| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Department of Medical Pharmacology and Physiology and 2National Center for Gender Physiology, University of Missouri School of Medicine, Columbia, Missouri; and 3Department of Pharmacology and Physiology, University of Rochester, Rochester, New York
Submitted 17 January 2007 ; accepted in final form 5 April 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Ps) were determined by training status. In the present study Ps to albumin was assessed in arterioles (n = 138) and venules (n = 24) isolated from hearts of male (N = 27) and female (N = 59) pigs in the exercise training group (EX). We evaluated the hypothesis that coronary microvessel exchange adapts to endurance exercise training not by altering basal Ps, per se, but by elevating Ps on exposure to ADO. In contrast, training resulted in a reduction of basal Ps in all arterioles, and in venules from males, with no change in venules from EX females. Exposure to ADO resulted in the predicted increase in Ps except for venules from EX males where Ps was reduced. Ps responses of arterioles to mediators of adenylyl cyclase (isoproterenol)- and guanylyl cyclase (atrial natriuretic peptide)-signaling pathways were attenuated in EX pigs relative to pigs in the sedentary group. The adaptation of EX arterioles involves an upregulation of a nitric oxide-dependent pathway since nitric oxide synthase inhibition blocks Ps by ADO. Thus adaptation of microvascular exchange capacity to endurance exercise training not only occurs but also involves multiple mechanisms that differ in arterioles and venules with their relative contribution to net flux being a function of sex.
-lactalbumin; albumin; heart; porcine; protein flux; sexual dimorphism; transvascular flux
The cell membrane permeant by-product of ATP hydrolysis, adenosine (ADO), is produced during exercise, and it is established that ADO acutely alters macromolecule permeability (Ps) of coronary arterioles isolated from the hearts of female pigs (26, 28). For these vessels, the magnitude and direction of the change in Ps (Ps) were a function of training status (28). Data from limited studies of pig coronary arterioles (28) and venules (18) implicated both exercise training status and sex as determinants of basal arteriolar Ps for the small protein, -lactalbumin, but not for the larger plasma protein, porcine serum albumin (PSA). The inference from these data was that the mechanisms regulating basal Ps are complex and that the regulation of basal Ps involves a different set of mechanisms from those determining Ps in response to metabolically relevant vasoactive agents, such as ADO. The purposes of the present study were to determine whether adaptation of coronary microvascular exchange, particularly that of albumin which ferries free fatty acids, to endurance exercise training is similar in arterioles and venules and, importantly, to establish whether adaptational changes in Ps are indeed influenced by the animal's sex. We hypothesized that basal Ps and Ps to ADO would be independent of sex but dependent on exercise training. To test the hypothesis, Ps was assessed in arterioles and venules isolated from the hearts of 16- to 20-wk treadmill-trained (EX) male and female pigs before and following suffusion with 10–5 M ADO and the data were compared with data generated using the same protocols on age- and sex-matched cage sedentary (SED) pigs (25, 27, 28).
In the aggregate, our results demonstrate clearly that the exchange characteristics of coronary arterioles differ from venules. Coronary arterioles were responsive to endurance exercise training, whereas coronary venules were sensitive to the animal's sex. The data were also consistent with the interpretation that multiple mechanisms regulate not only the transvascular flux of protein, but also, in the case of the heart, the transvascular flux of solutes, such as free fatty acids, carried by those proteins. Furthermore, the relative importance of the set of mechanisms to net flux involves sex hormones. Finally, these data demonstrate that differences in adaptation to physical training extend beyond the musculoskeletal system, involve more than blood flow distribution, and likely influence volume distribution and solute flux differently during exercise in males versus females. Preliminary results of these studies have been reported previously (18, 24).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
All animal care and research was conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals under the supervision and approval of the Office of Laboratory Medicine at the University of Missouri.
Endurance Training Protocol
The training and performance testing protocols have been described previously (34). Briefly, during a 1- to 2-wk pretraining period, Yucatan miniature swine (females, 20–40 kg, N = 81; and males, 23–45 kg, N = 38; ages, 13–16 mo) were exposed to the treadmill (Quinton Fitness Equipment, Bothell, WA; or Warren E. Collins, Braintree, MA) and taught to run. Veterinary students assigned to individual pigs remained with the animals throughout the training period. Following the 2-wk pretraining period, the animals were divided randomly into SED [22 females and 11 males, in addition to the 74 SED animals reported on previously (25)] and EX (59 females and 27 males) groups. The animals were housed with one SED and one EX pig of the same sex.
