Split-drop Technique Research Articles

Barrier effects of hyperosmolar signaling in microvascular endothelium of rat lung.

We determined the effects of hyperosmolarity on lung microvascular barrier properties by means of the split-drop technique in single venular capillaries of the isolated, blood-perfused rat lung. Using isosmolar and hyperosmolar test solutions (colloid osmotic pressure = 21 cm H2O), we quantified transcapillary flux at a fixed absorptive capillary pressure, and the capillary hydraulic conductivity (Lp). Loss of barrier function was indicated in flux reversal from isosmolar absorption to hyperosmolar filtration (P < 0. 01), and by hyperosmolarity-induced Lp increase (P < 0.01). Barrier recovery after a 1-min hyperosmolar exposure was delayed > 25 min. The flux reversal was blocked by the tyrosine kinase inhibitors genistein and MDC (P < 0.01). Genistein also inhibited the Lp increase (P < 0.01). Immunoblots of hyperosmolarity-exposed, cultured rat lung microvascular endothelial cells (RLMEC) and of endothelial cells freshly harvested from lungs given hyperosmolar infusions indicated a genistein-inhibitable enhancement of protein tyrosine phosphorylation. Immunoprecipitation studies indicated tyrosine phosphorylation of the mitogen activated protein kinases (MAPK) ERK1 and ERK2 and the adaptor protein Shc in lysates of RLMEC exposed to hyperosmolar conditions. We conclude that in lung venular capillaries hyperosmolarity deteriorates barrier properties, possibly by inducing tyrosine phosphorylation of endothelial proteins.

Ligation of endothelial alpha v beta 3 integrin increases capillary hydraulic conductivity of rat lung.

Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin alpha v beta 3. Vitronectin, SC5b-9, and SC5b-9-enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the alpha v beta 3 integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the alpha v beta 5 integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5-dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located alpha v beta 3 integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation.

Effects of hyperoncotic albumin on endothelial barrier properties of rat lung

We have determined the effect of concentrated albumin on lung endothelial hydraulic conductivity (Lp) by our split-drop technique. We anesthetized 39 rats (2% halothane and pentobarbital sodium, 30 mg/kg ip; Sprague-Dawley, 500 g), then isolated and blood perfused their lungs. At constant inflation pressure (5 cmH2O) and stopped blood flow, we viewed subpleural venules (diameter, 20 microns) by microscopy and video. By micropuncture, we first microinfused a venule for 6 min with albumin that was either isoncotic (4 g/dl) or hyperoncotic (6 g/dl) with respect to rat plasma. In the same venule we then injected and split an oil drop with 4 g/dl albumin. From the rates of movement of the split oil drop at different vascular pressures, we determined Lp for 4 g/dl albumin. In venules that were previously microinfused for 6 min with 4 g/dl albumin, Lp for 4 g/dl albumin did not differ from that of noninfused controls (5.4 +/- 0.8 x 10(-7) ml.cm-2.s-1.cmH2O-1). However, after a similar microinfusion of 6 g/dl albumin, the Lp immediately increased more than two times above baseline (P < 0.01) but returned to baseline after 15 min. Lp for 4 g/dl albumin was not affected by hyperoncotic preinfusions of either neutral dextran or immunoglobulin G. We conclude that concentrated albumin caused a reversible increase of the lung endothelial barrier conductivity.

Hydraulic conductivity of ischemic pulmonary venules

We report the first determination of lung endothelial barrier properties in ischemic, nonreperfused microvessels. We quantified the endothelial barrier in terms of hydraulic conductivity (Lp) in single pulmonary venules (diameter 20-50 microns) of isolated blood perfused lungs (dog, rat), held at constant inflation pressure (5 cmH2O) with a gas mixture containing 21% oxygen. Lp were determined by our split-drop technique in which an oil drop is first microinjected into a venule and then split by microinjection of a protein solution. Lp was interpreted from measurements of the rate of oil drop movement. Baseline Lp recorded in the first 30 min of perfusion averaged 3.4 +/- 0.9 x 10(-7) ml/(cm2.s.cmH2O). Then, in two separate groups of venules in which we established 1.3 +/- 0.1 h and 3.4 +/- 0.8 h of ischemia, we determined Lp which were, respectively, 145 +/- 6.5 and 308 +/- 13% above baseline (P < 0.05). We conclude that ischemia alone, in the absence of reperfusion, significantly deteriorates the lung endothelial barrier.

Soluble complex of complement increases hydraulic conductivity in single microvessels of rat lung.

