Pulmonary fluid extraction and osmotic conductance, sigmaK, measured in vivo

The change in aortic blood density in an in vivo rabbit preparation was measured to assess fluid movement at the pulmonary capillaries caused by infusion of hypertonic solution (NaCl, urea, glucose, sucrose, or raffinose in isotonic saline) into the vena cava over 20 s. The hypertonic disturbance increased the plasma osmotic pressure by 相似文献   

Effects of enteral infusion of hypertonic saline and nutrients on canine jejunal motor patterns

The aim of the study was to clarify the effects of hypertonic solutions on jejunal motility. The study focused on differential effects of hypertonic saline and nutrients. Motility of the canine proximal jejunum was recorded with closely spaced strain-gauge transducers. During fasting, hyperosmotic solutions (up to 1520 mosmol/liter) of saline or nutrients (1 kcal/ml) were infused into the proximal jejunum (0.5-1.5 ml/min) up to 6 hr. The hyperosmotic solutions stimulated jejunal motility. With both increasing osmolarity of saline or increasing energy load of nutrients, jejunal motility linearly declined. The reduction of motility was associated with a change in motor pattern from a propulsive to a more segmenting one. Hypertonic glucose evoked a significantly smaller level of motor activity compared with both saline (at given osmolarities) and an elemental diet (at given energy loads). Motility parameters were not different between glucose and maltose, although osmolarity of maltose was less than half (760 vs 1520 mosmol/liter). In contrast, a mixture of glucose-fructose exerted a smaller inhibition of jejunal motility than glucose. The hypertonic solutions of saline or nutrients were tolerated over 2 hr; with hypertonic saline retrograde power contractions with or without vomiting occurred, whereas with hypertonic nutrients vomiting was preceded by strong inhibition of jejunal motility. Three conclusions can be derived from the present results: (1) The behavior of jejunal motility suggested that the motor activity was the result of both a local stimulation and an inhibitory feedback mechanism. (2) The different degree of inhibition between glucose and saline indicated that the nutrient itself played a major role in the inhibitory feedback regulation, whereas osmolarity was of minor importance. (3) Comparisons between different nutrients suggested a linkage between inhibitory control of motility and the absorptive capacity of the gut for the single nutrient.     

Glaxo/MRS Young Investigator Medal. Molecular studies on adenosine deaminase deficiency and hereditary haemorrhagic telangiectasia

Factorially arranged experiments were designed to study prefreeze packed cell volume (PCV) changes and associated percentages of motile and unstained bull sperm in simple macromolecule-free Tyrode's solution and egg yolk-Tris (EYT), varying in osmolarity, and with addition of rapidly permeating cryoprotectants, glycerol and 1,2-propanediol, and nonpermeating substances, sucrose and NaCl. The percentage of motile and unstained sperm was assessed after resuspending sperm in 300 mOsm/L Tyrode's solution. At 25 degreesC PCV increased in Tyrode's solution as osmolarity was decreased from 250 to 150 mOsm/L and decreased as Tyrode's solution was increased to 400 mosmol/L. The relationship of PCV to the reciprocal of the osmolarity was essentially linear over the range of 150 to 400 mOsm/L, but PCV did not decrease further in solutions ranging from 500 to 1000 mOsm/L. The percentage of motile sperm declined to zero in Tyrode's solution at 700 mOsm/L, but 40% of the sperm were still unstained in 1000 mOsm/L solutions. The addition of glycerol or 1,2-propanediol had little effect on PCV. With glycerol or 1,2-propanediol added to 308 mOsm/L Tyrode's solution to give a total of 1267 mOsm/L, there were 49 and 56% motile sperm, respectively, compared to 1% with NaCl added to give 787 mOsm/L. The PCV and percentage of motile sperm suspended in EYT responded to osmotic changes similar to those reported for Tyrode's solution at both 25 and 5 degreesC. Some sperm remained motile after initial exposure to 800 mOsm/L solutions. These findings may have application in improving bull sperm cryopreservation.     