During the first week of training, the EX group exercised at 8 km/h for 15 min (sprint) and 4.8 km/h for 20–30 min (endurance run). By week 8 the duration of each exercise training bout lasted for 85 min/day, 5 day/wk. The training regime consisted of a 5-min warm-up at 4 km/h, a 15-min sprint at 9.7 to 12.9 km/h, a 60-min endurance run at 6.4 to 9.7 km/h, and a 5-min warm-down at 3.2 km/h. This intensity of exercise was maintained for the next 12–20 wk. During the workout the animals were kept cool with convection and misted water. After completion of a training session, the animals were fed Purina pig chow; the amount was based on the animal's weight (36).
Training effectiveness was assessed by measuring cardiovascular and metabolic indexes in both EX and SED animals during both baseline and treadmill performance testing. The treadmill performance test consisted of four stages of exercise (34). During stage 1, pigs ran at 5 km/h and 0% grade for 5 min. Pigs ran for 10 min at stage 2 (speed = 5 km/h, and grade = 10%) and then for 10 min at stage 3 (speed = 6.9 km/h, and grade = 10%). Finally, pigs ran at stage 4 (speed = 9.7 km/h, and grade = 10%) until exhaustion.
Surgical Preparation
On the day of an experiment, the pig was sedated with ketamine (25 mg/kg im) and xylazine (Rompun; 2.25 mg/kg im), anesthetized with pentobarbital sodium (20 mg/kg iv), intubated, and then ventilated with room air. Following the placement of a catheter into an ear vein, heparin was administered (1,000 U/kg) and a left thoracotomy was performed. The heart was excised, its wet weight determined, and it was immersed into cold (4°C) mammalian Krebs solution (36). Other tissues, blood, and organs (brain, lung, liver, skeletal muscle, fat, skin, and eyes) were harvested for studies in multiple laboratories before final spinal transection.
Oxidative Enzyme Activity
After removal of the heart, samples were taken from the middle of the long, medial, lateral, and accessory heads of triceps brachii and deltoid muscles; frozen in liquid N2; and stored at –70°C until processed. Citrate synthase activity was measured from these tissues (56) and used to assess training status.
Microvessel Plexus Isolation and Cannulation
The right ventricular wall (5–7 x 2–3 cm) of the excised heart was removed into fresh Krebs solution containing 10 mg/ml PSA (Krebs-PSA) at 4°C (25) for transport and storage (not more than 15 min) before microvessel dissection. For dissection the tissue was submerged in fresh Krebs-PSA and pinned onto a closed-cell foam pad with Minuten pins (Carolina Biological, Burlington, NC) to maintain the tissue at a constant length. When venules were isolated from the tissue, they were removed first because of their anatomical location near the epicardial surface of the ventricle away from the arterial circulation. In the case of the venules, the plexus contains vessels of irregular, noncylindrical shapes described previously by Kassab et al. (31) as "rootlike" rather than "treelike" in topology. In the case of the arterioles, the plexus consisting of interconnected vessels (32) was then removed from the surrounding myocardium. The arterioles (<100 µm diameter, 
1,000 µm long) branched from larger vessels (>250 µm diameter), which, in turn, had originated from the right coronary artery or the left anterior descending artery.
The excised arteriolar (which could contain microvessels that spanned, in situ, from the epi- to the endocardium) or venular (primarily epicardial) plexus was secured with Minuten pins (
100 µm OD, Carolina Biological) to a 3-mm deep Sylgard (Dow Corning, Midland, MI) pad set on an inverted 5-cm-diameter organ culture dish (Falcon, 1008) at approximately its in vivo length. Finally, a microvessel was cannulated with a beveled glass theta micropipette (WPI, Sarasota, FL), resulting in an immediate perfusion through several branches of the plexus.
Solutions
Mammalian Krebs. All perfusion and suffusion solutions were prepared and used fresh daily. The Krebs base consisted of (in mmol) 141.4 NaCl, 4.7 KCl, 2.0 CaCl2·2H2O, 1.2 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 3.0 NaHCO3, and 1.5 Na-HEPES. The pH of the solution was 7.37 and 7.41 ± 0.01 at 4° and 37°C, respectively.
Krebs-PSA. PSA (Sigma, St. Louis, MO), used as the colloid in all solutions, was prepared from a stock solution of 60 mg/ml dialyzed previously (in 12–14,000 mol wt cut-off dialysis tubing; SpectroPor, Spectrum, Houston, TX) against 2 liters of Krebs solution in three sessions over 72 h 8°C. The dialysis procedure equilibrated the plasma protein solutions with the Krebs solution, removed small water soluble vasoactive contaminants, set the calcium content, and fixed the pH of the protein solution (3, 20). The protein content of the final solution was determined by absorbance spectroscopy and adjusted so that the perfusate and suffusate solutions contained 20 and 10 mg/ml, respectively.
