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1 Facultad de Medicina, Departomento de Fisiologia y Farmacologia, Universidad Autonona de San Luis Potosi, San Luis Potosi, San Luis Potosi CP 78210; and 2 Centro de Investigaciones Biologicas del Noroeste, La Paz, Baja California Sur, 23090 Mexico
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In isolated perfused guinea pig hearts, coronary flow causes a positive inotropic effect [positive coronary flow-induced effect (+CFIE)] that could be altered by dextrans (Dx) in the coronary perfusion solution. To test this possibility, Dx of 20, 40, 70, and 500 kDa were infused and found to modulate +CFIE; however, when Dx infusion was terminated, the effect persisted, i.e., was irreversible/nonwashable, suggesting that Dx may bind to luminal endothelial lectinic structures. This hypothesis was tested when Dx [with fluorescent traces (D*)] bound to the vessel wall was hydrolyzed by dextranase infusion and washout of D* fragments completely reverted the +CFIE, and it was found that bound D* to be displaced by free Dx required concentrations 50-100 times that used during binding. In addition, dose-response curves for Dx on +CFIE show that the higher the Dx molecular mass, the lesser the concentration required to have an effect. Because a large Dx molecule has a greater number polymeric glucose branches, it can bind to a larger number of endothelial lectinic sites, requiring a lower concentration to affect +CFIE. Our results suggest that luminal endothelial lectinic structures are part of the flow-sensing assembly.
shearing stress; protein polymerization; steady state; irreversibility; endothelial glycocalyx; endothelial lectins; dextranase; albumin; interstitial volume
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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BROUGHT-ON CHANGES IN CORONARY FLOW modulate cardiac oxygen and glucose metabolism and several functions, contraction amplitude, auricular-ventricular delay, diastolic time fraction, and spontaneous ventricular rhythm, through debatable mechanisms (7, 11, 13, 15, 19, 20, 25, 28).
One concept, "the luminal extracellular matrix deformation," derived from studies on isolated blood vessels, cultured endothelium, and isolated hearts, suggests that shearing stress acts on flow-sensing endothelial luminal membrane proteins (2, 3), causing the release of cardiotonic paracrine substances (2, 3, 6, 8, 10-12, 14, 16-20). In these studies, the physiochemical properties of several luminal endothelial membrane proteins were altered differentially with antibodies, glycosidic hydrolyzing enzymes, and lectins that polymerize specific luminal glycoproteins (4, 18, 19, 21, 22, 24, 25). These agents, which react specifically with luminal proteinic structures, differentially depressed diverse flow-induced responses through unknown mechanisms.
Another concept proposes that changes in coronary flow through capillary filtration alter interstitial fluid volume/pressure causing a parallel activation of myocyte stretch-activated ion channels (13, 15, 28). In these studies, amplitude and time contractile parameters were altered by changes in coronary flow, perfusion of highly hypertonic solutions, and a decrease in plasma oncotic pressure, which potentially alters interstitial volume (13, 15, 28).
Shearing stress is proportional to the product of two equally important parameters: flow × viscosity. Solutions of dextrans, highly branched glucose polymers (1), have been used to increase the viscosity of perfusion media (8, 20, 25); however, in these studies (19, 20, 25), an increase in viscosity was less effective as a stimulus than flow, suggesting that dextrans, in addition to enhancing viscosity, also diminish the effect of flow through binding to coronary luminal lectinic proteins, an osmolarity effect (13, 15, 28), or both. We decided to explore the possibility of binding of dextrans of different molecular weights at various concentrations.
In addition, in capillaries, there is a luminal stationary fluid layer determined by the luminal glycocalyx (4). Dextrans diffuse through this layer, and albumin influences their rate of motion (9, 30). Albumin could prevent the binding of dextran to the luminal glycocalyx, thus accelerating its free diffusion through the fluid stationary layer; therefore, in the presence of albumin, dextrans could not bind to the endothelial lumen, nor could they exert their modulatory effect on coronary flow-induced inotropism. We decided to test this second hypothesis.
