The role of a swelling-activated taurine transport pathway in the regulation of articular chondrocyte volume

Swelling articular chondrocytes by reducing osmolarity stimulates a taurine transport pathway, which is implicated in regulatory volume decrease (RVD) in various cell types. The present study investigated factors controlling the activity of this pathway in chondrocytes, in particular (1) the effects of the acute (seconds) and chronic (hours) exposure of chondrocytes to anisotonic media, and (2) whether there is a role for metabolites from the arachidonic acid cascade in activating the taurine transport pathway. For in situ and isolated chondrocytes, the point at which swelling-activated [14C]taurine efflux was stimulated (the "set-point") corresponded closely to the osmolarity of the incubation medium (180, 280 or 380 mosmol/l). However, the volume of chondrocytes isolated into these media and measured by confocal microscopy was not different ( congruent with 645 microm3). Activity of the swelling-activated taurine transport pathway was inhibited by REV5901 (an inhibitor of steps of the arachidonic acid cascade; K0.5 8+/-4 microM), NDGA (a general lipoxygenase inhibitor; K0.5 28+/-5 microM), or MK886 (an inhibitor of the 5-lipoxygenase-activating protein; 91% inhibition at 10 microM), but weakly by the more potent 5-lipoxygenase inhibitor REV5901 para (K0.5 350+/-100 microM). Addition of the leukotriene (LT) B4 or D4 receptor antagonists, CP-105,696 and L660,711 respectively, or of the leukotrienes LTB4, LTC4, LTD4 and LTE4 or lipoxins (hepoxylin A3 or B3) had no effect on the activity of the pathway in isotonic or hypotonic media. The role of the pathway in RVD was determined in isolated calcein-loaded chondrocytes using fluorescence imaging. RVD was observed and inhibited by REV5901 (50 microM) and by NDGA (75 microM). The data show that despite chronic exposure of chondrocytes to anisotonic media, the cells maintain a pre-determined volume that is the "set-point" for the activation of the taurine transport pathway following acute hypotonic challenge. This pathway appears to play a role in chondrocyte RVD, but its activation does not involve metabolites of the arachidonic acid cascade.     

Influence of volume expansion,serum sodium,and fractional excretion of sodium on urate excretion

The relative contributions of volume expansion and increased fractional excretion of sodium to the uricosuria of saline infusion were assessed in 19 subjects by volume expansion with rapid infusion of 21 of hypertonic saline (3%), isotonic saline (0.9%), or hypotonic saline (0.45%). Urate excretion increased 385 mug/min (P less than 0.01) with hypertonic, 145 mug/min (P less than 0.05) with isotonic saline, and 294 mug/min (P less than 0.001) with hyptonic saline. When 150 meq of sodium chloride was administered as appropriate volumes of hypertonic, isotonic of hypotonic saline, the magnitude or uricosuria was correlated with volume load (r = 0.66, P less than 0.002). fractional excretion of sodium correlated with infusion volumes for all studies taken together (r = 0.35, P greater than 0.1). The relationship between fractional excretion of sodium and fractional excretion of urate was entirely attributed to their correlation with infusion volume. Both post-pyrazinamide urate excretion and pyrazinamide suppressible urate excretion increased with volume expansion.     

Effects of ammonia in vitro on endogenous taurine efflux and cell volume in rat cerebrocortical minislices: influence of inhibitors of volume-sensitive amino acid transport.