Labeled protein.
The primary globular protein used to assess microvessel exchange was PSA (65,000 mol wt). In a selected subset of experiments, the smaller globular protein, -lactalbumin (14,000 mol wt, Sigma) was used in place of PSA (20, 21). In either case, one half of a perfusion theta pipette contained unlabeled protein; the second half contained the same concentration of probe protein labeled with a fluorescent tag [either tetramethylrhodamine isothiocyanate (TRITC) Isomer L (CalBiochem or Molecular Probes) or 2',4,5,6,7,7'-hexafluorfluorescein, Oregon Green 514 (Molecular Probes)] using methods detailed in previous work (20, 26). The final protein content of these solutions was 20 mg/ml for PSA and 23.5 (0.56 mM) for -lactalbumin, respectively. Given that TRITC-PSA was 0.17 mM, the total PSA concentration was 0.33 mM. The net oncotic pressure of these protein solutions ranged between 6 and 8 cmH2O.
ADO, isoproterenol, atrial natriuretic peptide, and NG-monomethyl-L-arginine.
ADO (Sigma) was dissolved in Krebs solution (10–3 M stock) and prepared in a 1:100 dilution into PSA. The final concentration was 10–5 M ADO in 10 mg/ml protein. Isoproterenol (ISO; Sigma) was diluted 1:100 into Krebs-PSA and labeled -lactalbumin from a 10–3 M stock in Krebs solution, making a final concentration of 10–5 M used in the reported experiments. Human atrial natriuretic peptide (1–28) (hANP; Bachem) with an identical amino acid sequence to porcine ANP was prepared from a stock of 10–6 M peptide in Krebs solution into Krebs-PSA and labeled -lactalbumin to a final concentration of 10–8 M. The solutions containing NG-monomethyl-L-arginine (L-NMMA, CalBiochem) were prepared from a 5 x 10–3 M stock solution in 0.9% (wt/vol) NaCl and diluted into either Krebs solution to a final concentration of 10–6 M or into Krebs plus ADO to a final concentration of 10–5 M ADO and 10–6 M L-NMMA.
Measurement of Microvessel Protein Flux
The method for assessing Ps to proteins in isolated mammalian microvessels and its limitations is described in multiple publications (25–28, 53, 60, 61). Briefly, the microvessels were perfused with either Krebs-PSA and fluorescently labeled solute (dye) or unlabeled solute (washout) solution using a system of manometers connected to hydraulic switches to control the choice of solution and the hydrostatic pressure in the vessel segment. With a switch between the pairs of manometers, perfusate in the vessel could contain just nonfluorescent washout solution, be changed rapidly to the dye solution for a time necessary to measure solute flux, or to the clear washout to reestablish the baseline, all within seconds. The relatively low perfusion pressures (14.9 ± 0.2 in arterioles and 10.3 ± 0.4 cmH2O in venules) used in these experiments minimized the contributions of convective coupling, or solvent drag, in the estimates of apparent permeability (Ps) (26, 53).
The perfused plexus was transilluminated and viewed at x10 or x20 magnification (numerical aperature, 0.32; UM 10, Leitz) with a fixed stage inverted microscope (Diavert, Leica, NJ) equipped with an adjustable magnifier (up to x2, providing up to x20 real magnification). The light path of the microscope was split 50/50 and projected simultaneously to a video system and to an analog microscope photometer (Leitz, PTI, or Solamere). In this system light intensity at the focal plane of the vessel was 5–11 mW/cm2 when using the N2 filter cube (Leitz) for the red fluorescent dye, TRITC, or the H2 filter cube (Leitz) for the green fluorescent dye, Oregon Green 514, in the light path. At these light intensities, fading of the fluorescent signal and/or light-induced changes in function are either absent or minimized (48, 50). Vessels were imaged using a black and white CCD camera (Dage-MTI 72, Michigan City, IN) fitted with an image intensifier (Dage-MTI Geni-Sys) or a low-light black and white camera (PTI, Brunswick, NJ) and displayed on two video monitors (projecting a field of view of 0.65 x 0.78 to 1.30 x 1.56 mm; Sony).
