Osmotic stability of blood platelets

1. Hypotonic solutions added to human platelet-containing plasma cause a transient decrease of absorbancy of light at 610 mmu which is followed by a gradual increase of absorbancy.2. When platelets are stored for 7 hr at 4 degrees C the absorbancy changes with variations of osmolarity and their aggregation with adenosine diphosphate (ADP) remain the same. However, the reversal of absorbancy declines during storage of platelet-containing plasma.3. Platelets are not aggregated by stearate. Platelets appear to be only slightly affected by stearate concentration higher than 0.8 mM, but oleate has no effect.4. Hypertonic solutions of NaCl and urea cause increase in absorbancy of platelet-containing human plasma. Hypertonic sucrose solutions produce no more change than isotonic solutions. Hypertonic NaCl produces permanent increases in absorbancy. In human platelet-containing plasma the increased absorbancy caused by hypertonic urea is transient and declines.5. The osmotic platelet changes occur in isolated platelets as well as in platelet-containing plasma.6. The absorbancy of frozen and thawed platelet-containing plasma is not significantly altered by hypotonic solutions but the absorbancy changes caused by hypertonic solutions are similar to that of unfrozen plasma.7. The immediate absorbancy changes caused by hypo- and by hypertonic solutions are the same at 5 degrees C and 30 degrees C and are therefore probably of a physical nature. The reversal of absorbancy and aggregation of platelets by added adenosine diphosphate have Q(10) > 1 and are therefore probably of a chemical-enzymic nature.8. Divalent cations and contact activation are not required for the osmotic platelet changes and 10(-3)M-Cu(2+) and Zn(2+) do not interfere. Inhibitors of oxidative phosphorylation, electron transfer, sodium, potassium activated adenosine triphosphatases and adenosine triphosphate do not inhibit reversal of absorbancy of platelets exposed to hypotonic solutions. Cyanide, 5 x 10(-3)M, fluoride, 1.23 x 10(-2)M, iodoacetamide, 10(-2)M, are moderately effective inhibitors. At hydrogen ion concentrations above pH 8, complete inhibition occurs.9. N-ethyl-maleimide, 10(-3)M, and mercloran inhibit completely reversal of absorbancy, indicating the necessity for sulphydryl compounds.10. Intact platelets are essential for the reversal of absorbancy after hypotonic swelling. Osmotic changes by hypotonic solutions are independent of ADP aggregation of platelets.     

Influence of intracranial osmotic stimuli on renal nerve activity in anaesthetized cats

In baroreceptor denervated cats one internal carotid artery (ICA) or the cerebral ventricular system (CVS) was perfused with isotonic, hypertonic and hypotonic sodium chloride solutions. Renal sympathetic activity (RSA) and blood pressure (BP) were recorded. ICA perfusion with isotonic sodium chloride (150 mM NaCl) produced no changes of RSA compared to control level,. RSA was increased from plus 30% to plus 350% in 44 tests out of 45 tests following hypertonic (425 mM NaCl) ICA perfusion. RSA was decreased following hypotonic (aqua dest.) ICA perfusion from minus 30% to minus 100% in 37 tests out of 50 tests. The degree of RSA changes was found to depend upon the osmolarity of the solutions. 425mM NaCl and aqua dest. produced greater RSA changes than 290 mM NaCl and 75 mM NaCl. CVS perfusion with isotonic sodium chloride produced a slight increase of RSA compared to control levels (plus 15%). Hypertonic sodium chloride produced a RSA increase from plus 15% to plus 135% in 10 tests out of 14 tests. Hypotonic sodium chloride produced a RSA decrease from minus 15% to minus 80% in 8 tests out of 14 tests. Changes of RSA following ICA perfusions and CVS perfusions were accompanied by changes of BP in the same direction. A quantitative correlation between delta RSA and delta BP could not be found. Results suggest that renal osmoregulatory response to osmotic stimuli in the carotid artery may not just arise in response to changing ADH levels but may also be induced by changes in RSA.     

Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC

The concentration of extracellular calcium plays a critical role in synaptic transmission and neuronal excitability as well as other physiological processes. The time course and extent of local fluctuations in the concentration of this ion largely depend on its effective diffusion coefficient ( D *) and it has been speculated that fixed negative charges on chondroitin sulphate proteoglycans (CSPGs) and other components of the extracellular matrix may influence calcium diffusion because it is a divalent cation. In this study we used ion-selective microelectrodes combined with pressure ejection or iontophoresis of ions from a micropipette to quantify diffusion characteristics of neocortex and hippocampus in rat brain slices. We show that D * for calcium is less than the value predicted from the behaviour of the monovalent cation tetramethylammonium (TMA), a commonly used diffusion probe, but D * for calcium increases in both brain regions after the slices are treated with chondroitinase ABC, an enzyme that predominantly cleaves chondroitin sulphate glycans. These results suggest that CSPGs do play a role in determining the local diffusion properties of calcium in brain tissue, most likely through electrostatic interactions mediating rapid equilibrium binding. In contrast, chondroitinase ABC does not affect either the TMA diffusion or the extracellular volume fraction, indicating that the enzyme does not alter the structure of the extracellular space and that the diffusion of small monovalent cations is not affected by CSPGs in the normal brain ionic milieu. Both calcium and CSPGs are known to have many distinct roles in brain physiology, including brain repair, and our study suggests they may be functionally coupled through calcium diffusion properties.     

Water intake during chronic preoptic infusions of osmotically active or inert solutions.

To further elucidate the role of the lateral preoptic area (LPO) as an osmoreceptive region, rats received chronic infusions (2 weeks) of low volumes (0.5 microliters/h) solutions of hypertonic sodium chloride (NaCl; 0.16 M), hypertonic potassium chloride (KCl; 0.16 M), hypertonic (0.32 M) or hypotonic (0.16 M) mannitol, isotonic saline, or water delivered bilaterally via subcutaneous osmotic minipumps attached to intracranial cannulae. All cannulae terminated within the anterior hypothalamus-preoptic region. Hypertonic NaCl and KCl increased water intake over preinfusion levels in the majority of animals tested. However, the effects were variable, including some sizable increases as well as decreases. Hypertonic mannitol decreased daily water intake in 15 of 25 rats and produced essentially no change in the average intake of the group. Isotonic NaCl produced smaller increases and decreases, while water produced larger changes in individual rats, but neither solution had a significant effect on the average intake of the group. None of the infusates significantly altered food intake.     

Two mechanisms that raise free intracellular calcium in rat hippocampal neurons during hypoosmotic and low NaCl treatment

Previous studies have shown that exposing hippocampal slices to low osmolarity (pi(o)) or to low extracellular NaCl concentration ([NaCl](o)) enhances synaptic transmission and also causes interstitial calcium ([Ca(2+)](o)) to decrease. Reduction of [Ca(2+)](o) suggests cellular uptake and could explain the potentiation of synaptic transmission. We measured intracellular calcium activity ([Ca(2+)](i)) using fluorescent indicator dyes. In CA1 hippocampal pyramidal neurons in tissue slices, lowering pi(o) by approximately 70 mOsm caused "resting" [Ca(2+)](i) as well as synaptically or directly stimulated transient increases of calcium activity (Delta[Ca(2+)](i)) to transiently decrease and then to increase. In dissociated cells, lowering pi(o) by approximately 70 mOsm caused [Ca(2+)](i) to almost double on average from 83 to 155 nM. The increase of [Ca(2+)](i) was not significantly correlated with hypotonic cell swelling. Isoosmotic (mannitol- or sucrose-substituted) lowering of [NaCl](o), which did not cause cell swelling, also raised [Ca(2+)](i). Substituting NaCl with choline-Cl or Na-methyl-sulfate did not affect [Ca(2+)](i). In neurons bathed in calcium-free medium, lowering pi(o) caused a milder increase of [Ca(2+)](i), which was correlated with cell swelling, but in the absence of external Ca(2+), isotonic lowering of [NaCl](o) triggered only a brief, transient response. We conclude that decrease of extracellular ionic strength (i.e., in both low pi(o) and low [NaCl](o)) causes a net influx of Ca(2+) from the extracellular medium whereas cell swelling, or the increase in membrane tension, is a signal for the release of Ca(2+) from intracellular stores.     

