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Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 1
    We have investigated the effects of various potential inhibitors on flow-dependent K+ permeability (PK) of single perfused mesenteric microvessels in pithed frogs.
  • 2
    Neither superfusion with a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (10 or 100 μmol l−1), nor the addition of indomethacin (30 μmol l−1) to both perfusate and superfusate reduced the positive correlation between PK and flow velocity (U).
  • 3
    In the presence of agents known to raise intracellular levels of adenosine 3′,5′-cyclic monophosphate (noradrenaline, 8-bromo-cAMP and a combination of forskolin and rolipram) the slope of the relation between PK and U was no longer significant, so that PK was no longer flow dependent.
  • 4
    These results confirm that the flow dependence of PK is a biological process and not an artefact of measurement and suggest a role for intracellular cAMP rather than nitric oxide or prostacyclin in the flow-dependent modulation of PK in frog mesenteric microvessels.

Increasing the blood flow increases transport of solutes between the blood and the tissues (for a review, see Renkin, 1984). The classical interpretation of this flow-dependent transport has been that flow increases the concentration gradient across the endothelial barrier or the surface area for exchange or both of these variables. The permeability coefficients themselves have been assumed to remain constant. Recent studies on isolated microvessels, however, have suggested that an increased rate of perfusion increases permeability itself (Yuan et al. 1992; Pallone et al. 1995; Turner & Pallone, 1997). In a previous study from our laboratory (Kajimura et al. 1998), we have shown that microvascular permeability to potassium ions (PK) increases linearly with the flow velocity (U) in single perfused mesenteric capillaries of pithed frogs. In the present study we describe experiments in which we have attempted to inhibit the phenomenon.

Yuan et al. (1992) demonstrated that an increase in flow increased the permeability to albumin in isolated perfused venules from the pig heart. They were able to block this increase in permeability by the nitric oxide synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Like nitric oxide (NO), prostacyclin is the other well-known mediator whose production is influenced by flow (Frangos et al. 1985; Rubanyi et al. 1986; Koller & Kaley, 1990). Thus the first part of the present study was to test the effects of the NOS inhibitor, L-NMMA, and a cyclo-oxygenase inhibitor, indomethacin, on the relation between PK and U.

When these agents were found to be ineffective, we then examined the effects of the agents which increase the intracellular concentration of adenosine 3′,5′-cyclic monophosphate ([cAMP]i) on the relation between PK and U. Numerous studies have found that elevated [cAMP]i attenuates increased microvascular permeability induced by various inflammatory mediators including those induced in frog mesenteric microvessels (He & Curry, 1993; Adamson et al. 1998; Fu et al. 1998). The experiments described here reveal that elevated [cAMP]i also inhibits the increase in PK which accompanies the increase in U.

Preliminary reports of our findings have been presented to the Microcirculatory Society (Kajimura et al. 1997) and The Physiological Society (Kajimura & Michel, 1998a).

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

General preparation

The experiments were carried out on mesenteric microvessels of male frogs (Rana temporaria and Rana pipiens), 6-7.5 cm in length, supplied by Blades (Edenbridge, Kent, UK). The brain and upper parts of the spinal cord of each frog were pithed and the mesentery was gently arranged on the surface of a polished Perspex pillar. This allowed transillumination of the mesenteric microvasculature. The upper surface of the mesentery was superfused continuously with frog Ringer solution at 16-18°C. The flow of superfusate was maintained at 3.5-4 ml min−1 and this kept the layer of fluid over the tissue at an approximately constant depth. The microvessels chosen for study were mostly venous capillaries (diameter, 18-35 μm) though some were true capillaries. The tissue was observed through a stereomicroscope (Wild Heerbrugg M8) with a CCTV camera (Hitachi) attached to the camera tube. The output from the camera was displayed on videomonitors and recorded.

