _m in control CHO and ΔF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from –40 ± 1.5 to –32 ± 1.6 mV, n = 41) and enhanced G _m strongly from 5.0 ± 0.9 to 36 ± 3.9 nS (n = 65). The conductance increase was mostly for Cl^–, because under stimulated conditions a reduction in bath Cl^–concentration depolarized these cells further and significantly from –26 ± 1.8 to –10 ± 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br^– > Cl^– > I^–(n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance C _m was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of ΔF508-CFTR in CHO cells induces a cAMP-activated Cl^–conductance. The activation of this large conductance obviously proceeds with little if any exocytosis. Received: 13 May 1997 / Received after revision: 1 July 1997 / Accepted: 7 July 1997     

Chloride and sodium transport across bovine tracheal epithelium: Effects of secretagogues and indomethacin

The regulation of ion transport in bovine tracheal epithelium was studied in vitro. In the absence of exogenous midifiers of ion transport, average values for transepithelial electrical potential difference (_t), short-circuit-current (I _sc) and tissue resistance (R _t) were 35.4 mV (lumen negative), 5.4 Eq·h^–1·cm^–2 and 187 ·cm² respectively; net Cl secretion (3.2 Eq·h^–1·cm^–2) and net Na absorption (1.3 Eq·h^–1·cm²) accounted for 82% of theI _sc. Amiloride reduced (₁) andI _sc, and increasedR _t. The values of (_t),R _t andI _sc obtained following addition of theophylline, epinephrine or prostaglandin E₁ (PGE₁) were not different from control values. Theophylline aldo did not alter Na and Cl fluxes but it increased tissue cAMP content 3-fold. Indomethacin did not affect (_t) but it increasedR _t and net Na absorption, and decreasedI _sc and net Cl secretion; it did not significantly reduce tissue cAMP. When added to indomethacin-treated tissues, epinephrine restoredI _sc,R _t and Na and Cl fluxes to control levels and increased tissue cAMP 3-fold. Similary, when PGE₁ was added to indomethacin-treated tissues,I _sc andR _t were restored to control levels.We conclude that: (1) bovine tracheal epithelium, like its canine counterpart, absorbs Na and secretes Cl; the two tissues differ, however, in two ways: the spontaneous rate of Na absorption is higher in bovine trachea and the spontaneous rate of Cl secretion cannot be further increased in bovine trachea by secretagogues; (2) Cl secretion and Na absorption in bovine trachea are normally regulated by endogenous prostaglandins; (3) although cAMP may mediate changes in ion transport, a strict correlation between tissue cAMP content and Na and Cl transport rates is not evident; and (4) Na absorptive and Cl secretory rates are reciprocally related suggesting that both processes are present in the same cells.     

Expression of Na+/Cl–/betaine and Na+/myo-inositol transporters, aldose reductase and sorbitol dehydrogenase in macula densa cells of the kidney

 It has been suggested that macula densa cells may be exposed to hyperosmotic stress. Since chronic exposure to hypertonic stress causes the amount of intracellular organic osmolytes to increase, the expression of transporters and enzymes that participate in the intracellular accumulation of organic osmolytes was examined using non-radioactive in situ hybridization in the macula densa region of control rats and furosemide-treated animals. Both the sodium- and chloride-dependent betaine transporter (BGT) and sodium-dependent myo-inositol transporter (SMIT) were expressed preferentially in macula densa cells and for both mRNAs the signal intensity was visibly reduced by furosemide. The enzymes aldose reductase (which mediates the conversion of glucose to sorbitol) and sorbitol dehydrogenase (which converts sorbitol into fructose) were expressed not only in macula densa cells but also in the surrounding tubular cells, and the expression was insensitive to furosemide. Thus it remains unclear whether the expression of BGT and SMIT is related to a putative hypertonic juxtaglomerular region. Received: 27 April 1998 / Accepted: 8 July 1998     