When labeled protein solution filled the lumen, fluorescence intensity (If) emanated from the perfused microvessel. A rectangular window (width 
3 vessel diameters), located in the light path between the vessel and the photometer, was used to limit and define the area over which the solute flux (Js, in mmol/s) was measured. Apparent solute permeability (Ps) was determined from the relation Js per unit surface area (S, in cm2) and constant concentration gradient (C, in mmol/ml): Ps = Js(SC)–1 = 0.25 D(dIf/dt)i·Io–1(Eq. 1), where I0 was the step change of fluorescence when the dye replaced the nonfluorescent washout solution in the lumen, (dIf/dt)i was the initial change in fluorescence intensity as solute moved across the vessel wall, and D was the internal diameter of the microvessel (in µm). As discussed later, the vessels were assumed to be circular in cross section and have a volume-to-surface area ratio of 0.25 D. All experimental protocols were performed at 15°C to minimize changes in diameter (25–28, 61) during the measurement of flux.
Experimental Protocols
Flux was measured at least twice in all vessels (arterioles and venules). From Js, Ps was assessed first to determine whether the exchange properties of the microvascular barrier differed with vessel type, endurance exercise training, and/or sex. The microvessels were then exposed to ADO in the suffusate, and Ps was assessed a second time to test whether a metabolic vasodilator known to increase during exercise influenced macromolecule exchange and whether the responses to the adenine nucleoside differed with training, vessel type, and/or sex.
Measures of Js commenced after a minimum of 5-min suffusion with 10–5 M ADO and continued for up to 90 min, depending on the rate of probe flux from the microvessels (e.g., "high" permeability vessels were exposed to basal and ADO perfusions for shorter times than "low" permeability vessels). Diameter was measured under both conditions.
Five additional protocols were carried out, requiring multiple measurements of flux following the determination of the basal Ps.
In protocol 1, the concentration response of Ps to ADO was assessed in arterioles isolated from the hearts of EX (N = 7) and SED [N = 7, data published previously in Wang et al. (61)] age-matched female pigs first in the absence (basal) and then following exposure to concentrations of ADO from 10–9 to 10–5 M. Up to five measures of Ps at increasing concentrations of ADO were made in individual arterioles.
In protocols 2 and 3, the adaptation of adenylyl cyclase (AC)- or guanylyl cyclase (GC)-dependent pathways in trained animals were probed using ISO (to activate AC-dependent signaling) or ANP (to activate particulate GC signaling). Ps was measured during perfusion of ISO (10–7 M) on arterioles of 16 (7 SED and 9 EX) pigs and during perfusion of ANP (10–9 M) in coronary arterioles from 12 age-matched (7 SED and 5 EX) pigs.
In protocol 4, to determine whether the response to nitric oxide (NO) synthase (NOS) activity was itself adapted to endurance exercise training, Ps in response to L-NMMA suffusion alone was assessed in arterioles from 18 age-matched female (9 EX and 9 SED) pigs.
In protocol 5, to determine whether the increase in Ps of EX arterioles, as opposed to the decrease in SED arterioles on exposure to ADO (25), involved a NO-dependent pathway, Ps was measured in arterioles from 23 age-matched females pigs (10 EX and 13 SED) that were suffused with 10–5 M ADO; each vessel was then suffused with 10–5 M L-NMMA plus 10–5 M ADO, and the Ps measurement was repeated.
In all the above protocols, the measures were paired, basal Ps was determined during perfusion with pipettes containing the labeled protein in Krebs-PSA, the test solution was then added to the solution suffused over the arteriole, and the Ps measurement was repeated. In the case of ISO or ANP, the perfusion pipette was withdrawn and the vessel was recannulated with a new pipette containing ISO or ANP plus the fluor-labeled protein before measures of flux were repeated.
Statistical Analyses
At the time of experimentation, the investigators were blind to the training status but not to the vessel type or sex of the pigs. Training status was revealed on completion of experiments on groups of 8 to 16 animals. Data from some of the age- and sex-matched SED pigs were reported recently in a companion article (25); the heart weight (HW)/body weight (BW) data as well as the concentration-response studies, NOS blockade, responses to ISO and ANP, or basal venular permeability to -lactalbumin have not been reported previously. Over a 7-yr period, data were obtained from 6 groups of 8–16 male and 12 groups of 8–16 female pigs. Only data from EX pigs, in which measures of basal coronary arteriole Ps and Ps following exposure to ADO, L-NMMA, ISO, or ANP were obtained, analyzed, and reported in this study. The effectiveness of training, for the animals from which Ps data are reported in this study, was determined by comparing data (means ± SE) from HW and BW, HW-to-BW ratios, and values for skeletal muscle oxidative enzyme activity between the SED and EX groups.