In this paper, we report systematic studies on the binding of dextrans to the coronary lumen, its modulation by albumin, and the effects on coronary flow-induced inotropism.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated Saline-Perfused Hearts
This preparation has been extensively described (19, 20). Briefly, from anesthetized male guinea pigs (350-400 g), the hearts were removed and retrogradely perfused via a nonrecirculating perfusion system. After a 25-min equilibration period at 10 ml/min, experimental measurements were initiated. Coronary flow was adjusted by varying the output of a variable-speed peristaltic pump. The perfusion medium was a Krebs-Henseleit solution containing 5 mM glucose and 5 mM pyruvate, equilibrated with 95% O2-5% CO2 (37°C, pH 7.4). Coronary pressure was continuously recorded, and the pressure was 46 ± 2.8 mmHg at a coronary flow of 10 ml/min. Heart wet weight averaged 1.49 ± 0.07 g. Venous effluents were drained out of the heart through the patent right atria and pulmonary artery and dripped freely. When needed, venous samples of this fluid were collected into tubes.Electrical Stimulation
Two small stainless steel wire clamps electrodes were affixed 2 mm apart on the epicardial tissue layer of the right atrial appendage (19, 20). Pacing was achieved by applying electrical square pulses at a rate of 4.5 ± 0.1 Hz (two times threshold, 2-ms duration).Measurements of Ventricular Contraction
Via the left atrium, a latex balloon attached to the end of a fluid-filled catheter was introduced into the left ventricle. The other end of the catheter was connected to a pressure gauge, and 0.2 ml of water was injected with a syringe into the balloon to adjust diastolic pressure to 10 mmHg. Developed ventricular pressure was continuously recorded. The frequency response of this pressure system may not be optimal to distinguish changes in the diastolic time fraction (15), change in pressure over time (dP/dt), and pressure development. Therefore, all changes in the amplitude of the developed ventricular pressure were taken as a qualitative indicator of the inotropic response.Preparation of Dextran, Albumin, and Dextran Plus Albumin Solutions and Determination of Their Viscosities
Concentrated stock solutions of dextrans of 20, 40, 70, and 500 kDa and bovine serum albumin were prepared and dialyzed at 4°C for >3 days against several changes of distilled water, and a dialysis membrane with a 14-kDa cutoff was used to remove small-molecular-mass contaminants. After dialysis, all stock solutions were stored frozen. Aliquots of each stock dextran solution were mixed with the salts and water necessary to prepare the Krebs-Henseleit solution. The final dextran concentration ranges were as follows: 0.55- 27 mM for 20 kDa, 0.26-5.0 mM for 40 kDa, 0.050-0.75 mM for 70 kDa, and 0.005-0.060 mM for 500 kDa. The viscosities of these solutions were measured with a Canon-Fenske-type viscosimeter at 37°C, and values were obtained in kinematic viscosity units, multiplied by the density of the solutions, and expressed in centipoise (×10
2 Pa · s).
Albumin stock (20%) was infused into the hearts at a final
concentration of 1%. The viscosities of the infused dextran, albumin, and albumin plus dextran solutions were measured. Albumin at 1% has no
effect on the viscosity of the Krebs-Henseleit solution (0.690 × 102 Pa) or of the infused dextran solutions.
Preparation of Dextrans Mixed With Tracer Amounts of Fluorescent Dextrans ("Spiked" Dextrans)
Dextran-FITC of 20- and 500-kDa dextran were freshly prepared the same day of the experiment and added in tracer amounts to normal dextran solutions of 0.55 mM 20-kDa dextran and 0.06 mM 500-kDa dextran, respectively. The final concentration ratios of dextran-FITC to normal dextran were 1:10,000 and 1:3,500 for 20- and 500-kDa dextran, respectively. Thus the main bulk of infused dextran ("spiked" dextran) was made up of nonfluorescent dextran.Preparation of Dextranase Enzyme
A small volume of a stock solution of dextranase (100 U/ml) was prepared the same day of the experiment. An effective concentration of 1 U/ml was infused into the hearts using an infusion pump.Experimental Protocols
Intracoronary retention of spiked dextrans, their displacement, and the associated inotropic response. To study dextran intracoronary retention, the hearts were perfused with either spiked 0.55 mM 20-kDa dextran or spiked 0.06 mM 500-kDa dextran for a 10-min period. The period of dextran perfusion was followed by a washout period of 10 min. During the dextran infusion and during the washout period, aliquots of the coronary sinus venous effluent were collected at 1-min intervals. Fluorescence was determined in all venous aliquots.