Rat cerebrocortical minislices were incubated with physiological saline in the absence or presence of 5 mM ammonium acetate ("ammonia") and/or inhibitors of osmosensitive amino acid transport: 50 microM niflumic acid and 100 microM N-ethyl-maleimide for 60 min, with medium changes after 20 min and 40 min. The efflux of endogenous taurine, glutamate and glutamine was assayed by high-performance liquid chromatography, and steady-state cell volumes were monitored in the slices with the [14C]inulin method. In the absence of ammonia, niflumic acid abolished taurine efflux but did not affect glutamate or glutamine efflux at all time-points, and increased cell volume at 20 min and 60 min. N-Ethyl-maleimide increased taurine, glutamine and glutamate efflux at 20 min and 40 min, inhibited taurine and glutamine efflux at 60 min, and increased cell volume at 20 min. Ammonia strongly stimulated taurine (by 380% at 20 min), and only moderately glutamate (30% at 20 min) or glutamine efflux (76% at 20 min). Ammonia increased cell volume above the control level at all time-points. Niflumic acid inhibited, but did not abolish ammonia-dependent taurine and glutamine efflux, and did not change glutamate efflux. The effects of ammonia + niflumic acid on cell volume did not differ from the effects of each compound separately. N-Ethyl-maleimide inhibited ammonia-dependent efflux of all three amino acids except for stimulation of glutamate efflux at 20 min. N-Ethyl-maleimide + ammonia decreased the cell volumes more than did each compound separately. It is concluded that although ammonia-induced taurine efflux is accompanied by an increase in cell volume, the underlying mechanism is not simply a cell volume regulatory response normally observed in hypoosmotic stress. Increased efflux of taurine, which is an inhibitory amino acid and a cell membrane protectant, may serve to counteract the deleterious effects of increased excitatory transmission accompanying acute hyperammonemic insult.     

Role of G-proteins in the regulation of organic osmolyte efflux from isolated rat renal inner medullary collecting duct cells

 Hypotonic shock (change of osmolality from 600 mosmol to 300 mosmol by lowering NaCl concentration) increases the release of organic osmolytes from isolated inner medullary collecting duct (IMCD) cells in the following sequence: taurine > betaine > sorbitol > myo-inositol > glycerophosphorylcholine (GPC). The role of G-proteins in regulating the hypotonicity-induced efflux was analysed by exposing cells to various concentrations of a G-protein inhibitor, pertussis toxin (PTX; 20–200 ng/ml), and a G_iα-protein stimulator, mastoparan (10–50 μM). PTX diminished the hypotonic release of sorbitol and betaine by 43.2±9.5% and 32.2±7.8% (n = 5), respectively. Efflux of GPC, myo-inositol and taurine was not significantly altered. Mastoparan (10 μM) increased osmolyte release under isotonic conditions such that release of betaine was increased 3.8-fold and that of sorbitol 2.1-fold, while GPC, myo-inositol and taurine effluxes were only slightly augmented. Under hypotonic conditions, mastoparan stimulated betaine release (1.86±0.2-fold, n = 5) but not that of sorbitol. As tested in connection with sorbitol and betaine release, the effect of mastoparan was abolished by PTX, but not the A23187-evoked sorbitol release. Like mastoparan, arachidonic acid increased the release of sorbitol and betaine under isotonic conditions, but under hypotonic conditions it only increased the release of betaine. As to the role of intracellular Ca²⁺, hypotonic shock evoked an intracellular Ca²⁺ peak which could be prevented by PTX. Mastoparan increased intracellular Ca²⁺ under isotonic conditions, whether the extracellular Ca²⁺ concentration was low or high. The results indicate that G-proteins are involved in regulating sorbitol and betaine efflux from IMCD cells. The G-proteins regulating sorbitol release are probably involved in generating the proper intracellular Ca²⁺ signal. Betaine efflux, which is independent of intracellular Ca²⁺, might be regulated by a G-protein-stimulated release of arachidonic acid. Thus, probably several G-proteins are involved in controlling organic osmolyte efflux from IMCD cells. Received: 2 April 1996 / Received after revision: 30 June 1996 / Accepted 25 July 1996     

Reversibility of Effects of Very Hypotonic Fluids on in Vitro Frog Gastric Mucosa: A Functional and Morphological Study