Increase in plasma sodium enhances natriuresis in response to a sodium load unable to change plasma atrial peptide concentration

The influence of plasma sodium concentration in the control of sodium excretion was investigated in conscious, water-diuretic dogs. NaCl was infused for 60 min as a hypertonic or isotonic solution at a rate of 60 mumol NaCl min-1 kg-1 body wt. Plasma sodium concentration rose only during hypertonic infusion (P less than 0.05). Sodium excretion increased markedly with both infusions (hypertonic, from 2.4 +/- 0.6 to 105 +/- 27 mumol min-1; isotonic, from 3.9 +/- 1.3 to 58 +/- 17 mumol min-1). Fractional sodium excretion increased more during hypertonic than during isotonic infusion. Hypertonic infusion decreased diuresis from 3.1 +/- 0.5 to 1.3 +/- 0.6 ml min-1, while isotonic infusion elicited an increase from 3.9 +/- 0.5 to 7.2 +/- 0.7 ml min-1. Plasma renin activity and plasma aldosterone decreased markedly in both series (P less than 0.05), the relative changes in the two series being very similar. Central venous pressure increased (2.8 +/- 0.7 to 4.5 +/- 1.0 mmHg) during isotonic infusion but not significantly during hypertonic infusion. Arterial pressure, heart rate and plasma levels of atrial natriuretic peptide and catecholamines did not change measurably in either series. It is concluded that simultaneous increases in extracellular volume and sodium concentration cause a larger natriuretic response than a change in volume alone, and that a 40-fold increase in sodium excretion may occur without measurable changes in plasma atrial natriuretic peptide concentration.     

Inhibition of adsorptive endocytosis and transcytosis in pulmonary microvessels

In isolated perfused rat lungs we have examined the effect of inhibition of receptor-mediated endocytosis and of intracellular acidic compartments activity on adsorptive and fluid-phase uptake and transport of macromolecules by capillary endothelium. Cationized and native ferritin were used as electron-dense tracers of adsorptive and fluid-phase transport, respectively. Luminal endothelial vesicles were labeled with tracers at 4 degrees C for 2 minutes and the density of ferritin particles in endothelial multivesicular bodies and in the capillary basement membrane, after a 60-minute chase with tracer-free perfusate at 37 degrees C, was determined by electron microscopic morphometry. Fluid-phase transport of native ferritin in 60-minute perfusions at 37 degrees C was also investigated. Adsorptive endocytosis was inhibited in chase experiments with hypertonic (800 mOsm) perfusate. Endothelial acidic compartments activity was decreased by adding either dextran sulfate or chloroquine to the chase media. We found that hypertonic solutions, dextran sulfate, and chloroquine inhibit cationized ferritin accumulation in multivesicular bodies and in the extravascular space. Hypertonic buffer or dextran sulfate have no effect on native ferritin uptake and transport in either pulse-chase or continuous perfusions. Since adsorptive or receptor-mediated mechanisms play a role in the transendothelial movement of plasma proteins, and other plasma constituents, our findings suggest a general role of the endothelial acidic compartments in modulating microvascular permeability to macromolecules.     