An average of a minimum of five measures of Js/SC at a single hydrostatic pressure was used to represent Ps for an individual arteriole or venule. Data from individual microvessels (n) were averaged to represent a single value for each heart (N). In the majority, only a single arteriole and/or venule was sampled per heart. Although the distributions of values for Ps to -lactalbumin of arterioles tended to be skewed, the means ± SE are reported for clarity, and nonparametric statistics were used in the statistical comparisons (StatView SE + Graphics, Abacus Concepts, Berkeley, CA). Repeated-measures two-way ANOVA with Games/Howell (nonparametric) post hoc test (5, 6, 10) was used to test for differences in values of Ps with exercise training and sex. The response to ADO was calculated as the ratio of P
(test) to P
(basal). The same approach was used when ADO was replaced by ISO, ANP, L-NMMA, or ADO + L-NMMA. In the figures showing Ps, the scales are log10 to represent appropriately the magnitude of decreases and increases in permeability [i.e., a twofold decrease (a ratio of 0.5) has the same magnitude as a twofold-increase (a ratio of 2) with regard to a change in Ps from the prestimulation levels]. A significant change from basal levels was determined using paired Student's t-test or Wilcoxon's signed-rank test, as appropriate. Ps with ADO was analyzed using repeated-measures ANOVA and using a Bonferroni/Dunn correction when there were more than two groups.
Power analyses (44) indicated that seven animals were required per solute to minimize both type I and type II errors. Consequently, for the venular data using -lactalbumin as the solute, only the mixed data (males and females) are reported since the sample size was too small to test whether the pig's sex influenced the results. A significance level of P < 0.05 was set before calculating the power analysis and performing the experiments.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Body weights did not differ with training status or sex on the day of euthanasia (Table 1). The hearts from the EX animals, however, weighed more than those from SED pigs without regard to sex (P < 0.01). Consistent with the HW and BW results, the HW-to-BW ratio was increased with training (P < 0.01). Interestingly, the HW-to-BW ratio was lower in SED females compared with that in SED male pigs (4.6 ± 0.1 vs. 5.2 ± 0.2 for SED females and males, respectively, P < 0.05), but this sex difference was no longer evident after training (5.5 ± 0.1 vs. 5.7 ± 0.2 for EX females and males, respectively).
|
As illustrated in Fig. 1A, resting heart rate (HR) was higher in female than in the male pigs in both experimental groups before (Pre) training. This difference was maintained, as expected, between female and male SED pigs after 16 wk. In contrast, HR was reduced for both sexes, and posttraining and the female/male difference were maintained. Both the male and female pigs improved their duration of exercise in the performance test; duration and magnitude of improvement did not differ with sex (Fig. 1B). Changes in HR in response to acute exercise in the stress test (HRPretraining – HRPosttraining, Fig. 1C) were observed following endurance exercise training before the performance test (rest) and at most stages of the exercise testing protocol. Of interest, the training-induced reduction in HR was most evident for the female pigs at all stages of work. Posttraining bradycardia was greater for males than for females at rest, and the change in HR levels in EX males was not significant beyond the S2 workload of the performance test. For the EX females significant reductions in HR, relative to their pretest levels, were observed at all four levels of work.
|
In EX pigs, Ps was measured under basal conditions on 138 coronary arterioles having an internal diameter of 40.4 ± 1.5 µm (mean ± SE; range 6–126 µm) and on 24 venules with an average diameter of 76 ± 9 µm (mean ± SE; range 26 to 126 µm, major axis). The majority of these arterioles would be classified as A4 (32), and the predominant venule classification would be V4 (31).
Overall, the EX group resulted in a reduction of P
compared with SED pigs. Basal arteriole P was 4.8 ± 0.4 x 10–7 cm/s (mean ± SE; n = 56) in EX pigs, which is significantly lower (P < 0.01) than P in SED animals [6.3 ± 0.6 x 10–7 cm/s; n = 61 (25)]. Arteriolar basal permeability did not differ between males and females; P = 0.9 (Fig. 2). Similarly, in EX males, basal venule P was significantly lower (8.1 ± 3.0 x 10–7 cm/s; n = 7) than in SED animals [22.2 ± 3.5 x 10–7 cm/s; n = 24 (25)]. In contrast, for venules from EX females, P was 21.6 ± 6.1 x 10–7 cm/s (n = 11), a value that was not different from venules of SED animals' P[22.2 ± 3.5 x 10–7 cm/s; n = 24 (25)]. Thus venular P in EX pigs differed significantly with sex (P < 0.001). Although basal arteriole P was significantly less than venule P(P < 0.001) for EX females, the arteriovenous difference for P was not demonstrable (P = 0.42) in coronary microvessels from EX males.