Solutions and reagents

Frog Ringer solution was used as the bathing solution for the dissection of the mesentery and for the superfusates. Its composition was (mmol l−1): 111 NaCl, 2.1 KCl, 1.0 MgCl2, 1.1 CaCl2, 0.195 NaHCO3, 5.5 glucose, buffered with 2.3 Hepes and 2.7 NaHepes (Sigma). The pH was adjusted to 7.2 by the ratio of Hepes acid to base. The perfusate consisted of Ringer solution containing bovine serum albumin (BSA; A-7638, Fraction V, Sigma) at 50 mg ml−1. In all except high-K+ perfusates, Evans Blue (5 mmol l−1) was added to colour the solution. At this concentration 98 % of the dye should be bound to the BSA (Levick & Michel, 1973). Evans Blue perfusates were dialysed in 8000 molecular weight cut-off dialysis tubing (Spectro/Por; Spectrum, Los Angeles, CA, USA) against three 2 l changes of Ringer solution of equal osmolarity over a 24 h period at 15°C. High-K+ solutions (20 mmol l−1 K+) were prepared by replacing 17.9 mmol l−1 NaCl with equimolar KCl.

Reagents were prepared as concentrated stock solutions with appropriate vehicles. These stocks were kept at -20°C and used within 2 weeks.

Indomethacin (CalBiochem) was initially dissolved in 95 % ethanol at a concentration of 30 mmol l−1 as a concentrated stock. On the day of the experiment the first dilution, one-tenth dilution, was made into BSA-free Ringer solution. The second dilution was then made using BSA-Ringer solution to bring the final concentration of indomethacin to 30 μmol l−1. The final ethanol concentration of the test perfusate was 0.1 % (v/v) (17 mmol l−1).

The perfusates containing forskolin (LC Laboratories, Woburn, MA, USA) and rolipram (Alexis, San Diego, CA, USA) were prepared as 25 and 50 mmol l−1 stock solutions, respectively, in ethanol. The final ethanol concentration of the test perfusate containing both forskolin (5 μmol l−1) and rolipram (10 μmol l−1) was 0.04 % (v/v) (6.8 mmol l−1).

Noradrenaline (L-(-)-norepinephrine-(+)-bitartrate), 8-bromo-cAMP and L-NMMA were obtained from CalBiochem and Ringer solution was used as a vehicle for these reagents.

Measurement and calculation of PK

A detailed description of the method used to measure PK and U in a single perfused microvessel has been published previously (Kajimura et al. 1998). Briefly, each microvessel was cannulated with a bevelled double-barrelled micropipette made from θ-tubing. One barrel of the pipette was filled with a normal-K+ solution (2.1 mmol l−1 K+) and the other was filled with a high-K+ solution (20 mmol l−1 K+). The tubes leading from the two barrels of the pipette were connected through an electric rotary valve (Omnifit Ltd, Cambridge, UK) to two water manometers. This arrangement allowed alternate perfusion with the normal-K+ solution or the high-K+ solution. The heights of the water columns of the two manometers were adjusted so that the normal-K+ solution was being perfused but the high-K+ solution was not. To do this, one solution (the normal-K+ solution) was coloured with Evans Blue (5 mmol l−1), therefore making the interface between the normal- and high-K+ solutions visible. The interface between the two solutions at the tip of the perfusion pipette was carefully observed to prevent either the normal-K+ solution from entering the other barrel or the high-K+ solution from perfusing the vessel.

After the interface was adjusted, the electric rotary valve, which functioned as a cross-over tap between the two manometers, was switched so that the higher pressure was applied to the high-K+ solution causing it to flow through the microvessel. After 2 s, the rotary valve was returned to its initial position. The intraluminal [K+] was monitored by two K+-sensitive microelectrodes. The two microelectrodes, designated as e1 and e2, respectively, were located downstream from the perfusion pipette at points 500-800 μm apart. The more proximal microelectrode, e1, was at least 250 μm downstream from the cannulation site. Potassium indicator potentials were acquired at the rate of 200 Hz using Chart software (Cambridge Electronic Design, Cambridge, UK) running on a Pentium 90 computer.

Increases and decreases in perfusion velocity (U) were achieved by raising and lowering the pressure applied to the perfusion pipette. Each change in perfusion pressure involved adjustment to both manometers so that the colourless (high-K+) perfusate filled its barrel of the micropipette down to the tip when the vessel was being perfused with normal (Evans Blue-containing) Ringer solution. In most experiments flow was increased in a series of steps and then lowered so that measurements of PK were made at low perfusion velocities at the beginning and end of each sequence and measurements at high velocity in the middle. In some experiments, measurements of PK at high and low U were alternated.