We determined the effect of sera enriched with the soluble complex of complement (SC5b-9), on hydraulic conductivity (Lp) of single pulmonary venules (diameter 20-30 microns). Sera free of anticoagulants and blood cells were prepared from rat and human blood. Lp were determined by our split drop technique in isolated, blood-perfused lungs prepared from anesthetized rats (2% halothane; Sprague Dawley, 500 g; n = 73). Zymosan-activated (ZAS) and control sera were used for Lp determinations. In ZAS prepared from human serum, SC5b-9 concentration was > 300 micrograms/ml (control: < 1 microgram/ml) as determined by ELISA. At baseline, Lp averaged 3.4 +/- .4 x 10(-7) ml/(cm2.s.cm H2O), but it increased by 217 +/- 32% with undiluted ZAS (P < 0.05). The Lp increase correlated significantly with different ZAS dilutions for rat serum and with SC5b-9 concentration for human serum. Lp did not increase significantly with ZAS prepared from heat-treated sera, C6- and C8-deficient sera; or with ZAS in which SC5b-9 had been depleted by immunoprecipitation. The ZAS-induced increase of Lp was blocked completely by venular preinfusion with the arginine-glycine-aspartic acid (RGD) tripeptide (1 mg/ml, 10 min). We report for the first time that: (a) SC5b-9 increases lung endothelial Lp; and (b) the increase of Lp is attributable to an integrin-dependent mechanism.

Thiazide-sensitive Na-Cl cotransport mediates NaCl absorption in amphibian distal tubule.

To find out the mechanism(s) underlying NaCl absorption in the distal tubule of Necturus, we devised a variant of the split-drop technique. Following injection an oil column, subsequently split by a NaCl solution isotonic to plasma, a double-barrelled microelectrode (conventional/selective to Na+ or to Cl- ions) recorded Na+ (alpha Na) or Cl- (alpha Cl) activity and transepithelial potential (Vte). Paired control/low-Na+ solutions yielded reabsorptive half-times (t1/2) of 0.68 +/- 0.11 min and 7.6 +/- 1.8 min respectively; corresponding Vte values were -22.2 +/- 4.0 mV and -7.6 +/- 1.9 mV. t1/2 values of control versus low-Cl- solutions were 0.77 +/- 0.32 min and 6.5 +/- 1.7 min respectively, whereas respective Vte values were not different from one another: -23.8 +/- 4.3 mV versus -18.8 +/- 5.5 mV. Nominally K(+)-free solutions or bumetanide, 10 mumol/l, did not alter t1/2 or Vte, with regard to the paired control. Amiloride, 5 mumol/l or 2 mmol/l, failed to decrease t1/2 or to lower Vte; apparently, the role of a Na+/H+ antiport does not contribute significantly to NaCl absorption. Furosemide, 0.1 mmol/l, reduced t1/2 by 54% with regard to the control state. Determination of t1/2 as a function of increasing hydrochlorothiazide concentrations revealed apical high- and low-affinity sites, estimated at 0.56 mumol/l and 0.115 mmol/l respectively. Taken together these observations indicate that NaCl absorption is predominantly carried out by an electroneutral Na(+)-Cl- cotransport.

Segmental barrier properties of the pulmonary microvascular bed

We determined liquid flux across single pulmonary microvessels of dog, ferret, and rat by our split-drop technique (J. Appl. Physiol. 64: 2562-2567, 1988). Data are reported from 58 lungs excised under halothane or pentobarbital sodium anesthesia and then blood perfused. We stopped blood flow at known vascular pressures and then micropunctured microvessels to inject oil, which we split with albumin solution. From measurements of vessel diameter and split oil drop length, we calculated Jv, the liquid transport rate per unit surface area [x 10(-6) ml/(cm2.s)]. At constant vascular pressure, Jv was not significantly different after different periods of oil-endothelium contact and at different sites within a single vessel. From measurements of Jv at different vascular pressures, we determined Lp, the hydraulic conductivity [x 10(-7) ml/(cm2.s.cmH2O)], and Pzf, the zero filtration pressure. From determinations of Pzf at different albumin concentrations, we quantified sigma alb, the albumin reflection coefficient. Lp and Pzf did not differ among venules of the same lung. However, in venules, Lp was 40% higher and sigma alb 25% lower than in arterioles (P less than 0.01). We conclude that 1) micropuncture procedures incidental to our split-drop technique do not progressively deteriorate the experimental microvessel and 2) in lung, permeability is higher in venules than in arterioles.