PROCEDURES USED TO DISPLACE INTRACORONARILY RETAINED DEXTRAN AT THE END OF THE WASHOUT PERIOD. First, dextranase was infused for a period of 10 min, and venous effluent samples were continuously collected. During the first 1.5 min of dextranase infusion, venous aliquots were collected at intervals of 5 s; thereafter, the sample collection interval was 1 min. Second, a high concentration of nonspiked dextran was infused, and venous samples were continuously collected. During the first 1.5 min of infusion of high dextran concentration, venous aliquots were collected at 5-s intervals; thereafter, samples were collected at 1-min intervals. Finally, 1% albumin was continuously infused, and venous aliquots were continuously collected. During the first 1.5 min of infusion of albumin, aliquots were collected at 5-s intervals; thereafter, samples were collected at 1-min intervals. The 10-min infusion period of albumin was followed by a 1-min washout period; at that point, dextranase was infused for a period of 10 min, and venous effluent samples were collected. During the first 1.5 min of dextranase infusion, venous aliquots were collected at 5-s intervals; thereafter, the sample collection was continued at 1-min intervals. STUDIES TO AFFECT THE INTRACORONARY RETENTION OF DEXTRAN BY ALBUMIN. During sustained 1% albumin infusion, either spiked 0.55 mM 20-kDa dextran or spiked 0.06 mM 500-kDa dextran were infused in the presence of albumin. Ten minutes after the initiation of perfusion of albumin, the spiked dextrans were infused for 10 min, followed by a 12-min washout period with Krebs-Henseleit-albumin solution. Thereafter, dextranase at 1 U/min was infused for 10 min. Venous aliquots were collected starting at the initiation of spiked dextran infusion and until the end of the infusion of dextranase; samples were collected at 1-min intervals except during the first minute of dextranase infusion, when they were collected at 5-s intervals. Fluorescence was determined on all venous samples.Measurement of Fluorescence Intensity
Fluorescence was determined in venous samples using excitation and emission wavelengths of 511 and 537 nm, respectively.Inotropic effects of coronary flow under control condition and in the presence of dextrans. For the different molecular mass dextrans, the effect of two concentrations were studied. Before the perfusion of dextran, a control ventricular contraction-coronary flow curve was determined. Each flow value was maintained constant for a period of 5 min, and the steady-state contraction was determined. The contraction amplitude increased with coronary flow and reached a maximum at a flow of 13 ± 1 ml/min. This contraction amplitude was defined as 100%. The increase in contraction amplitude was not associated with a decrease in diastolic pressure or in the duration of contraction. Thereafter, the perfusing Krebs-Henseleit solution was replaced by one containing a given dextran concentration, and the ventricular contraction-coronary flow curve was repeated. Thereafter, the heart was perfused with Krebs-Henseleit solution alone for a washout period, and the ventricular contraction-coronary flow curve was repeated. Both the curve during dextran infusion and the washout curves were compared with their own control curve, i.e., each heart was its own control. All contraction amplitudes were expressed as a percentage of the maximal contraction amplitude induced by coronary flow under control conditions. The dextran concentrations tested were 0.55 and 2.76 mM for 20-kDa dextran, 0.263 and 1.0 mM for 40-kDa dextran, 0.15 and 0.75 mM for 70-kDa dextran, and 0.016 and 0.06 mM for 500-kDa dextran. For each concentration, six hearts were utilized.
Concentration-dependent inotropic effect of dextrans of different molecular mass at a constant coronary flow. These experiments were performed at a constant coronary flow (10 ml/min); the contraction amplitude of hearts perfused with Krebs-Henseleit solution alone was taken as 100%, and all other contraction amplitude values were expressed as a percentage of this value. Thereafter, the heart was perfused with increasing concentrations of a given molecular mass dextran. The infusion period for each concentration lasted 10 min, until a steady-state response was achieved. Each dextran infusion period was followed by a 5-min washout period by perfusion with Krebs-Henseleit solution; the next higher dextran concentration was infused, and the cycle was repeated. This protocol was applied for each of the differently sized dextrans. For every dextran size, six hearts were used.