The method of Crone et al. (1978) was used to estimate permeability. Briefly, a bolus of high-K+ solution flowed along a single microvessel and the intraluminal [K+] was recorded at two points by K+-sensitive microelectrodes (e1, e2) separated by the length of the vessel over which the permeability was to be determined. If C1 and C2 are the K+ concentrations at e1 and e2, respectively, as the K+-rich bolus passes the electrodes and if C0 is the K+ concentration in the control perfusate and superfusate, then:

  • image(1)

where r is the capillary radius, τ is the transit time of the bolus between e1 and e2 and α is a factor which relates the K+ concentration in the pericapillary space to its concentration in the capillary. Previously, we have found that α has a value equal to 0.53 for frog mesenteric capillaries (Kajimura et al. 1998).

Fabrication of K+ ion-sensitive electrode

The electrodes were made according to the method described by Voipio et al. (1994). Single-barrelled pipettes (quartz with filament; o.d., 1.2 mm; i.d., 0.60 mm; Sutter Instrument Co., Navato, CA, USA) were pulled on a micropipette puller (Model P-2000; Sutter Instrument Co.). Micropipettes were mounted horizontally on a brass holder, placed in a Petri dish, and baked at 200°C. After 30 min, approximately 50 μl of N,N-dimethyltrimethylsilylamine (Fluka Chemicals, Dorset, UK) was added to the Petri dish. Baking continued for a further 1 h. This silanization process made the glass surface hydrophobic and ensured good contact between the glass and the lipophilic ion exchanger. They were then backfilled with a small amount of a liquid ion exchanger (potassium ionophore I - cocktail A, Fluka Chemicals) and filled with an electrolyte solution (0.5 mol l−1 KCl). Electrodes were manufactured on the day of the experiment.

Statistical analysis

Values are reported as means ±s.e.m. throughout. To compare and contrast the average values of slopes and intercepts between two groups, the Wilcoxon signed-rank test (paired comparison) and Mann-Whitney U test (unpaired comparison) were used. In unpaired comparisons, our earlier results from 43 microvessels (Kajimura et al. 1998) were used as control values (PK= (0.0122 ± 0.0016)U+ (8.99± 1.08)). To compare slopes and intercepts within a single experiment, Student's t test was used. The level of significance was set at < 5 %.

We believe there are at least two arguments for using the measurements reported in our previous study as control data for the unpaired measurements which we describe here. First, the relations between PK and U which were determined as controls in those experiments of the present series where it was possible to make paired comparisons (i.e. the effect of L-NMMA) were entirely consistent with the earlier series. Second, the data reported by Kajimura et al. (1998) were based on groups of experiments made at different times of the year. Subsets of data obtained at one time of year did not differ significantly (in terms of the relation between PK and U) from either the whole population of data or other subsets (e.g. January data vs. June data). Thus, there is no reason to believe that use of the data from our previous study could be compromised by seasonal variation.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Effects of inhibition of NOS and cyclo-oxygenase on the relation between PK and U

L-NMMA.

Figure 1 shows the results of an experiment on a single capillary in which PK was determined over a range of perfusion velocities (U), first under control conditions and then after L-NMMA (10 μmol l−1) had been present in the superfusate for 15 min. It is seen that there was a clear correlation between PK and U both before and after the addition of L-NMMA to the superfusate. Although, in this experiment, the intercept and the slope were slightly lower after addition of L-NMMA, neither of these changes was significant (t test).

image

Figure 1. Effect of L-NMMA on relation between PK and U

Paired measurements of PK in a single vessel before and after the addition of L-NMMA (10 μmol l−1) to the superfusate are shown as a function of U. Nine determinations of PK were made under control conditions (○). PK strongly correlated with U (r= 0.95,P < 0.01; continuous line). The same vessel was then superfused with Ringer solution containing 10 μmol l−1 L-NMMA for 15 min. Another nine determinations of PK were made on this vessel with a similar range of U (□). The strong positive correlation between PK and U was maintained (r= 0.92,P < 0.01; dashed line). The two slopes were not significantly different (t test).

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For the eight paired experiments of this kind where the superfusate concentration of L-NMMA was 10 μmol l−1, mean values for PK (μm s−1) before the addition of L-NMMA were related to U by the expression:

  • image(2)

and after the L-NMMA (10 μmol l−1) superfusion by:

  • image(3)

In only one out of the eight vessels investigated was the positive correlation between PK and U lost in the presence of L-NMMA. In each of the other seven experiments, a clear positive correlation between PK and U was seen. After the treatment with L-NMMA, neither the slopes (P > 0.09) nor the intercepts (P > 0.40) were significantly different from the controls (n= 8, Wilcoxon signed-rank test).