|
-lactalbumin, a smaller, monomeric protein possessing a shape and charge similar to albumin at physiological pH, was determined. -Lactalbumin has been used in multiple studies and shown to be without apparent influence on exchange barrier function (19–21, 25–28). As with PSA, basal arteriole P
(10.7 ± 0.9 x 10–7 cm/s; n = 82) did not differ with sex (P = 0.29). As anticipated, P, for the smaller solute, was significantly greater than that of the larger protein PSA (P < 0.001), and similar to the results with PSA P in arterioles from EX pigs was significantly less than that of SED pigs [12.1 ± 1.4 x 10–7 cm/s; n = 76 (25)].
In the venules basal P was 30.2 ± 8.9 x 10–7 cm/s (4 females and 2 males). Although the small sample size precluded drawing conclusions concerning sex-related differences, P of venules was greater than P of arterioles (P < 0.01).
Barrier Properties Following ADO Exposure in EX Pigs
The cumulative concentration-response curve (Fig. 3) illustrates that P in coronary arterioles from EX pigs (3 females and 4 males) increases from basal levels at concentrations of ADO >10–9 M. For this sample size, the increase was significant at 10–7 M, the EC50 was 2.3 x 10–8 M, and a maximal response (70% over basal levels) was reached at 10–6 M. For all of the subsequent studies, Ps was tested at a concentration of 10–5 M ADO. For comparison, the concentration-response for coronary arterioles (4 females and 3 males) isolated from SED pigs (61) is given as the broken gray line in Fig. 3.
|
in three of the four experimental groups (all EX arterioles and venules from EX females) (Fig. 4).
|
Ps response of arterioles from EX females (2.27 ± 0.34-fold, n = 35, P < 0.01) and EX males (1.65 ± 0.29-fold, n = 18, P < 0.05) failed to reach significance (P = 0.17). Venules from the hearts of EX females also responded to ADO with an increase in P(1.31 ± 0.09-fold, n = 7, P < 0.05), whereas the same treatment of venules from EX males resulted in a decrease in P(–0.15 ± 0.10-fold, n = 7, P = 0.01). This sex difference was previously observed in venules from SED pigs (25) where there was an increase in SED female venule P(1.4 ± 0.2-fold, n = 15, P < 0.05) and a decrease in SED male venule P[–0.21 ± 0.06, n = 13, P < 0.01, (25)]. Importantly, the responses of venules differed from those of arterioles, whether from EX (P < 0.05) or SED (P < 0.05) pigs (25), indicating that some aspect of the training-induced adaptation of coronary venules, but not arterioles, is sex dependent. Does Adaptation to EX Involve Different Second Messenger Pathways?
Most of the evidence indicates that vascular ADO receptor activation is mediated by activation of AC following binding to ADO A2A receptors (8). Our working hypothesis is that AC-mediated signaling mechanisms reduce Ps, whereas those mediated by GC will increase Ps (22, 40–42). Therefore, if in the coronary microvasculature ADO regulates exchange via exclusive activation of A2A receptors, then we predicted that Ps in EX animals would fall from basal levels in response to ADO as occurred in arterioles from SED animals (25, 27). Instead, in arterioles from the EX pigs, Ps increased following exposure to ADO. Previous studies in this laboratory have demonstrated both message and protein expression for three of the four ADO receptor subtypes (A1, A2A, and A2B) in both coronary arterioles and venules (61). The signaling mechanisms for the A1 and A2B receptor subtypes result in decreases and/or increases in AC, respectively (9, 54), via their respective Gi- and Gs coupling (8). Consequently, the net change in Ps that we observe could be a mixed response reflecting the interplay of the different second messenger systems, depending on the relative abundance and activities of the ADO receptor subtypes (54). We speculate that their affinity, abundance, and/or efficacy is modified in the EX adaptation.
As a first step to investigating whether the cyclase system activities were indeed modified by EX per se, we chose two agents thought to work exclusively by a single cyclase: ISO, which on binding to the 
-adrenergic receptor activates AC (2, 33, 43, 47), and ANP, the receptor of which is a particulate GC (15, 29). In previous studies, we and others have shown that Ps of intact vessels can decrease in response to ISO (1, 17, 24) and increase in response to ANP (23, 40, 51). Given that the basal Ps levels and the responses of the arterioles of the SED and EX pigs were independent of sex, the data from female and male pigs were pooled according to training status.