For each dextran concentration, the response achieved represented a true steady-state response because the same amplitude was reached whether the dextran concentration was achieved through successive steps or in a single step. This single-step protocol was performed in a group of experiments using one heart for only one dextran concentration. The steady-state responses obtained were the same for the two protocols. In these experiments, in six different groups of hearts, the ratios between ventricular dry weight to wet weight were determined. Each heart was perfused with a given concentration of a dextran for a period of 10 min, followed by a 5-min washout period by perfusion with Krebs-Henseleit solution. At this point, the hearts were removed from the perfusion system, and ventricles were dissected out weighed and dried. There were five hearts per group. In group 1, no dextran was perfused (control). In groups 2 and 3, 70-kDa dextran was perfused at 0.1 and 1 mM, respectively. In groups 4 and 5, 40-kDa dextran was perfused at 0.6 and 6 mM, respectively; in group 6, 20-kDa dextran was perfused at 5.5 mM.Statistics
Values are expressed as means ± SE. In all experiments, each heart was its own control; responses under control conditions and during specific manipulations were compared in the same heart. Statistical significance was determined using a paired t-test with Bonferroni correction factors for multiple comparisons. P
0.05 was considered to be
statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Intracoronary Retention of Spiked Dextrans, Their Displacement, and the Associated Inotropic Response
The time course of the inotropic effects and the corresponding venous fluorescence during the periods of arterial infusion and washout of spiked 20- and 500-kDa dextrans are shown in Figs. 1-4. During dextran infusion, venous fluorescence gradually increased and plateaued at the same level as the arterial fluorescence (100%). During the following 10-min washout period, the venous fluorescence gradually dropped to reach practically zero levels.
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The corresponding inotropic effects during the dextran infusion and washout periods showed that infusion of 0.55 mM 20-kDa dextran (Figs. 1A, 2A, and 3A) caused a positive inotropic effect that persisted during the washout period. In the case of infusion of 0.06 mM 500-kDa dextran, the inotropic effect was negative and also persisted during the washout period (Figs. 1B, 2B, and 4A).
After the washout periods of the spiked 20- and 500-kDa dextrans, the sustained administration of dextranase caused a rapid large release of venous fluorescence, which gradually dropped to zero levels (Fig. 1, A and B). The release in venous fluorescence was paralleled by a reversal of the inotropic response toward control levels (Fig. 1, A and B). Thus the dextran inotropic effect results from its binding to vessel surface because breakdown of retained dextran into small moieties caused a complete reversal of the effects.
Intracoronarily retained spiked dextrans were also displaced upon infusion of high concentrations of normal dextrans. After the washout period of spiked 20- and 500-kDa dextrans, a sustained administration of 27 mM 20-kDa dextran (Fig. 2A) or 6 mM 500-kDa dextran (Fig. 2B) caused a sudden rise of venous fluorescence, which gradually dropped to zero levels. The displacing dextran concentrations were 50- and 100-fold higher concentrations than those used of spiked 20- and 500-kDa dextrans, respectively. Attempts to displace bound fluorescence using nonspiked 0.55 mM 20-kDa dextran and nonspiked 0.06 mM 500-kDa dextran did not caused fluorescence release (data not shown). Thus a spiked dextran perfused at a given concentration requires a much greater concentration of nonspiked dextran to be displaced into the venous effluent. The infusion of high concentrations of nonspiked dextran in both cases was associated with a predictable negative inotropic effect (Fig. 2, A and B).