A calcium-dependent chloride current in insulin-secreting βTC-3 cells

 Ca²⁺-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting β cells in response to high plasma glucose. A Maxi K⁺ channel has received the most attention, while a low-conductance Ca²⁺-activated K⁺ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca²⁺-dependent Cl^–current [I _Cl(Ca)] in insulin-secreting pancreatic β cells. The reported I_Cl(Ca) could be activated under physiological Ca²⁺ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca²⁺ influx: their current/voltage (I/V) relations followed that of the ”L-like” Ca²⁺ current (I _Ca) present in these cells; they were blocked completely by the removal of external Ca²⁺ or application of Cd²⁺ at concentrations sufficient for complete block of I _Ca; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of I _Ca showed a weak linear voltage dependence, while at voltages positive to activation of I _Ca it followed the voltage dependence of I _Ca. This Ca²⁺-dependent current reversed at –21.5 mV and when the external Cl^–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation for a Cl^–-selective current. Cl^–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was a Ca²⁺-dependent Cl^–current [I _Cl(Ca)]. From substitution of the external Cl^–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for I _Cl(Ca): I ^–>NO₃ ^–>Br^–>Cl^–>Acetate^–. Received: 10 October 1996 / Received after revision and accepted: 19 December 1996     

Mechanism of calcium ionophore stimulated Cl secretion from frog skin glands

The aim of the present study was to investigate the mechanism by which the calcium ionophore A23187 stimulates Cl and water secretion from exocrine glands in the frog skin. The Cl secretion was visualized as changes in short-circuit current (SCC) in skins where the Na absorption was blocked by amiloride applied to the apical membrane. Measurements of A23187 stimulated ion fluxes showed that the ionophore induced a net secretion of Cl, Na and K. The active Cl secretion was enhanced more than the Na and K secretion, resulting in a net secretion of negative ions which closely resembled the A23187-stimulated SCC. The effect of A23187 was abolished in skins pretreated with indomethacin, implying the involvement of prostaglandins in the response. Furthermore, the effect of A23187 was inhibited in the presence of quinacrine, indicating that the activation of the cyclooxygenase pathway is dependent on phospholipase A₂ activity. In addition, the A23187, but not the arachidonic acid stimulated Cl secretion was abolished in the presence of trifluoperazine, suggesting that the effect of the ionophore may be mediated via a Ca²⁺-calmodulin-dependent step located before the activation of the cyclooxygenase. The net water flow and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and time-course of the water secretion were similar to the changes observed for the Cl secretion. The A23187 stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophyllin, indicating that the effect of A23187 was caused by an increase in the intracellular cAMP level in the gland cells. From the present data it is suggested that the calcium ionophore stimulates the Cl and water secretion from frog skin gland not by a direct effect of Ca²⁺-ions per se, but in an indirect manner by stimulating the prostaglandin synthesis, which probably results in an increase in the cAMP level in the gland cells.     

Role of anions in the regulation of cell volume and intracellular pH in perfused rat mandibular salivary gland

 To investigate the role of Cl^– in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pH_i) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl^– was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl^– on the Na⁺-K⁺-2Cl^– cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl^– by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pH_i consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO₃ ^– omission, 100 μM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pH_i was due to Cl^–/HCO₃ ^– exchange while the secondary rise resulted from activation of Na⁺/H⁺ exchange. Although replacement of Cl^– by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl^– replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl^– concentration contribute to the up-regulation of the exchanger following muscarinic stimulation. Received: 12 November 1996 / Received after revision: 11 August 1997 / Accepted: 18 August 1997     

The plasma contact system in atopic asthma

An in vitro test for the rate of appearance of kallikrein in plasma due to contact system activation by dextran sulfate at 0 degree C was applied to plasmas of 19 atopic asthma patients and 19 age- and sex-matched controls without atopy. The average prekallikrein activation rate was markedly higher in the plasmas of the atopic patients. Mean endogenous heparin levels were also elevated.