Extracellular adenosine 5'-triphosphate induces Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchange mechanism

In the present study, we examined the effect of extracellular adenosine 5'-triphosphate (ATP) on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. ATP at 1 mM caused a release of 3.6+/-0.08% of the total cellular content. The 45Ca2+ efflux from the cells was also stimulated by adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma s), alpha, beta-methylene-ATP and adenosine 5'-diphosphate (ADP), but not by adenosine 5'-monophosphate (AMP) or adenosine. The effect of ATP was inhibited by a known purinergic P2-receptor antagonist, but not by a P1-receptor antagonist. From these results, it is conceivable that the effect of ATP on Ca2+ efflux from cardiomyocytes is mediated through P2-purinoceptors. It was also observed that ATP caused a rise in [Ca2+]i to almost 200 nM. The ATP-stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. Moreover, ATP caused a 22Na+ influx into the cells of about 2.0-fold over the basal value. These result suggest that ATP stimulates extracellular Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane P2-purinoceptors which may couple to Na+/Ca2+ exchange.     

Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils

The ubiquitous isoform of the Na+/H+ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59(fgr) and p56/59(hck) were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.     

Addition of an osmotic agent to liver preservative solutions in a model of in vitro preservation of hepatocytes

To avoid hypoxic cell swelling during liver preservation followed by reduced perfusion in the reoxygenation period, osmotic substances such as mannitol, sucrose and raffinose, and the impermeant anion lactobionate are used in established liver preservation solutions. The various osmotic agents were investigated at concentrations of 60, 140, 260 and 300 mM, the solutions being kept isotonic by substitution with sodium and potassium chloride to 300 mosmol/l. Cultures of adherent pig hepatocytes were incubated in an in vitro model of cold hypoxia (4 degrees C, PO2 < 0.1 mmHg) for 24 h and reoxygenated with standard culture medium for 3 h. After each incubation period, light microscopy was performed to estimate cell viability and detachment rate. LDH and GOT liberation were also measured. To estimate the change in cell volume, isolated hepatocytes were incubated in suspension for 24 h of cold hypoxia. The cell volumes were compared after centrifugation and measurement of the pellet and the solute levels. Rising concentrations of osmotic substances resulted in increasing liberation of LDH and GOT. The levels of LDH and GOT release from cultures incubated with 60 mmol/l sucrose or raffinose were comparable to those in a preservation solution of "extracellular" ion composition. Addition of mannitol to the preservation solution resulted in cell damage. At high concentrations, sucrose did not affect the hepatocytes as much as raffinose. While mannitol can permeate the hepatocytes and lead to cell swelling, a cell-shrinking effect was observed when sucrose was used, and even more pronounced cell shrinking was seen with raffinose, to which the hepatocyte membrane is known to be permeable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of low-osmolality nutrient media on growth and culturability of Campylobacter species

The growth and culturability of Campylobacter jejuni NCTC 11351 and other campylobacters were examined in media having different osmolalities at a range of temperatures (4, 25, and 42 degreesC). The medium osmolalities used ranged from the osmolality of full-strength nutrient medium (modified campylobacter broth having an osmolality of around 254 mosmol) down to 96 mosmol. The following two methods were used to produce media having different osmolalities: dilution of the nutrient medium with distilled water and reformulation of the medium such that the concentrations of various osmolytes were altered while the nutrient content of the medium was unchanged. The results obtained with the two experimental methods were similar, indicating that there was an osmotic threshold effect, such that none of the campylobacters examined (C. jejuni NCTC 11351 and ATCC 33291, Campylobacter lari, and Campylobacter coli) grew in media having osmolalities around 130 mosmol and at temperatures below at 42 degreesC. Conversely, growth occurred in media having osmolalities of around 175 mosmol and above. Osmolar concentrations can be expressed in terms of osmolarity or osmolality. Osmolality is easier to evaluate, is the more commonly used term, and was used in the current study. In nutrient media having low osmolalities (i.e., 130 mosmol and below), the number of CFUs per milliliter declined rapidly regardless of the temperature, and no cells were recovered after 24 h. However, at nongrowth temperatures (25 and 4 degreesC) in higher-osmolality media (175 mosmol and above) a significant population was recovered throughout the experiment (up to 96 h). In low-osmolality nutrient media, the cellular morphology was principally coccoid, while in the early stages of growth in full-strength media the morphology was predominantly rodlike. We propose that the formation of coccoid cells in these experiments was the result of osmotic stress in low-osmolality media. This osmotic effect was apparent regardless of the osmolyte used to reformulate the medium (NaCl, KCl, Na2SO4, NH4Cl, and glucose were used).     

Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with cystic fibrosis

BACKGROUND: Patients with cystic fibrosis are known to have decreased mucociliary clearance. It has previously been shown that inhalation of a 7.0% solution of hypertonic saline significantly improved mucociliary clearance in a group of adult patients with cystic fibrosis. The aim of this study was to measure the response to increasing concentrations of inhaled hypertonic saline. METHODS: Ten patients (seven men) of mean (SE) age 22 (4) years and mean forced expiratory volume in one second (FEV1) 52.0 (6.7)% predicted completed the study. Mucociliary clearance was measured using a radioaerosol technique for 90 minutes after the interventions which comprised 0.9% NaCl + voluntary cough (control), 3.0% NaCl, 7.0% NaCl, and 12% NaCl. RESULTS: There was a significant increase in the amount of activity cleared from the right lung with all concentrations of hypertonic saline (HS) compared with control. The amount cleared at 90 minutes on the control day was 12.7% (95% confidence interval (CI) 9.8 to 17.2) compared with 19.7% (95% CI 13.6 to 29.5) for 3% HS, 23.8% (95% CI 15.9 to 36.7) for 7% HS and 26.0% (95% CI 19.8 to 35.9) for 12% HS. The improvement in mucociliary clearance was not solely due to coughing as the number of coughs recorded on the control day exceeded that recorded on any other day. The hypertonic saline did not induce a clinically significant change in FEV1. CONCLUSIONS: Within the range of concentrations examined in this study, the effect of hypertonic saline appears to be dose dependent. Inhalation of hypertonic saline remains a potentially useful treatment for patients with cystic fibrosis.     

Small heat shock proteins and protection against ischemic injury in cardiac myocytes

BACKGROUND: Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and alphaB-crystallin protect against ischemic injury. METHODS AND RESULTS: Our aim was to examine whether the overexpression of hsp27 and alphaB-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or alphaB-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45+/-7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, alphaB-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29+/-6.6%) and neonatal (32+/-5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29+/-9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34+/-4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes. CONCLUSIONS: The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.     

Use of the MTT assay in adult ventricular cardiomyocytes to assess viability: effects of adenosine and potassium on cellular survival

This study used the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay to assess cell viability in isolated quiescent adult guinea-pig ventricular myocytes exposed to different insults or cardioprotective conditions, including adenosine and hyperkalemic-cardioplegia. Optical density (OD), reflecting intracellular reduction of MTT into formazan pigment formation, was a function of the number of viable cells (coefficient of linear correlation approximately 0.99), with MTT reduction preferentially carried out by rod-shaped cardiomyocytes (absorbance at 1.009 +/- 0.013 and 0.006 +/- 0.001 OD units for populations containing 50 and 0% of rod-shaped cells). Following prolonged mechanical (pressure of 1 lb/min for 40 min), chemical (10% DMSO or ethanol) or hypoxic injury (N2-saturated solution), the MTT reductase activity reflected reduction in the number of viable cells by 87%, >50%, and 77%, respectively. In cardiomyocytes exposed to a 40 min hypoxia (with CO2), the MTT reductase activity was 0.056 +/- 0.009 in the absence, and 0.074 +/- 0.008 OD units in the presence of adenosine (1 mM), i.e. adenosine reduced the number of non-viable cells. Also, the MTT assay revealed that the effect of potassium-containing solutions (16 and 32 mM K+) on cellular viability may depend on the extent of insult imposed on cardiomyocytes; i.e. a approximately 24% and 49% increase under mild hypoxia (0.03% CO2), or an 18% decrease in cell viability under severe hypoxia (N2) in pre-injured cells. Thus, the MTT assay used to assess viability of isolated adult cardiomyocytes revealed a direct cytoprotective effect of adenosine and hyperkalemic-cardioplegia by promoting cell survival under certain conditions in vitro.     