Response of the Small Intestine to the Application of a Hypertonic Solution

The morphologic and functional alterations caused by a commonly used hypertonic radiographic dye (Hypaque®-50%) were compared with changes observed during the absorption of 150 mM saline in closed segments of the ileum of 2- to 3-kg rabbits. Hypertonic dye caused a rapid decrease in height and width of the villi, a decrease in height of the epithelial cells and closure of the intercellular space. Concomitantly, the tissue fluid content of the bowel wall and the volume of venous outflow from the segment of ileum decreased, presumably in response to the osmotic gradient between ileal lumen and blood. The fluid added to the luminal contents was hypotonic and contained sodium, potassium, chloride and bicarbonate. In contrast, the ileum exposed to 150 mM saline had prominent intercellular spaces between adjacent epithelial cells and absorbed the solution at isotonic conditions. These studies indicate that production of diarrheal fluid by this hypertonic solution is different from that reported for enteric pathogens.     

Responses of neurons in the rat anterior hypothalamic periventricular zone to osmotic stimulation and angiotensin II in vitro.

Extracellular recordings were made in vitro from neurons in slices of the rat periventricular zone surrounding the anterior portion of the third ventricle. Spontaneous unit activity of these neurons was decreased in 39.5% of the neurons tested, increased in 32.9%, and biphasically changed (i.e., excitation-inhibition) in 10.5%, in response to bath application of hypertonic artificial cerebrospinal fluid (aCSF) containing elevated levels of NaCl (330 mOsm). Similar responses were observed following bath application of hypertonic aCSF prepared with additional mannitol. Angiotensin II in the perfusion medium dramatically increased spontaneous unit activity in 73.7% of neurons tested, while, unexpectedly, 5.3% were inhibited. These results suggest that some neurons in the periventricular zone are themselves osmosensitive, and may be involved in the regulation of water balance.     

Comparison between hypertonic and isotonic saline-induced sputum in the evaluation of airway inflammation in subjects with moderate asthma

Background Hypertonic saline-indueed sputum has recently been used for the evaluation of airway inflammation in asthma. Objective To assess the effect of hypertonicity on airway inflammation. Methods We compared the inflammatory cell composition of hypertonic saline-induced sputum with that of isotonic saline-induced sputum in 21 asthmatic subjects and, at baseline and 30min after each sputum induction, we measured bronchial hyper-responsiveness to methacholine as an indirect marker to detect increased airway inflammation. On two different days, the patients inhaled hypertonic saline (3–5% NaCl) or isotonic saline (0.9% NaCl) for 30 min via an ultrasonic nebulizer, while monitoring FEV₁. Sputum was collected for inflammatory cell analysis. Results There was no difference in inflammatory cell percentages obtained with the two methods. Eosinophils were >1% in 20 subjects after hypertonic saline and in 16 subjects after isotonic saline, but this difference was not statistically significant. Intraclass correlation coefficients for sputum inflammatory cells obtained with the two methods were +0.642 for eosinophils, +0.644 for neutrophils. +0.544 for lymphocytes and +0.505 for macrophages. Hypertonic saline induced bronchoconstriction in a significantly greater number of subjects than isotonic saline. Also, hypertonic saline increased bronchial responsiveness to methacholine. while isotonic saline did not. Conclusion We conclude that hypertonicity does not affect sputum cell composition, suggesting that inflammatory cells in hypertonic saline-induced sputum are probably preexisting and not acutely recruited in the airways by the hypertonic stimulus. However, the bronchoconstriction and the increase in bronchial hyper-responsiveness after hypertonic saline inhalation may imply the release of inflammatory mediators. This fact must be considered in the evaluation of soluble markers of inflammation in hypertonic salineinduced sputum.     