When a higher concentration of L-NMMA (100 μmol l−1) was applied, no further reduction in the slope of the relation between PK and U was found. In fact there was a small, non-significant increase in mean values for the slope from 166 (± 55) × 10−4 to 228 (± 77) × 10−4 after the addition of L-NMMA (n= 5,P > 0.22, Wilcoxon signed-rank test). A significant positive correlation was found in all five vessels before and during the treatment with L-NMMA.

Indomethacin.

We also examined the effect of indomethacin because prostacyclin has been suggested as a mediator of flow-dependent responses in some microvascular beds. In these experiments, indomethacin (30 μmol l−1) was added to both the perfusate and superfusate and after 25-30 min the relation between PK and U was examined. In each of the six vessels tested, a strong positive correlation between PK and U was found with values for PK similar to those determined under control conditions. Mean values of PK (μm s−1) for the six vessels could be summarized by the expression:

  • image(4)

This was not significantly different from the control relation (n= 6, Mann-Whitney U test, P > 0.53).

Effects of agents which raise [cAMP]i on the relation between PK and U

Noradrenaline.

We investigated the effect of noradrenaline because it has been known for many years that catecholamines and β2-agonists decrease the plasma leakage caused by a variety of inflammatory mediators. Noradrenaline was added both to the perfusate (100 μmol l−1) and to the superfusate (10 μmol l−1) for approximately 25-30 min. At this time, PK was estimated over a range of perfusion velocities and when the measurements were analysed, there was no longer a significant correlation between PK and U in any of the six vessels investigated. In the single experiment shown in Fig. 2, no variation of PK was seen despite a ninefold variation of U. The mean value of the slope, 18 (± 11) × 10−4, was significantly different from that of the control slope (n= 6, Mann-Whitney U test, P < 0.01).

image

Figure 2. Effect of noradrenaline on relation between PK and U

Determinations of PK plotted against corresponding values of U made on a single venous microvessel when the perfusate and superfusate contained noradrenaline. The continuous line is the regression line (PK= -0.0002U+ 8.56; n= 12). The dashed line represents the mean relation between PK and U based on experiments on 43 vessels under control conditions (Kajimura et al. 1998).

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8-Bromo-cAMP

. As noradrenaline is known to increase the [cAMP]i through activation of β-receptors, we studied the effect of raising [cAMP]i. When we perfused microvessels with a cell-permeant cAMP analogue, 8-bromo-cAMP (2 mmol l−1), for approximately 15 min, the correlation between PK and U was no longer clear. The correlation coefficients determined in the four vessels investigated were outside the level of significance. The regression equation between PK and U was:

  • image(5)

The values of the slope were significantly different from the control (n= 4, Mann-Whitney U test, P < 0.05). The intercepts of the relations between PK and U were higher than in control experiments though the differences were not significant (P > 0.11). Consistent with the increased intercepts (which implied increased PK at low U) we noticed that whereas the Evans Blue albumin of the control perfusate was rarely seen to leak into tissues in control experiments, it leaked through the vessel wall in all experiments where the perfusate contained 8-bromo-cAMP. We therefore sought another method of raising intracellular cAMP levels.

Forskolin and rolipram.

Intracellular levels of cAMP can be raised by stimulating adenylate cyclase with forskolin to increase its rate of synthesis and inhibiting its breakdown by cAMP phosphodiesterase with rolipram. We therefore examined the relation between PK and U in nine vessels perfused with solutions containing forskolin (5 μmol l−1) and rolipram (10 μmol l−1). Each vessel was perfused with the forskolin-rolipram-containing solution for 25-30 min before the first estimate of PK was made. In eight out of the nine vessels tested in this way, the positive correlation between PK and U was abolished. Figure 3 shows an experiment of this kind (n= 11,r= 0.13). For all nine vessels, the mean regression equation was:

  • image(6)
image

Figure 3. Effect of forskolin and rolipram on relation between PK and U

Determinations of PK plotted against corresponding values of U made on a single venous microvessel perfused with a solution containing forskolin (5 μmol l−1) and rolipram (10 μmol l−1). The continuous line is the regression line (PK= 0.0001U+ 9.20; n= 11). The dashed line represents the relation between PK and U under control conditions and is identical to the dashed line in Fig. 2.