Functional and morphological properties of the in vitro frog gastric mucosa were studied during and after exposure to very hypotonic (? 25 mOsM) solutions. Within 20 min the acid secretory rate decreased to zero, but it returned to normal levels after isotonic fluids had been restored. The potential difference (PD) dropped within the first minutes after the exposure to hypotonic solutions, and became inverted. Following the return of isotonic conditions the PD increased to levels higher than in the controls. The electrical resistance increased about 10–fold during the hypotonic period, but decreased to near normal values when isotonic conditions were restored. By light and electron microscopy the cells of the hypotonic mucosae appeared greatly swollen, and the alterations were assessed by morphometric methods. The gland lumina were almost obliterated, and the lamina propria was reduced to about 60% of its former volume. After the return to isotonic conditions normal morphology was restored. It is conceivable that the great increase in resistance during the hypotonic period was caused by the occlusion of the gland lumina. Quantitative analyses of the Na, K, and C1 tissue concentrations indicated a large loss of these ions during the hypotonic state. Presumably the epithelial cells in the hypotonic mucosae avoid bursting by rapidly letting large numbers of ions exit, which results in a cellular osmolarity close to that of the bathing fluids.     

Volume regulation in mouse pancreatic islet cells as studied by a new technique of microperifusion

A new technique was designed to analyse whether pancreatic islet cells are able to regulate their volume in anisotonic media. The projected cell area of individual cells was continuously observed, and the corresponding volume calculated during microperifusion with media of different osmolarities. In isotonic medium (317 mosmol) the cell volume was stable during perifusion and decreased by 17 or 25% when the osmolarity was increased (sucrose) to 417 or 517 mosmol. Reducing the medium osmolarity to 285 mosmol resulted in a volume reduction of about 7%. No evidence for cell volume regulation was observed in these media. However, reducing the medium osmolarity to 262 mosmol induced an immediate and rapid cell swelling of approximately 14%, after which the initial cell volume was regained within 9 min. The data suggest that the pancreatic islet cells are equipped with mechanisms for regulatory volume decrease that appear to be activated when the cell volume is increased above a certain limit.     

The relationship between hypotonically-induced taurine and K fluxes in trout red blood cells

Hypotonic swelling of teleost erythrocytes activates multiple transport systems leading to the regulatory decrease of cell volume. We have examined using pharmacological manipulation the swelling-induced taurine flux pathway in red blood cells of the rainbow trout and its relationship to swelling-induced K flux pathways. We show that the activation and deactivation of taurine flux is rapid and that the flux is a sigmoidal function of cell volume. N-ethylmaleimide (NEM) and the non-specific protein kinase inhibitor, staurosporine, both inactivated the hypotonically-induced taurine flux with concentrations eliciting half-maximal inhibition (IC50s) of 212 and 17 micromol(-1), respectively. The low taurine fluxes under isotonic conditions were unaffected. By contrast, the tyrosine kinase inhibitor, genistein, partially inhibited taurine flux under both isotonic and hypotonic conditions. The specific phosphatase inhibitor, calyculin A, had no inhibitory or stimulatory effect under either condition whilst the less-specific phosphatase inhibitor, ortho-vanadate, reduced taurine flux only under hypotonic conditions. In these respects the regulatory control of the taurine pathway differs from the Cl-dependent K flux. However, NEM and staurosporine also inhibited the Cl-independent K flux, both with similar IC50s to those observed for taurine fluxes. This supports the idea of the hypotonically-induced taurine flux and the Cl-independent K flux sharing the same transport pathway.     