Hypertonic and isotonic saline solutions in dehydration therapy in neonate calves: comparison of clinical profile and serum and urinary concentrations of electrolytes

Sixteen male Holstein calves, from 1 to 9 days old, were used in the study. The purpose was to compare the effect of both hypertonic (NaCl at 7.2%, 2,400 mOsm/l) and isotonic (NaCl at 0.9%, 300 mOsm/l) saline solutions associated with oral rehydrating solution, using the serum biochemical profile, serum and urinary osmolality, and electrolytic renal clearance and excretion in neonate calves with osmotic diarrhea-induced dehydration. Calves were randomly distributed into four groups: group 1, normal; group 2, treatment with hypertonic saline plus oral solution; group 3, treatment with isotonic saline plus oral solution; and group 4, with no treatment (diarrhea control group). Animals with no treatment presented aqueous diarrhea, severe hyponatremia, hypochloremia, hypokalemia, decrease of rhythm of glomerular filtration, hyperphosphatemia, and azotemia. The use of small volumes of hypertonic saline solution in a single dose restored the plasma volume, serum sodium and chloride concentrations, and rhythm of glomerular filtration. When compared to isotonic saline, hypertonic saline brought about a less marked hemodilution and reestablished serum potassium concentration and rhythm of glomerular filtration. The use of large volumes of isotonic saline solution is associated with a higher rhythm of glomerular filtration, larger hemodilution, and urinary losses of sodium and chloride. We conclude that a rapid infusion of small volumes of hypertonic saline solution with oral rehydrating solution immediately increases plasma volume, serum osmolality, sodium and chloride seric concentrations, rhythm of glomerular filtration, urinary flow, restoring the volume of extracellular fluid after 24 h constituting a practical and economical alternative to the use of large volumes of isotonic saline solution.

Estimation of Isotonic Point of Incubation Medium For Two-Cell Mouse Embryo

Osmolarity of Dulbecco’s medium at which the volume of two-cell mouse embryo remained similar to that of intact embryo was determined. The method is based on comparison of kinetic curves describing the volume of embryonic cell in solutions of different osmolarity. The blastomere volume was measured by quantitative laser microtomography after fixed osmotic stress intervals. It was found that Dulbecco’s saline with 125 mM NaCl solution is an isotonic solution for two-cell mouse embryo. This concentration corresponds to 290 mOsm, which is lower than osmolarity (~310 mOsm) of media routinely used for culturing of differentiated cells or biological fluids, e.g. blood plasma.     

Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2

We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.     

Nickel inhibition of the osmotic-sensitive ionic cellular channels

The effects of nickel on cell volume regulation were studied in isolated proximal renal tubules from Carassius auratus (goldfish). Variations in cell volume were measured as changes in the external tubular diameters as a function of time of exposure to NiCl2 (Ni(II] in both isotonic and hypotonic solutions. Renal tubules were first incubated in isotonic fish Ringer's solution (290 mOsm) with and without 0.05, 0.10, 0.50, and 1.00 mmol/L Ni(II) for three minutes. After this period, the bath solution was rapidly changed to hypotonic NaCl-poor Ringer's solution (110 mOsm). Renal cells exposed to Ni(II) (0.05 to 1.00 mmol/per L) did not undergo cell swelling in isotonic solutions. However, in a dose related manner, Ni(II) inhibited the volume regulatory process of cells exposed to hypotonic solutions. The effect of Ni(II) on cell volume regulation was specific to the osmoregulatory phase; that is, while there was no inhibition of the hypotonically-induced cell swelling (osmometric phase), there was a significant dose-dependent inhibition of the cellular volume regulatory decrease that follows (osmoregulatory phase). It is postulated by us that nickel inhibition of the osmoregulatory process is secondary to an inhibition of the osmotic sensitive ionic channels.     