Studies to Affect the Intracoronary Retention of Dextran by Albumin
The effects of albumin were tested in two different ways: 1) through administration of albumin after the washout of spiked dextrans (Figs. 3A and 4A), and 2) through administration of spiked dextran during a sustained infusion of albumin (Figs. 3B and 4B).The effects of a sustained infusion of 1% albumin were tested after the washout period of spiked dextrans (20 kDa; Figs. 3A and 4A). Albumin caused a slow and small rise of venous fluorescence, which gradually dropped to negligible levels. The small transient release in venous fluorescence values were lower than those induced by infusion of dextranase and were associated in small nonsignificant return of the dextran-inotropic effects toward control levels (Figs. 3A and 4A). To test that the albumin infusion did not significantly displace the spiked dextran retained by the heart, dextranase was infused for 10 min. Administration of dextranase caused a rapid large release of venous fluorescence (Figs. 3A and 4A), which was paralleled by a reversal of the inotropic response toward control levels.
Administration of spiked dextrans during the sustained infusion of albumin is shown in Figs. 3B and 4B. As dextran infusion proceeded, venous fluorescence gradually increased and plateaued at the same level as the arterial fluorescence (100%). In the following 10-min washout period, venous fluorescence gradually dropped to reach zero. In contrast to the effects observed in the absence of albumin, there were no inotropic effects during the infusion of spiked 20-kDa dextran or spiked 500-kDa dextran and the following washout period. Thereafter, administration of dextranase was associated with a small release of venous fluorescence, which was not associated with an inotropic effect (Figs. 3B and 4B). Thus the spiked dextrans administered in the presence of albumin were poorly retained in the coronary vessels and did not exert an inotropic effect.
Viscosities of Solutions of Dextrans of Different Molecular Masses
The viscosity of a dextran solution increased linearly as the concentration rose, and the straight line slope was inversely related to the molecular mass of the dextran (Fig. 5A). To allow the lines corresponding to high-molecular-mass dextrans to be distinguished from each other at low concentrations, the abscissae maximal value represented is limited to 5 mM and not 30 mM. All the straight lines at zero dextran concentration intersected the y-axis at a value of 0.691 ± 0.005 cP. The value of 0.691 cP is the viscosity of Krebs-Henseleit solution at 37°C and compared well with value of 0.696 cP reported for H2O at 37°C (13a). To represent the relationship between the slope of the straight lines and the molecular weight of the dextran, the slope values (ordinates) were plotted against the dextran molecular mass (abscissae) (Fig. 5B). To define more clearly the relationship of slope to molecular mass, two additional dextrans sizes were used: 148 and 260 kDa. Figure 5B shows that the plot of the dextran molecular mass-slope relationship is as a complex exponential function. It is evident that the viscosity of a dextran solution is determined by two variables: the concentration and the molecular mass.
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Inotropic Effects of Coronary Flow Under Control Conditions and in the Presence of Dextrans
The inotropic effects of coronary flow and its modification by two different concentrations of each dextran are shown in Figs. 6 and 7. Under control conditions, when coronary flow increased within a range of 5-15 ml/min, the ventricular contraction was stimulated (open squares). These curves were modified when the perfusion solutions contained dextran.
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Figure 6A shows the modification of the inotropic effects of coronary flow when the perfusion medium contained 20-kDa dextran at concentrations of 0.55 and 2.76 mM. Perfusion with 0.55 mM dextran caused an upward displacement of the curve with respect to the control. However, when perfusion of the dextran solution was stopped and replaced by Krebs-Henseleit solution, the ventricular contraction amplitude remained at the same amplitude even after 2 h of washout. Similarly, the perfusion of 2.76 mM dextran caused a smaller upward displacement of the curve with respect to the control. Again, when perfusion of the dextran solution was stopped and replaced by Krebs-Henseleit solution, the curve did not return toward control values (data not shown). In both cases, the effects of perfusion with dextran were irreversible, and even after a 2-h washout period the effect persisted.
Figure 6B shows the modification of the inotropic effects of coronary flow when the perfusion medium contained 40-kDa dextran at concentrations of 0.263 and 1.0 mM. Dextran at 0.263 mM caused an upward displacement of the curve with respect to the control, which remained elevated during washout (data not shown). In contrast, perfusion of 1.0 mM dextran did not have an effect, and during the washout period the curve obtained was the same as that during dextran perfusion.