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The values for the slopes were significantly lower than the controls (n= 9, Mann-Whitney U test, P < 0.001). In these experiments (by contrast with those where the vessels were treated with 8-bromo-cAMP), there was no obvious leakage of Evans Blue albumin from the control perfusate.

Altering the sequence of the changes in U did not influence the values of PK. Furthermore, when similar values of U were used for the first and last measurements in a microvessel, the values of PK were also similar indicating little change in the value of PK in those experiments.

In all the forskolin and rolipram experiments, the perfusate contained ethanol at a concentration of 6.8 mmol l−1 (0.04 % v/v). To test whether the presence of ethanol itself influenced the relation, a control experiment was carried out where the perfusate contained ethanol (6.8 mmol l−1) in the absence of forskolin and rolipram. In each of the three microvessels tested, a clear positive correlation between PK and U was found. Mean values of PK (μm s−1) for the three vessels could be expressed as:

  • image(7)

Neither the slopes (P > 0.85) nor the intercepts (P > 0.67) were significantly different from the controls (n= 3, Wilcoxon signed-rank test).

The effects of various agents on the flow dependence of PK are summarized in Fig. 4.

image

Figure 4. Summary of effects of various agents on the flow dependence of PK in frog mesenteric microvessels

Mean values for the slopes of the relations between PK and U are given as a measure of the flow dependence of PK. The open column on the left represents the mean slope determined from measurements in 43 vessels under control conditions (Kajimura et al. 1998). Data obtained from experiments in which 10 μmol l−1 and 100 μmol l−1 L-NMMA were added to superfusates have been pooled (n= 13). The values for n for the other groups of experiments were: indomethacin, 6; noradrenaline, 6; 8-bromo-cAMP, 4; and a combination of forskolin and rolipram, 9. *P < 0.05vs. Control.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

The experiments described in this paper extend our observation that the PK of perfused frog mesenteric microvessels increases with the velocity at which they are perfused, by demonstrating that this correlation is lost when the vessel is perfused with agents that are known to increase intracellular levels of cAMP. Perhaps the most significant conclusion to be drawn from this is that, since it can be inhibited by specific pharmacological agents, the relation between PK and U is a biological phenomenon and is not an artefact of measurement. This leads us to consider the possible mechanisms responsible for it. Changes in flow velocity are presumably detected by receptors which are sensitive to shear stress or shear rate and located either within or at the surface of the microvascular endothelium. Stimulation of these receptors may then activate a cascade of reactions which ultimately give rise to the increase in PK. While our experiments reveal nothing about the receptors and little about the consequences of stimulating them, they do appear to eliminate the involvement of both NO and prostacyclin as important members of the signalling cascade. Furthermore, although increased intracellular levels of cAMP might act in several ways to prevent microvascular permeability from increasing, the values of PK which we have estimated at high perfusion velocites raise important questions about the pathways which are responsible for raising potassium permeability under these conditions.

Lack of effects of L-NMMA and indomethacin on the PK-U relation

The failure of L-NMMA to influence the relation between PK and U contrasts with the report of Yuan et al. (1992) who found that L-NMMA abolished the flow dependence of albumin permeability in venules from pig heart. We, ourselves, were surprised by our finding. Since NO release from the endothelium appears to be an important step both in flow-induced vasodilatation of large vessels (Griffith, 1994) and in mediator-induced increases in permeability of small vessels (including those of the frog mesentery), it seemed likely that NO would be a component of the signalling cascade of flow-dependent permeability. When we found that L-NMMA failed to affect the changes in PK with U, we examined the effects of indomethacin, as Koller & Kaley (1990) have shown that, in the microcirculation of the rat cremaster muscle, prostacyclin rather than NO is responsible for flow-induced vasodilatation. Indomethacin also failed to influence the relation between PK and U.

We now believe that the failure of L-NMMA to influence the flow dependence of PK reflects a difference in the signalling cascades between frog and mammalian endothelia. We have recently shown that PK varies directly with flow in rat mesenteric venules but here the flow dependence is abolished with NOS inhibitors (Kajimura & Michel, 1998B).