In arterioles isolated from 16 (7 SED and 9 EX) pigs, basal P was assessed, and the microvessels were then recannulated with pipettes containing 10–5 M ISO. Exposure to ISO, as expected, reduced Ps in all microvessels (Fig. 5A). The magnitude of the response was greater (P < 0.05) in those from SED pigs than those from the age-matched EX counterparts [0.39 ± 0.08-fold (P < 0.01) vs. 0.59 ± 0.05-fold (P < 0.01) for SED and EX pigs, respectively], suggesting that the attenuation of AC-mediated responses may constitute an additional adaptation to endurance training.
|
from basal levels (7 SED and 5 EX pigs, Fig. 5B). Again, there was a significant difference in the magnitude of the response with training in that the response of arterioles from SED pigs (2.08 ± 0.62-fold; P < 0.01) significantly (P = 0.02) exceeded that from EX animals (1.31 ± 0.08-fold; P < 0.01). Thus the arteriolar permeability response in trained animals appears consistent with an attenuation in GC-dependent signaling. Our data thus show that the overall response of adaptation to exercise is complex, involving alterations in the contributions of both of these signaling pathways; the resulting effect on Ps will reflect their relative balance but is likely to include an overall lower permeability phenotype.
Contribution of NO to the ADO-Induced Increase in Arteriole Ps
To further explore possible adaptations in signaling pathways modulating Ps in exercise-adapted animals, we asked whether the increase in arteriole Ps produced by ADO involved an NO-dependent component (7, 45), as we and others (24, 49) have shown that the ADO-induced increase in Ps has an NO-sensitive component, and it has also been shown that exercise adaptation includes an increase in endothelial NOS expression (34, 35, 37, 38, 62).
In arterioles from EX animals, inhibition of NOS by an addition of 10–5 M L-NMMA to the suffusate produced a significant 25 ± 9% reduction in arteriole P(P < 0.05, N = 9, Fig. 6A), confirming that there is a NO-dependent component to the basal Ps in these vessels. In a second set of EX arterioles, suffusion with 10–5 M ADO first resulted, as expected, in a 32 ± 13% increase (P < 0.05) in Ps; on subsequent exposure to 10–5 M L-NMMA in combination with 10–5 M ADO, Ps fell to 86 ± 11% of control levels (N = 10, Fig. 6B). The reduction in Ps from basal levels did not differ (P = 0.78) between L-NMMA alone (Fig. 6A) and L-NMMA + ADO (Fig. 6B), indicating that ADO affects Ps via a NO-dependent pathway.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-lactalbumin. The single exception was the venules from EX males where P was reduced. In addition, the sex-dependent responses of the venules of SED pigs persisted after and were not modified by EX training. Overall, our data demonstrate that coronary microvascular exchange capacity is altered in adaptation to 16 wk of endurance exercise training. The mechanisms responsible for this adaptation depend, to varying extents, on microvessel location (arterioles vs. venules), as well as the sex of the adult animals used in this study.
Method Limitations and Assumptions
An assumption fundamental to the calculations of solute flux is that the isolated coronary microvessels are, during perfusion and determination of flux, circular in cross section and have a volume-to-surface area ratio of 0.25 D (Eq. 1). Preliminary work in this study, and detailed to a greater degree by studies of rat and mouse skeletal muscle preparations (53), suggests that the assumption of a circular cross section under the conditions of these experiments is valid for coronary arterioles. Venules, especially those in the coronary circulation, are less likely to have a circular cross section and instead are relatively flat such that the minor axis, b, is not equal to the measured major axis, D (31, 58). The consequence of the noncircularity will be in the reporting of the absolute value of Ps rather than in the direction and/or the magnitude of the responses to test substances since the geometrical terms, in both the numerator and denominator, will be cancelled out (58). Thus it is possible, given the wider availability of techniques (e.g., confocal microscopy), to determine both axes in vivo with greater accuracy; hence, the absolute values of venule Ps will be lower than reported in this or previous studies (18, 24, 25, 27, 49, 61).
Influence of Training Status and Sex on Basal Coronary Microvascular Ps to Macromolecules
Basal P of both arterioles and venules changed in adaptation to endurance exercise training, in a manner that was independent of the animal's sex. Basal Ps of arterioles from both males and females and of venules from males was reduced following 16 wk of EX relative to their SED counterparts. Contrary to our hypothesis, basal Ps of the arterioles from EX pigs was reduced relative to their SED counterparts. Similarly, in the venous coronary microvasculature, endurance exercise training resulted in a reduction in Ps (pooled data from the males and females) relative to comparable vessels from SED pigs. Thus, overall, coronary microvessels are less leaky in trained animals and represent an adaptation that clearly supports the maintenance of vascular volume in performance-adapted subjects.