Hypotonic stress activates an intermediate conductance K+ channel in human colonic crypt cells

AIM: To investigate the effect of hypotonic stress on human colonic crypts cells in terms of ion channel activity and intracellular Ca2+ concentration. METHODS: Single crypts were isolated from biopsies taken during colonoscopy. The patch clamp technique was used (in the cell-attached mode) to observe the activity of ion channels during hypotonic stress. Calcium measurements were made using the fluophores Fluo 3 or 4. RESULTS: The intermediate conductance (29 pS), Ca2+ -sensitive, K+ channel (also known as KCNN4) previously described (Sandle et al. 1994) was seen in 54 of 149 patches (36%) when the crypts were bathed in normal extracellular solution (290 mOsm kg(-1)). Forty-one patches could be used for further analysis. Activation of one or several 29 pS channels was seen in 15 of 41 patches (39%) after 30 s to 4 min of exposure to hypotonic solution (160 mOsm kg(-1)). The open probability increased from 0.0043 in control solution to 0.44 at 5 min of hypotonic stress. When the crypts were exposed to hypotonic solution, an increase in intracellular Ca2+ could be seen. The increase in intracellular Ca2+ emanates mainly from intracellular stores. CONCLUSION: The 29 pS K+ channel takes part in volume regulation in human colonic crypt cells. The activation of this channel is mediated through an increase in intracellular Ca2+.     

Excretion of total solute, sodium and potassium in the saliva of the rat parotid gland

Summary The rat parotid has been considered a salivary gland that produces saliva isotonic to plasma and with high sodium concentrations at all flow rates. We studied the excretion of sodium, potassium and total solute in the unstimulated and pilocarpine stimulated parotid of adult rats. The effects of retrograde injection of ouabain into the duct system of the gland were also investigated. Use of microanalytical methods enabled us to study the composition of the saliva at all flow rates.The saliva was isotonic to plasma at resting flow rates. Minimal increase in flow rate was associated with a precipitous drop in osmolarity to as low as 86 mOsm/l. Further increase in flow rate up to 135 mg/min/gram wet gland tissue, was followed by gradual increase of osmolarity towards isotonicity. The changes in osmolarity were mainly caused by changes in the sodium concentration. The potassium concentration of the saliva was 3–4 times that of plasma and showed little variation with changing flow rates.Retrograde injection of ouabain into the duct system of the gland up to the acini has no effect on the flow rate but partially inhibited the ability of the gland to elaborate hypotonic saliva.These findings demonstrate that, contrary to what has so far been accepted, the rat parotid is similar to the parotid of man and dog in the excretion of water and electrolytes. In addition, this work supports the hypothesis that the parotid saliva is formed by secretion of a plasma-like fluid in the acini-intercalated duct region, which is subsequently modified by active sodium reabsorption in excess of water in another site (or sites) of the duct system of the gland.Supported by N.I.H. Grant Nr. AM 06806-04 and by Deutsche Forschungsgemeinschaft.From the Dept. of Pediatrics, University of Wisconsin, Madison Wisconsin, U.S.A. Supported by the National Cystic Fibrosis Research Foundation.     

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.     

The effect of hypoosmolarity on the electrical properties of Madin Darby canine kidney cells