Microvascular permeability and raised [cAMP]i

Our finding that increases in PK with U become insignificant when intracellular levels of cAMP are raised is consistent with many reports of cAMP tightening the barrier properties of endothelium. In single frog mesenteric microvessels, He & Curry (1993) reported that the increased hydraulic permeability induced by either ionomycin or ATP could be inhibited by perfusing the vessel with 8-bromo-cAMP for 15 min prior to applying these mediators. This inhibitory effect of cAMP occurred in spite of the increase in intracellular calcium concentration which followed the application of ionomycin. More recently, Adamson et al. (1998) have reported that, in frog mesenteric microvessels, the reduction in baseline hydraulic permeability, induced by perfusion with forskolin and rolipram, is associated with a small but significant increase in the number of tight junctional strands in the intercellular clefts.

If raising the [cAMP]i within microvascular endothelium increases the number of junctional strands in the intercellular clefts, it is reasonable to propose that the increments of PK with increasing U are the result of a reduction in the number of junctional strands, thus raising the fraction of the intercellular cleft available for exchange. While changes of this kind could be involved, there are quantitative difficulties in accepting them as being entirely responsible for the increase in PK with U. These difficulties arise from the absolute values of PK. If the intercellular clefts were the only pathways which open to raise PK as U increases, then PK should reach a maximum when there are no longer any junctional strands impeding the diffusion of K+ through the intercellular clefts. This maximum value of PK can be calculated from the diffusion coefficient (D) of potassium in free solution and the dimensions of the intercellular clefts in frog mesenteric microvessels, i.e.

  • image(8)

where L is the length of open cleft per unit area of the endothelium, w is the width of the cleft, and Δx is the distance through the cleft from the lumen of the vessel to the endothelial basement membrane. Detailed ultrastructural studies of the intercellular clefts of frog mesenteric capillaries (Mason et al. 1979; Bundgaard & Frøkjær-Jensen, 1982; Clough & Michel, 1988) suggest that L is 2000 cm cm−2, w is 20 nm and Δx is 0.4 μm. Taking these values together with a value of 2 × 10−5 cm2 s−1 for D leads to the calculated maximum permeability of the clefts to K+ being 20 × 10−4 cm s−1. This figure is regularly exceeded by our estimates of PK at high perfusion velocities (e.g. see Fig. 1). While it is possible that, by incorporating too large a factor (α) into our calculation of PK to account for the rise in pericapillary concentration of K+, we may have overestimated the true value of PK, this does not resolve the dilemma as values of PK greater than 20 × 10−4 cm s−1 are calculated even when this correction is ignored (i.e. when α= 0). This leads us to conclude that the increase in PK with increasing U cannot be accounted for entirely by a reduction in the length of the junctional strands increasing the fraction of the intercellular cleft available for exchange.

PK could be increased if Δx, the distance through the cleft, was reduced as perfusion velocity increased. Clough & Michel (1988) found that some of the variation in the hydraulic permeability between individual frog mesenteric microvessels could be accounted for by a relation between hydraulic permeability and mean estimates of (1/Δx). If Δx were reduced to a minimum value of 0.1 μm, the maximum value of PK could increase to 80 × 10−4 cm s−1, providing, of course, that the junctional strands were reduced to mere points of contact between adjacent cells. The hypothesis could be tested by comparing the ultrastructure of the interendothelial clefts in vessels perfused at high perfusion velocities prior to fixation with those perfused at low perfusion velocities.

If it emerges that the increase in PK with U cannot be accounted for by reductions in Δx, alternative pathways through the endothelium will have to be considered. These would include the formation of fenestrae, the opening of transendothelial channels through the vesiculo-vacuolar organelles (Dvorak et al. 1996) or the opening of intercellular or transcellular gaps (Neal & Michel, 1995) in the endothelium.

The opening of gaps in the endothelium, whether they be intercellular or transcellular, is usually associated with an increased leakage of macromolecules and Yuan et al. (1992) reported increased permeability to serum albumin in response to increases in the perfusion rate of isolated coronary venules. In our experiments, however, there was no observable increase in the leakage of Evans Blue albumin (which was present in all normal-K+ perfusates) when U was raised. Since this would have been expected if albumin permeability increased with increasing flow it would seem that, if the permeability to macromolecules does increase in frog mesenteric vessels at high perfusion velocities, the increase is small. At this stage we are left to speculate on the nature of the pathways for K+ which are opened as flow increases.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

This work was supported by The Wellcome Trust grants (038904/7/93/1.27) (C. C. M.). We thank Dr S. D. Head for his help in developing the technique.