Figure 7A shows the inotropic effect of coronary flow under control conditions and the effect of perfusion with 0.15 and 0.75 mM of 70-kDa dextran. Perfusion with 0.15 mM dextran displaced the curve downward with respect to the control, and this inhibitory effect persisted during the washout period. Perfusion with 0.75 mM dextran also caused a greater downward displacement of the curve with respect to the control, and the depressing effect persisted during the washout period (data not shown). Figure 7B shows the inotropic effect of coronary flow under control conditions and the effect of perfusion with 0.016 and 0.06 mM of 500-kDa dextran. Perfusion with 0.016 mM dextran displaced the curve downward with respect to the control, and this inhibitory effect persisted during the washout period (data not shown). Perfusion with 0.06 mM dextran also caused a greater downward displacement of the curve with respect to the control, and the depressing effect persisted during the washout period.
The results shown in Figs. 6 and 7 show complex concentration- and molecular mass-dependent effects of the dextrans. These results prompted us to explore whether the different molecular mass dextrans act in a dose-dependent manner.
Concentration-Dependent Inotropic Effect of Dextrans of Different Molecular Masses at a Constant Coronary Flow
The concentration effects for each dextran size were biphasic (Fig. 8). As the concentration was increased, the ventricular contraction amplitude rose and reached a maximum; thereafter, the contractile response gradually diminished to become smaller than the control amplitude. The higher the molecular mass of the dextran, the lower were the concentrations required to produce its effects.
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Changes in viscosity appear not to be associated with the effects of
dextrans 1) because the solutions of the various dextran sizes tested had viscosity values within the same range of
0.691-2.16 × 102 Pa · s
(in Fig. 8, only the 2 highest concentrations of 20-kDa dextran were
higher, 2.90 and 5.10 cP); 2) because of the fact that the
dextran effects persisted during the washout period when the viscosity
of the perfusion solution returned to control values; and 3)
because the addition of dextran in the presence of albumin did not bind
to the vascular lumen despite the fact that it caused the same increase
in viscosity without an associated inotropic effect.
The osmolarity increase by dextran was not a determinant of the effects. The addition of NaCl to the perfusion media to elevate osmolarity by 35 mosmol/l was without an effect on the contraction amplitude. The maximum attained osmolarity produced by the highest concentration of 20-kDa dextran was 27 mosmol/l.
The dry weight-to-wet weight ratios determined in hearts perfused with no dextran, 0.1 mM 70-kDa dextran, 1 mM 70-kDa dextran, 0.6 mM 40-kDa dextran, 6 mM 40-kDa dextran, and 5.5 mM 20-kDa dextran were 0.1495 ± 0.0087, 0.1496 ± 0.0075, 0.1483 ± 0.0098, 0.1501 ± 0.0049, 0.15498 ± 0.0103, and 0.1503 ± 0.097. No differences were observed between these values, suggesting that surface-bound dextrans are not associated with an osmotic effect.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Our results show that intracoronarily infused dextrans bind irreversibly to the coronary vascular lumen in a concentration- and molecular mass-dependent manner and that this binding causes modulation of coronary flow-induced inotropism. The luminally bound dextran is in a steady-state equilibrium with the loading concentration, and the inotropic effective concentration range for a dextran is inversely proportional to its molecular mass.
Infused Dextrans Are Confined to the Intravascular Space
Taylor and Granger (27) perfused dextrans intracoronarily to dog hearts, studied their diffusion into the interstitial space, and determined an upper dimensional limit for a capillary pore diameter of 0.024-0.032 µm that compares with that determined by Simionescu et al. (26). The dextran diameters (27) we infused are at or above the upper limit of the endothelial pore range. Thus all these dextrans were confined intravascularly, and their inotropic effects likely result from an action on the blood vessel luminal surface.Binding of Dextrans to Coronary Endothelial Luminal Lectinic Structures
The fact that dextrans during infusion remain confined to the intravascular lumen and exert an inotropic effect that is irreversible and nonwashable suggests that dextrans bind and remain bound to the intravascular luminal surface. We reasoned that hydrolyzing bound spiked dextran by intracoronary infusion of dextranase should result in the release of small dextran fragments into the venous effluents and a parallel reversal of the dextran inotropic effects toward control levels. Dextranase caused these effects.The strong binding of dextran to the vascular lumen suggests it may be of a lectinic nature, i.e., the bonds are strong with slow dissociation kinetics, where equilibrium favors formation of the dextran-lectin complex (5, 29). This implies that for the spiked dextran to be displaced the dextran concentrations must be greater than the loading concentration. To displace luminally bound spiked dextran into venous effluents required concentrations 50-100 times greater than the loading dextran concentration. This indicates a strong binding between dextran- and glucose-recognizing lectinic endothelial structures. Proteins with lectinic properties have been identified on the vascular lumen (5, 29).