Taurine efflux is a cell volume regulatory process in proximal renal tubules from the teleost Carassius auratus

The potential role of taurine transport associated with volume regulation in renal tissue and isolated proximal renal tubules was studied in the teleost Carassius auratus (goldfish). The cellular taurine content in renal tissue fragments incubated in isosmotic solution (290 mOsm) (7.8 ± 0.9 (SD) μmol g wet wt^-1) decreased by 60% following exposure to hyposmotic medium (100mOsm). The rate coefficient for [¹⁴C]taurine efflux in renal tissue and in isolated proximal renal tubules was strongly stimulated following hyposmotically or urea-activated cellular swelling. The stimulated basolateral taurine efflux pathway exhibited channel-like functional characteristics since (a) [¹⁴C]taurine influx was stimulated in parallel with the osmolality-dependent taurine efflux and (b) this efflux could not be stimulated by high medium taurine concentrations (40 mM) applied 10 min following the osmolality reduction (trans-stimulation test). Administration of the 5-lipoxygenase inhibitor ETH 615-139 (20μM) during hyposmotic stimulation inhibited regulatory volume decreases but had no effect on taurine efflux. In addition, hyposmotically induced taurine efflux was slightly but significantly inhibited by the cyclooxygenase inhibitor indomethacin (10 μM). The taurine efflux was also dependent on both extra- and intracellular Ca²⁺. It is concluded that taurine is likely to coparticipate with KCI as an osmoeffector during RVD in Carassius proximal renal tubule cells. Cellular swelling seems to activate a basolateral taurine transport pathway with functional properties of a channel. This efflux mechanism appears to be partly regulated by Ca²⁺. Such a transport pathway could play a role in the cell volume regulatory mechanisms participating during transepithelial solute and water transport.     

Effects of osmotic stress on dextran diffusion in rat neocortex studied with integrative optical imaging.

Effects of osmotic stress on dextran diffusion in rat neocortex studied with integrative optical imaging. This study investigated how dextran (Mr = 3,000) diffused in rat cortical slices when the osmolarity of the bathing artificial cerebrospinal fluid was altered by varying the NaCl content. The apparent diffusion coefficient, D*, was measured in the neocortex region using fluorescent molecules and the integrative optical imaging (IOI) method. The main results were: 1) the value of D* in rat neocortex in the isotonic (300 mOsm) artificial cerebrospinal fluid at 34 degrees C was D* = 0.68 +/- 0. 01 x 10(-6) cm2 s-1 (mean +/- SE, n = 78) and it could be changed within minutes by varying the extracellular osmolarity. 2) Hypotonic stresses up to -100 mOsm decreased D* by 35% and were fully reversible when the slices were returned to the isotonic medium. Further hypotonic stress to -150 mOsm caused further decrease in D* but after removal of the stress, D* overshot its control value. 3) Hypertonic stress of +50 mOsm increased D*, but the maximum reversible increase in D* was only 15%. Further hypertonic stress (to +200 mOsm) did not cause any further increase in D* and, after removal of the stress, D* undershot the control value. The changes in D* are thought to be related to volume changes of cells in tissue: hypotonic solutions caused cell swelling, resulting in reduced extracellular space and compressed extracellular matrix so that the dextran diffusion was more hindered. Hypertonic solutions had the opposite effect. Recordings of extracellular field potentials in the hippocampal CA1 region demonstrated that, on return to the isotonic solution after exposure to an extreme hypotonic or hypertonic stress, the neurons retained their ability to generate synaptic responses.     

渗透压、细胞容积与鼻咽癌细胞增殖

目的:研究渗透压、细胞容积与鼻咽癌细胞增殖的关系。方法:用MTT法检测在不同渗透压培养条件下低分化鼻咽癌细胞(CNE－2Z)的增殖能力,流式细胞仪测定细胞周期分布,活细胞图像分析测量细胞容积,台盼蓝拒染法检测细胞存活率。结果:高渗(370、440mOsmol/L)培养增大细胞容积和促进细胞增殖,细胞容积分别增大8.7%、27.8%,增殖率分别提高22.2%和33.9%;而低渗(160、230mOsmol/L)培养减小细胞容积和抑制增殖,细胞容积分别减小12.8%和4.1%,增殖率分别降低34.0%和15.6%;细胞容积与细胞增殖率呈正相关。非等渗长期培养条件下,细胞周期各时相分布没有显著差异。低渗培养降低细胞生存率。结论:胞外渗透压、细胞容积与鼻咽癌细胞增殖密切相关,低渗培养可能通过减小细胞容积、促进细胞死亡而抑制细胞增殖。     

Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687 mosmol l⁻¹) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced in cells expressing a constitutively active G protein, Rac (RacV₁₂A₃ cell), indicating that Rac acts upstream to caspase-3 activation. The stress-activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol to the nucleus. Inhibition of p38 in Rac cells reduced the activation of both p53 and caspase-3. After 60 min in hypertonic medium the rate constants for K⁺ and taurine efflux were increased, particular in Rac cells. We suggest the following sequence of events in the cell shrinkage-induced apoptotic response: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation.     

Vasopressin-induced taurine efflux from rat pituicytes: a potential negative feedback for hormone secretion

Previous work on the whole neurohypophysis has shown that hypotonic conditions increase release of taurine from neurohypophysial astrocytes (pituicytes). The present work confirms that taurine is present in cultured pituicytes, and that its specific release increases in response to a hypotonic shock. We next show that vasopressin (VP) and oxytocin (OT) also specifically release taurine from pituicytes. With an EC₅₀ of ∼2 n m , VP is much more potent than OT, and the effects of both hormones are blocked by SR 49059, a V_1a receptor antagonist. This pharmacological profile matches the one for VP- and OT-evoked calcium signals in pituicytes, consistent with the fact that VP-induced taurine efflux is blocked by BAPTA-AM. However, BAPTA-AM also blocks the taurine efflux induced by a 270 mosmol l⁻¹ challenge, which per se does not evoke any calcium signal, suggesting a permissive role for calcium in this case. Nevertheless, the fact that structurally unrelated calcium-mobilizing agents and ionomycin are able to induce taurine efflux suggests that calcium may also play a signalling role in this event. It is widely accepted that in hypotonic conditions taurine exits cells through anionic channels. Antagonism by the chloride channel inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) suggests the same pathway for VP-induced taurine efflux, which is also blocked in hypertonic conditions (330 mosmol l⁻¹). Moreover, it is likely that the osmosensitivity of the taurine channel is up-regulated by calcium. These results, together with our in situ experiments showing stimulation of taurine release by endogenous VP, strengthen the concept of a glial control of neurohormone output.     

Components of taurine efflux in rat brain synaptosomes.

The efflux of [³⁵S]taurine from isolated rat brain synaptosomes was studied in a superfusion system. The spontaneous efflux of taurine was slow and could be described as comprising two first-order rate components, of which the slower (t_1/2 = 77.0min) represents release from intrasynaptosomal spaces. Only a high taurine concentration (10 mmol/1) in the medium enhanced the efflux of intrasynaptosomal taurine, which indicates a rather stable intrasynaptosomal taurine compartment. Depolarizing concentrations of potassium ions stimulated taurine efflux and also induced the appearance of a new ‘intermediate’ efflux component. This component was absent when calcium ions were omitted from the medium. It is therefore suggested that the component may originate from the emptying of synaptic vesicles.     

Alterations in electrical and mechanical activity in Langendorff-perfused guinea pig hearts exposed to decreased external sodium concentration with or without hypotonic insult