The present study has been performed to test for the effect of hypotonic extracellular fluid on the electrical properties of Madin Darby canine kidney (MDCK)-cells. The volume of suspended MDCK-cell is 1,892±16 fl (n=8) in isotonic (298.7 mosmol/l) extracellular fluid. Exposure of the cells to hypotonic (230.7 mosmol/l) extracellular fluid is followed by cellular swelling to 2,269±18 fl (n=4) and subsequent volume regulatory decrease to 2,052±22 fl (n=4) within 512 s. Volume regulatory decrease is abolished by quinidine (1 mmol/l) and by lipoxygenase inhibitor nordihydroguaiaretic acid (50 mol/l). The potential difference across the cell membrane averages –53.6±0.9 mV (n=49) in isotonic extracellular perfusates. Reduction of extracellular osmolarity depolarizes the cell membrane by +25.7±0.8 mV (n=67), reduces the apparent potassium selectivity of the cell membrane, from 0.55±0.07 (n=9) to 0.09±0.01 (n=26), and increases the apparent chloride selectivity from close to zero to 0.34±0.02 (n=21). Potassium channel blocker barium (1 mmol/l) depolarizes the cell membrane by +15.2±1.1 mV (n=13). In the presence of barium, reduction of extracellular osmolarity leads to a further depolarization by +14.0±1.4 mV (n=12). Addition of chloride channel blocker anthracene-9-COOH (1 mmol/l) leads to a hyperpolarization of the cell membrane by –6.7±2.2 mV (n=11). In the presence of anthracene-9-COOH, reduction of the extracellular osmolarity leads to a depolarization by +22.4±1.7 mV (n=11). Application of 1 mmol/l quinidine depolarizes the cell membrane to –6.6±0.5 mV (n=8) and virtually abolishes the effect of reduced extracellular osmolarity on cell membrane potential. Nordihydroguaiaretic acid (50 mol/l), a substance known to inhibit lipoxygenase, increases steady state cell membrane potential in isotonic extracellular fluid to –58.8±1.8 mV (n=10) and blunts the depolarizing effect of hypotonic extracellular fluid (+5.4±1.5 mV,n=10). In conclusion, exposure of MDCK-cells to hypotonic media depolarizes the cell membrane by activation of a conductive pathway, which is insensitive to both barium and anthracene-9-COOH. The conductive pathway is possibly activated by leucotrienes.Parts of this work were presented at the 8th International Symposium on Biochemical Aspects of Kidney Function, Dubrovnik, 1986.     

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.     

The functional state of the isolated rabbit kidney perfused with autologous blood

This report describes the technique and procedure for perfusing an isolated rabbit kidney with 25 ml heparinized autologous blood in a closed circuit including a pump and an oxygenator. The duration of the operative ischaemia was 5-8 min; the perfusion lasted 2.5 hours. An additional infusion was made to compensate for urinary losses. Renal blood flow increased progressively from 2.01+/-0.1 to 2.65+/-0.22* ml/g kidney weight (kw) per min (*P<0.05). Between the first (P1) and the last (P4) urine collection period the glomerular filtration rate (GFR) fell from 288+/-25 to 217+/-38* microl/g kw per min, urine flow from 5.58+/-1.13 to 4.91+/-0.75 microl/g kw per min, Na+ excretion from 1.07+/-0.19 to 0.63+/-0.12* micromol/g kw per min, K+ excretion from 0.46+/-0.03 to 0.28+/-0.05* micromol/g kw per min, P excretion from 2.5+/-0.2 to 2.0+/-0.5 microg/g kw per min, Ca excretion from 0.4+/-0.1 to 0.12+/-0.05* microg/g kw per min, creatinine excretion from 6.94+/-0.32 to 5.68+/-0.54 microg/g kw per min, glucose excretion from 18.2+/-3.2 to 1.6+/-0.5* microg/g kw per min, the free water clearance (CH2O) from -6.57+/-0.85 to -5.10+/-1.31 microl/g kw per min and urine osmolality from 600+/-52 to 590+/-105 mOsm/kg, urea excretion from 0.75+/-0.16 to 0.95+/-0.13 micromol/g kw per min. Excretion of glucose, P or Ca was observed only above a given plasma threshold value, and no transport maximum was found for glucose or P. Ca reabsorption paralleled the Na reabsorption. The proximal tubule pressure, measured within the 1st h of perfusion, was 12.5+/-1.1 mm Hg. Histological examination at the end of the perfusion showed dilatation of the tubules as in the non-perfused kidneys, and the presence of numerous bacteria. Hypertonic urine (380-1110 mOsm/kg) was observed in the presence of vasopressin, in the latter's absence the urine was hypotonic urine (206-278 mOsm/kg). There was no correlation between renal plasma flow and the GFR. CH2O increased with increasing filtered Na+ load. In conclusion, the blood-perfused, isolated rabbit kidney has a fairly constant functional capacity for approximately 2 h.