From the studies of Vink and Duling (30), we predicted that albumin could prevent the binding of dextran to the endothelial luminal glycocalyx. The implication is that infusion of spiked dextrans in the presence of albumin prevents their inotropic effects as a result of no binding to the vascular lumen. The lack of dextran binding was confirmed because ulterior infusion of dextranase failed to displace spiked dextran fragments into the venous effluents. Thus albumin through an unknown mechanism reduces the binding affinity between dextran and glycocalyx lectinic sites.
Coronary Flow-Induced Inotropic Mechanism Is Modulated by Dextran Binding to the Luminal Endothelial Membrane: Two Possible Mechanisms of Dextran Effects
First, it is necessary clarify that the frequency response of the intraventricular pressure system we used may not be good enough to distinguish changes in dP/dt and pressure development and, therefore, all changes in amplitude were interpreted as changes in the inotropic state.Dextrans are glucose-branched polymers that are composed of a main chain and a number of branches, and the branch length is proportional to the dextran molecular mass (1). Each of the large number of branches can potentially bind with a high affinity to multiple glucose-recognizing lectinic proteins on the endothelial luminal surface (29), and as a result these polymers irreversibly modulate the coronary flow-induced inotropic mechanism in a molecular mass- and concentration-dependent manner. However, the mechanisms behind the cardiotonic effect of flow and the mechanism of the effect of dextran are still unknown and are interdependent.
The luminal endothelial matrix as flow-sensing assembly. MECHANISM OF THE BIPHASIC CONCENTRATION EFFECT OF A DEXTRAN. Flow may be detected by dextran-binding structures in the endothelial luminal surface (2, 14, 19), and dextrans affect their responsiveness to flow. To explain why a dextran concentration-inotropic effect is biphasic, we propose that as the perfused concentration of a dextran rises, its glycosidic chains bind irreversibly to an increasing number of endothelial lectinic sites. In the low concentration range, few of the branches of a dextran molecule would bind to luminal sites and most of its branches remain free. The free branches of a bound dextran could amplify the deforming forces of flow on the endothelial luminal glycocalyx, enhancing coronary flow-induced inotropism. In the high concentration range, as the perfused concentration of dextran gradually increases, so will the number of luminal lectinic sites cross-linked with dextran branches. This increasing surface polymerization could render the endothelial luminal glycocalyx less susceptible to flow, gradually reducing the flow-induced inotropism. This sequence from low to high dextran concentrations could result in the biphasic effect of a dextran.
MECHANISM OF THE INVERSE RELATIONSHIP BETWEEN THE EFFECTIVE CONCENTRATION OF A DEXTRAN AND ITS MOLECULAR MASS. A similar reasoning could be proposed. A dextran molecule, upon binding to a lectinic site, creates a microenvironment of elevated concentration of polymerized glucose moieties. The spatial dimensions of the microenvironment is proportional to the dextran molecular mass. A dextran of 500 kDa has a greater number of glucose moieties per mole distributed in more and larger branches than a 20-kDa dextran. Consequently, the smaller number of 500-kDa dextran molecules per unit volume compared with 20-kDa dextran could bind to the same number of lectinic sites on a given surface area. For example, the concentration range for the 500-kDa curve is 500 times smaller than that of the 20-kDa curve (Fig. 8), i.e., smaller concentration increments of 500 kDa cause the same change of the coronary flow-induced response. It should be noted that 500-kDa dextran is only 25 times larger than 20-kDa dextran; however, 500-kDa dextran exerts its effects at concentrations about 500 times smaller. As a general rule, it appears that when two dextrans of molecular masses W1 and W2 have effective concentration ranges of
C1 and C2,
respectively, the ratio of W1 to
W2 is 1-2.5 orders of magnitude smaller
than the inverse ratio of C2 to C1. These results and their interpretation support the notion that luminal endothelial membrane structures are part of the cellular flow-sensing assembly that can be altered by dextran binding.