In order to examine electrical and mechanical effects of hyponatremia and hypotonicity, relevant to those in patients with ‘water intoxication’ syndrome, Langendorff-perfused guinea pig hearts were exposed to reduced NaCl concentrations (hypotonic [NaCl]₀-reduction) under the monitoring of left ventricular developed pressure (LVDP) and epicardial ECG. In some hearts, hyponatremia (from 140 to 80 mEq/l) was compensated for by adding mannitol to maintain osmolarity at a constant level (isotonic [NaCl]₀-reduction) or tetraethylammonium chloride to maintain both osmolarity and chloride concentrations at a constant level (isotonic [Na⁺]₀-reduction). Progressive isotonic [NaCl]₀-reduction increased LVDP, which was abolished in the presence of KB-R7943, a novel inhibitor of Na⁺/Ca²⁺-exchange. LVDP was reduced in hypotonic [NaCl]₀-reduction in which myocardial water content was increased. PQ interval and QRS duration were prolonged with both hypotonic and isotonic [NaCl]₀-reduction and these changes tended to be more pronounced with hypotonic than with isotonic [NaCl]₀-reduction. Similar ECG changes were also evident with isotonic [Na⁺]₀-reduction. Gd³⁺ (1–5 μM), a blocker of stretch-activated nonspecific cation channels, had no substantial effects on the electrical or mechanical changes seen with hypotonic [NaCl]₀-reduction. In conclusion, isotonic [NaCl]₀-reduction produced a positive inotropism by modulating Na⁺/Ca²⁺-exchange, whereas hypotonic [NaCl]₀-reduction led to negative inotropism, due in part to hypotonic myocardial swelling. In addition, [Na⁺]₀-reduction, irrespective of the concomitant [Cl⁻]₀ or osmotic changes, depressed atrioventricular as well as intraventricular conduction.     

Osmotic regulation of neuronal activity: a new role for taurine and glial cells in a hypothalamic neuroendocrine structure

Maintenance of osmotic pressure is a primary regulatory process essential for normal cell function. The osmolarity of extracellular fluids is regulated by modifying the intake and excretion of salts and water. A major component of this regulatory process is the neuroendocrine hypothalamo-neurohypophysial system, which consists of neurons located in the paraventricular and supraoptic nuclei. These neurons synthesize the neurohormones vasopressin and oxytocin and release them in the blood circulation. We here review the mechanisms responsible for the osmoregulation of the activity of these neurons. Notably, the osmosensitivity of the supraoptic nucleus is described including the recent data that suggests an important participation of taurine in the transmission of the osmotic information. Taurine is an amino acid mainly known for its involvement in cell volume regulation, as it is one of the major inorganic osmolytes used by cells to compensate for changes in extracellular osmolarity. In the supraoptic nucleus, taurine is highly concentrated in astrocytes, and released in an osmodependent manner through volume-sensitive anion channels. Via its agonist action on neuronal glycine receptors, taurine is likely to contribute to the inhibition of neuronal activity induced by hypotonic stimuli. This inhibitory influence would complement the intrinsic osmosensitivity of supraoptic neurons, mediated by excitatory mechanoreceptors activated under hypertonic conditions. These observations extend the role of taurine from the regulation of cell volume to that of the whole body fluid balance. They also point to a new role of supraoptic glial cells as active components in a neuroendocrine regulatory loop.     

Effect of extracellular volume expansion on renal cortical and medullary Na+−K+-ATPase

Summary The role of renal Na⁺–K⁺-ATPase in the acute changes in sodium reabsorption caused by isotonic volume expansion was evaluatedin vivo andin vitro in the rat and the dog. Duringin vivo volume expansion with isotonic saline in the rat, renal medullary Na⁺–K⁺-ATPase specific activity increased, while the simultaneously determined cortical Na⁺–K⁺-ATPase specific activity and kinetics remained unchanged. Furthermore, experimentsin vitro failed to demonstrate a circulating inhibitor of renal Na⁺–K⁺-ATPase both in plasma dialysates from volume-expanded rats and in plasma dialysates concentrated 20-fold by ultrafiltration from volume-expanded dogs. These results suggest that the decreased proximal tubular reabsorption of sodium during volume expansion is not mediated by inhibition of renal cortical Na⁺–K⁺-ATPase. The acute increment in medullary Na⁺–K⁺-ATPase observed could represent an adaptive response to increased sodium reabsorption by the loops of Henle, and raises the possibility that this enzyme may participate in relatively rapid adjustments in the transport of sodium by the renal tubule.     

Cell volume-induced changes in K+ transport across the rat colon.