Improving the palatability of oral rehydration solutions has implications for salt and water transport: a study in animal models

It is believed that improving the taste of oral rehydration solutions (ORSs) might lead to greater patient acceptability. A pilot trial showed that replacing glucose with sucrose and increasing the citrate concentration at the expense of chloride improves palatability. However, the transport implications of such modifications are not known. Three hypotonic experimental ORSs (Suc/cit-ORS, 211 mosmol/kg; Suc/Cl-ORS, 224 mosmol/kg; and Glu-ORS, 224 mosmol/kg) were compared with a standard European ORS (Euro-ORS, 265 mosmol/kg) by in vivo perfusion of entire rat small intestine in normal adult rats and rotavirus-infected neonates. All ORSs were of identical sodium, potassium, chloride, and citrate content except that in the Suc/cit-ORS, chloride was removed in favor of increased citrate, and the chloride concentration in Euro-ORS was higher than in the others. Suc/cit-ORS and Suc/Cl-ORS had glucose partially replaced by sucrose while Glu-ORS and Euro-ORS contained only glucose. In normal small intestine, water absorption was greater from Glu-ORS than Suc/cit-ORS or Euro-ORS, although water absorption was similar from Suc/cit-ORS and Suc/Cl-ORS. In the rotavirus model, Glu-ORS produced more water absorption than Euro-ORS or either sucrose ORS. In both models, Suc/cit-ORS caused sodium and chloride secretion. Glucose absorption was similar from all ORSs. These findings indicate that attempts to improve ORS palatability by adding sucrose or increasing citrate at the expense of chloride would incur a significant penalty in terms of salt and water absorption.     

Acute effects of cell isolation on InsP profiles in adult rat cardiomyocytes

The isolation and culture of adult rat cardiomyocytes was shown to cause major changes in the contents of [3H]-labeled inositol phosphates and inositol phospholipids. Undigested heart tissue contained high levels of [3H]Ins(1,4,5)P3 (5364+/-800 ct/min/g tissue, 80+/-12 ct/min/mg protein) and mass content averaged 13.8 nmol/g tissue or 208+/-36 pmol/mg protein (mean+/-S.E.M., n=4). After collagenase digestion, [3H]Ins(1,4,5)P3 was undetectable and the mass content of Ins(1,4,5)P3 had decreased to 0.8+/-0.2 pmol/mg protein (mean+/-S.E.M., n=4, P<0.01). [3H]Ins(1,4)P2 was reduced by 80% and [3H]PtdIns(4,5)P2 by 90%. These profiles remained essentially unchanged when the isolated cells were maintained in culture for up to 24 h, even though the inositol phosphate response remained sensitive to norepinephrine. Similar to findings in intact tissue, the inositol phosphate response to norepinephrine in these cells was inhibited by neither U-73122 (5 microM) nor by neomycin (5 mM). By 48 h in culture, the relative levels of [3H]Ins(1,4,5)P3 and [3H]Ins(1,4)P2 had increased in relation to the total inositol phosphate content and responses appeared to better reflect intact tissue. However, while retaining insensitivity to neomycin, cells at 48 h were fully sensitive to U-73122 (5 microM). These data demonstrate that altered inositol phosphate responses are observed in adult cardiomyocytes from the time of isolation and that while the profiles change over time in culture, a pattern similar to that in intact heart is not re-established.     

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132+/-6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA (P < 0.001) and IR-BNP (P < 0.05) levels in 2-month old rats. There was also a 1.5-fold (P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% (P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% (P < 0.05) and 39% (P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% (P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.     

Vasopressin response to 2-deoxy-D-glucose in the rat

The effect of 2-deoxyglucose (2DG) upon plasma arginine vasopressin (AVP), measured by a specific and sensitive RIA, has been studied in unanesthetized male Sprague-Dawley rats. Injection of the drug ip at a dose of 100 mg/kg BW caused a 10-fold rise in plasma AVP to 23.4 +/- 4.9 pg/ml (mean +/- SD). This effect was associated with a rise in plasmas osmolality to 301.8 +/- 3.9 mosmol/kg, but this change was accounted for totally by a rise in plasma glucose to 17.3 +/- 3.0 mM and in plasma urea to 3.4 +/- 0.4 mM. The only effective solute, plasma sodium, fell to 135.0 +/- 0.8 mM. Idiogenic osmoles did not change. Blood volume was reduced by about 5%, while PRA and mean arterial pressure did not change. Thus, the rise in plasma AVP caused by 2DG was not mediated by osmotic or hemodynamic stimuli. Treatment with propranolol (2.5 mg/kg BW) 15 min before 2DG completely abolished the AVP response. These results indicate that 2DG stimulates AVP release by a primary mechanism different from that which mediates the effect of insulin-induced hypoglycemia on AVP.