Changes in intestitial fluid volume as a cause of coronary flow-induced inotropism. This idea proposes that a rise in coronary flow through capillary water filtration increases interstitial fluid volume/pressure and that this deforming force activates myocyte stretch-activated ion channels (13, 15, 28). In these studies, the associated changes in interstitial volume induced by perfusion of highly hypertonic solutions, possibly by changes in coronary flow and a transient decrease in plasma oncotic pressure, were assumed to parallel changes in amplitude (13, 28) and time-contractile parameters (15). One could propose that after the perfused dextran is removed, dextran bound to the endothelial lumen sustains an osmotic effect that reduces interstitial fluid volume and in turn contraction amplitude. Such reasoning could be applied only to the descending branch of the biphasic dextran concentration-contraction curve but not in the ascending branch. Furthermore, 1) the dry weight-to-wet weight ratios measured for various dextran sizes, concentrations, and contraction amplitudes show no differences between them; and 2) Vargas and Johnson (28) added 100-400 mM sucrose to Ringer solution to induce transient hyperosmotic effects paralleled by negative inotropic effects. How the loading concentrations of solute/dextrans we used, two to four orders of magnitude smaller than those of Vargas and Johnson's study (28), result in luminal solute accumulations capable to sustain a reduction of interstitial volume is not clear. Perhaps the difference resides in the size of the solute employed. Nevertheless, it is likely that there are several mechanisms behind the coronary flow-induced inotropism that operate simultaneously.
Viscosity May Not Be a Necessary Parameter for Flow to Act As a Stimulus
It has been assumed that flow acts on the endothelial luminal surface through shearing stress, which is proportional to the product of flow × viscosity (2, 3, 8, 14, 18, 19, 23). In our experiments, at a constant flow (Fig. 8), viscosity was changed (range of 0.691-5.7 × 102
Pa · s) through either a change in dextran
concentration or molecular mass and seem to be irrelevant. A possible
interpretation could be that dextrans are not biologically inert
(31) and interact with the endothelial luminal
wall. However, in the presence of albumin, infusion of
dextrans did not bind to the endothelial lumen, and viscosity rose to
the same value as with dextran alone. This indicates that it is
possible to increase viscosity without luminal binding of dextran and
still lack the expected inotropic effect. However, it is possible that
our system may not be used to define capillary shearing stress due to
network geometric complexity.
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ACKNOWLEDGEMENTS |
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This work was supported by Consejo Nacional de Ciencia y Tecnologia (CONACYT) Grants 25963N, 83853, and G34998N (to C. Gonzalez-Castillo).
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Rubio, Departamento de Fisiologia y Farmacologia. Facultad de Medicina, UASLP, Av. Venustiano Carranza No. 2405, Col. Los Filtros, San Luis Potosi CP 78210, Mexico (E-mail: rrubio{at}uaslp.mx).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 2, 2003;10.1152/ajpheart.00323.2002
Received 2 May 2002; accepted in final form 16 December 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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) and venous
fluorescence (VF; 
) during the perfusion period of
spiked dextran, washout (wash), and during dextranase (1 U/ml)
infusion. A: contraction amplitude and VF rose during the
infusion of 0.55 mM 20-kDa dextran (D 20). During the washout period,
contraction amplitude remained elevated, whereas VF decayed toward zero
levels. After the washout period and infusion of dextranase, there was
a transient rise in VF associated with a reversion of the inotropic
effect (n = 6). B: contraction amplitude and
VF rose during the infusion of 0.06 mM 500-kDa dextran (D 500). During
the washout period, contraction amplitude remained reduced, whereas VF
decayed toward zero levels. After the washout period and infusion of
dextranase, there was a transient rise in VF associated with a
reversion of the inotropic effect (n = 6).
Right ordinate, contraction amplitude as a percentage of the
control; left ordinate, VF as a percentage; abscissae, time
(in min). The fluorescence of the arterial perfusion solution
containing spiked dextran was defined as 100%.