The effect of cell swelling and cell shrinkage on K+ transport across the rat colonic epithelium was studied by measuring unidirectional fluxes, uptake and efflux of 86Rb+, a marker for K+. Exposure to a hypotonic medium stimulated the secretory, serosa-to-mucosa flux of K+, whereas exposure to a hypertonic medium inhibited the absorptive, mucosa-to-serosa flux of K+ in the distal, but not in the proximal colon. Neither manoeuvre had any effect on the uptake of K+ across the apical or the basolateral membrane. Cell swelling induced a sustained increase in the apical and basolateral K+ efflux from both colonic segments, whereas cell shrinkage reduced the efflux. Ba2+ (10(-2) mol l(-1)) inhibited the swelling-induced stimulation of the apical, quinine (10(-3) mol l(-1)) that of the basolateral K+ efflux in the distal colon. Incubation of the tissue in Ca2+-free buffer or La3+, which blocks Ca2+-influx into the epithelium, strongly reduced the basal K+ efflux across the basolateral membrane. The same was observed with brefeldin A, a blocker of the transport of newly synthesized proteins out of the endoplasmatic reticulum. Swelling-induced K+ efflux, however, was not reduced. In the presence of colchicine, an inhibitor of the polymerization of microtubules, swelling evoked only a transient increase in mucosal efflux, which, especially in the proximal colon, fell after 6 min to the level of the isotonic control period. These results demonstrate that the cell volume is involved in the regulation of transepithelial K+ transport across the rat colonic epithelium and suggest a role of the cytoskeleton in the control of a part of the volume-sensitive K+ channels.     

Evidence for the involvement of K+ channels and K+-Cl– cotransport in the regulatory volume decrease of newborn rat cardiomyocytes

In order to delineate ion transport mechanisms involved in volume homeostasis of freshly isolated newborn rat ventricular myocytes, we investigated the effects of ion substitutions and pharmacological maneuvers upon (1) isotonic volume, (2) hypotonically induced initial swelling, and (3) the subsequent regulatory volume decrease (RVD), as determined by electronic cell sizing. Cardiomyocytes exposed to hypotonic medium (176 mosmol/l) swelled by 51+/-1% of isotonic volume, and they underwent a partial regulatory volume decrease (RVD), reaching a maximum regulation after 30 min (51+/-1% of initial swelling), with a half-time (t1/2) of 6+/-1 min (n=60). RVD was associated with significant cardiomyocyte K+ loss (12+/-4% at 5 min and 15+/-2% of isotonic control after 30 min: n=6, P<0.001), 71% of which was Cl- dependent (P<0.05). Within the 30-min experimental time frame, ouabain, a Na+/K+ pump inhibitor, had no significant effect on RVD (despite an inhibitory trend), cell swelling or on isotonic volume (n=6). Bumetanide (50 microM), a Na+-K+-Cl- co-transport blocker, induced a significant reduction of isotonic cell volume (3+/-2%, n=6. P<0.05), potentiated initial swelling by 16+/-1% (n=8, P<0.02), and it partially inhibited RVD (24+/-11% at 30 min, n=6), whereas Na+ omission had no significant effect on isotonic cell volume, cell swelling or RVD. The effects of bumetanide on initial swelling and RVD were prevented by gadolinium ion (10 microM), a stretch-activated cation channel blocker (n=5). Quinidine (500 microM), a non-selective Ca(2+)-activated potassium channel blocker with no side-effects on K(+)-Cl(-) cotransport, did not modify initial cell swelling, but inhibited RVD (50+/-3% at 5 min, n=9, P<0.01; 22+/-3% at 30 min), an effect which was cancelled by external Ca2+ chelation with EGTA (n=5), and reproduced by tetraethylammonium (TEA, 20 mM), another K+ channel blocker. 4,4'-Diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS, 100 microM), a non-selective swelling-activated Cl- channel blocker with marginal side-effects on K(+)-Cl(-)cotransport, did not modify initial swelling, but inhibited RVD to the same extent as quinidine (42+/-3% at 5 min, and 23+/-3% at 30 min, n=15, P<0.05), whereas hypotonic Cl(-)-free solution had no effect on isotonic volume, but potentiated initial swelling by 16+/-2% (P<0.05) and fully inhibited RVD (n=5, P<0.001). R(+)-[(2-n-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inde n-5yl)-oxy] acetic acid) (DIOA, 80 microM), a K(+)-Cl- cotransport blocker (with inhibitory potency toward Ca(2+)-activated K+ channels), inhibited 87+/-5% of the RVD process at 5 min (P<0.001) and 56+/-16% at 30 min (P<0.001), whereas it had a small effect on isotonic volume (+4%, P<0.01) and initial cell swelling (+2%, N.S.; n=9). In contrast to quinidine, DIOA was able to inhibit Ca(2+)-omission-resistant RVD (full inhibition at 5 min, and 56+/-9% at 30 min; P<0.01, n=5). In conclusion, our results suggest that at least three distinct ion transport mechanisms are involved in the RVD in newborn rat cardiomyocytes: (1) K+ and Cl-channels, (2) K(+)-Cl- cotransport, and (3) Na(+)-K(+)-Cl- co-transport.     