Hydraulic conductivity of lung venules determined by split-drop technique

The split-drop method has been used to determine filtration rate per unit surface area in the single pulmonary venule. In isolated perfused lungs of nine dogs, blood flow was stopped at different vascular pressures. By means of a double-micropuncture technique under stereomicroscopy, an oil drop was injected in a subpleural venule. The oil drop was then split with a solution of albumin (5.6 g/100 ml) in Ringer lactate. As the Ringer-albumin solution filtered, the distance between the menisci of the split oil drop (split-drop length) decreased. The split-drop geometry and the rate of change of split-drop length were recorded. The calculated venular filtration rate per unit surface area related linearly with vascular pressure (P less than 0.05). The slope of the line equaled venular hydraulic conductivity, which averaged 2.9 +/- 0.02 x 10(-7) ml/(cm2.s.cmH2O). Hydraulic conductivity is lower in lung than in systemic venules.

Induced development of proximal tubular NaKATPase, basolateral cell membranes and fluid reabsorption.

We have previously demonstrated that adrenal corticoid hormones (ACH) induce NaK-ATPase activity in immature proximal tubular (PT) cells in rats. We have now determined the effect of betamethasone (beta) and aldosterone (aldo) on the size of PT basal and lateral cell membranes (BLM), PT fluid reabsorption (J v (a] measured in vivo with the split drop technique and PT NaKATPase activity in 20-day-old (young) and 40-day-old (adult) rats. Serum levels of ACH were the same in young and in adult rats, but adrenalectomy caused a significantly larger fall in NaKATPase activity in young than in adult rats. BLM surface area (micron 2/micron -3 cell volume), J v (a) and NaKATPase were significantly lower in young than in adult rats. Three-day treatment with high doses of beta (60 micrograms X 100 g-1 or aldo (40 micrograms X 100 g-1) significantly increased BLM surface area. J v (a) and NaKATPase activity in young but not in adult rats. Three days after adrenalectomy, J v (a) was significantly depressed in adult rats. There was a significant correlation between the ACH-dependent changes in NaKATPase activity and J v (a). Short-term treatment (2-3 hours) with high doses of ACH did not significantly increase NaKATPase activity and J v (a) in young rats.

The dissociation between renal HCO3- reabsorption and H+ secretion in the skate, Raja erinacea.

The rate of H+ secretion from cell to lumen was studied in the proximal nephron segment of the skate,Raja erinacea. The split drop technique was used, and the rate of acidification (from pH 7.5 to 5.8) was estimated by the time of the color change of the buffer dye, bromcresol purple (pK 6.2). The secretory cells (in each liter of their volume) produced 40·10−6 mol H+/min; calculated for the whole fish the rate of H+ excretion is about 0.15·10−6 mol/min. This rate is about the same as observed in vivo in the free swimming elasmobranch; and about 1/5 to 1/10 that calculated for the production of H+ from the uncatalyzed hydration of CO2, since elasmobranchs have no renal carbonic anhydrase. Addition of large amounts of NaHCO3 (17 fold) to the bromcresol purple did not change the time requested for the color change of this buffer dye; addition of phosphate prolonged the time significantly. It is concluded that luminal HCO3− in this species is very rapidly reabsorbed by a process not involving proton production by tubular cells.

Kidney function in rats with experimental DOCA-induced hypertension

Rats were made chronically hypertensive by unilateral nephrectomy, repeated injections of desoxycorticosterone-acetate and offering saline as drinking fluid (DOCA-rats). Unilaterally nephrectomized animals (Nephrex-rats) were used for control. Compensatory renal hypertrophy was greater in DOCA-rats than in Nephrex-rats. Diameter and length of the proximal convolute in the DOCA-group increased more than in Nephrex-rats. GFR per unit kidney weight in the DOCA-group remained unchanged as compared to untreated Wistar-rats but increased significantly in Nephrex-animals. the water and sodium load of each nephron had doubled in both groups. Nevertheless, there were no substantial changes in the fractional water and sodium reabsorption in the different segments of the nephron of DOCA-rats as compared to normal animals. In Nephrex-rats fractional sodium and water reabsorption were increased in the proximal tubule and diminished in the loop of Henle and in the distal tubule. In Nephrex-rats an indirect correlation was found between blood pressure and both water reabsorption and the transit time of Lissamine green in superficial loops of Henle but no such correlation could be found in the DOCA-group. Determination of the specific reabsorption capacity of the proximal tubules during free-flow or using the split-drop technique yielded identical values in all animals studied.