Suppressive action of prolactin on renal response to volume expansion.

The effects of prolactin on rat renal sodium and water handling during volume expansion were studied using clearance techniques. Both control and experimental adult male Wistar rats were prehydrated with an oral water load of volume equal to 2.5% body weight (BW). At least 3 h later, a continuous intravenous infusion of ovine prolactin (NIH-P-S8), 7.1 mug/h per 100 g, was started in the experimental group. After a 1-h steady-state period, the rats were given an intravenous expansion infusion of either hypotonic saline (2.5% BW), isotonic saline (2.5% and 7.5% BW), or blood (2.5% BW). In all control hypotonic and isotonic saline-expanded animals, within 1 h the rats excreted a volume of urine equal to over 50% of the volume of saline infused. The diuretic and natriuretic responses to saline expansion of prolactin-treated rats were significantly smaller than controls. In contrast to the effects of prolactin on the renal response to saline infusions, it did not alter the natriuretic or diuretic response to blood infusion. Prolactin may be counteracting the effects of physical factors on the regulation of sodium reabsorption in the proximal tubule.     

Cellular taurine release triggered by stimulation of the Fas(CD95) receptor in Jurkat lymphocytes

 One of the hallmarks of apoptosis is cell shrinkage which appears to be important for cell death. The mechanisms mediating cell volume decrease have, however, not been addressed. Mechanisms employed by swollen cells to decrease their cell volume include activation of ion transport pathways, such as ion channels and KCl cotransport, and release of cellular osmolytes, such as taurine, sorbitol, betaine and inositol. The present study has been performed to test for release of taurine. To this end Jurkat human T-lymphocytes were loaded with [³H]taurine and apoptotic cell death induced by triggering the Fas(CD95) receptor with monoclonal crosslinking antibody. Triggering the Fas(CD95) receptor led to a release of 60±5% of cellular taurine within 90 min. The release did not occur prior to 45 min. The release coincided with cell shrinkage as evidenced from forward scatter in FACS analysis and preceeded DNA fragmentation according to propidium iodide staining. The delay of taurine release was not influenced by exchange of medium and thus was not due to extracellular accumulation of a stimulator. The Fas(CD95)-induced taurine release, cell shrinkage and DNA fragmentation were blunted by lowering of ambient temperature to 23°C. Following pretreatment of cells with Fas(CD95) antibody at 23°C rewarming led to rapid taurine release, cell shrinkage and DNA fragmentation, indicating that the temperature-sensitive step is distal to the mechanisms accounting for the delay. Osmotic cell swelling led to an immediate release of taurine. In conclusion, Fas(CD95) triggering leads to delayed taurine release through a temperature-sensitive mechanism. Received: 4 March 1998 / Received after revision and accepted: 23 March 1998