Consistent with the conclusion that it is the venular elements of the coronary exchange microcirculation that are sex sensitive, we found that basal P of venules from the EX females was >80% higher than comparable venules taken from the hearts of males (P < 0.05). We found that venules from male EX animals were less than a third as leaky as all the other vessel groups: Ps of venules from females remained unchanged by training in contrast to the decrease in Ps in the venules from males. No other apparent features, including the diameters (P = 0.73), of the venules differed between groups. Although the mechanism(s) underlying the adaptation in Ps of venules of males remains to be determined, there are clear functional implications of these findings. Assuming that there are no sex-dependent differences in coronary network architecture, it is clear from the measured Ps values that net macromolecule flux will be greater in exercise-trained females compared with males under basal, nonexercising conditions. Given that there is literature (55, 59) indicating that hydrostatic pressures are lower in the exchange microvasculature of females compared with age-matched males, the net effect may thus be to minimize differences in macromolecular flux between the sexes under resting conditions.
Training Status and Sex Alter the Coronary Microvascular Permeability Gradient to Macromolecules
In characterizing the permeability properties of vessels making up the exchange microvascular network, it is generally assumed that there is a gradient of permeability properties such that the vessels at the arterial, high-pressure end of the vascular network are tighter (less permeable) than those at the venous, low-pressure end of the network. In the present studies, this was true for three of the four experimental groups (SED females and males and EX females, independently of whether permeability increased or decreased on exposure to ADO). EX males, to our surprise, did not fit this paradigm. Two aspects of the data were notable. First, the basal atrioventricular (AV) gradient in the EX males did not reach significance before exposure to ADO (P= 5 ± 1 in arterioles vs. 8.1 ± 3 x 10–7 cm/s in venules; P = 0.08). Second, the gradient was abolished, if not reversed, following ADO application (P= 9.2 ± 1 in arterioles vs. 6.5 ± 2 x 10–7 cm/s in venules; P = 0.10). In both cases, the feature that distinguished EX males from the EX females and SED pigs of either sex was the fact that their coronary venules had permeability properties similar to arterioles, and in response to ADO, permeability fell from basal levels. Thus our study shows that, in EX males, it was the venules that adapted to become "tighter" compared with arterioles in the same network or to venules in EX female pigs. Clearly, it will be important to investigate the mechanisms underlying this unexpected adaptational response in the male animals.
A reduction in permeability on exposure to ADO has been observed in a variety of endothelial monolayer model systems (13, 46), as well as in the isolated coronary arterioles from SED animals of both sexes (this study and Ref. 25), in isolated coronary venules from SED males (this study and Ref. 25) and also in skeletal muscle arterioles and venules isolated from juvenile female rats (60). We note that the AV gradient observed by Wang and Huxley (60) in the skeletal muscle microvessels under basal conditions was abolished in the presence of ADO. As in the present study, this was a consequence of the Ps in the venous vessels being lower than expected rather than the Ps in arteriolar vessels being higher than expected. This ability for venous vessels to modulate their permeability by becoming "as tight as arterioles" provides support to the conclusion that mechanisms underlying venular responses differ significantly from those regulating an exchange across the walls of coronary arterioles.
We considered whether the absence of the AV gradient in the EX males might serve to limit solute flux across the intact microvasculature during EX. As was shown in Fig. 1, HR differs between EX females and males and, given that an exchange in the myocardium occurs primarily during diastole, we speculated that a reduction in venous permeability would help to limit net transport in the EX males relative to females. However, the training bradycardia was actually greater in the EX females than in the males during all stages of the performance test (Fig. 1); hence, we would expect overall solute flux to be greater in females also. Thus it remains to be determined whether adaptation to EX with respect to net coronary exchange involves more than the changes we have seen in microvessel barrier properties. Other candidates likely to contribute to the net flux of solute include differences in coronary microvessel anatomy, hence, surface area for exchange; differences in extracellular matrix composition, thereby, influencing both transfer and gradients within the matrix; lymphatic function, again influencing solute gradients in the compartment outside of the vascular space; and differences in microvascular hydrostatic pressures, thereby influencing convective transport of the macromolecules, to mention a few possibilities.
Our use of AC- and GC-dependent pathways to probe their adaptation in regulation of permeability responses indicated that both of these signaling pathways had been modulated in response to exercise training. Exchange capacity mediated by both pathways was attenuated in microvessels from exercise-adapted animals: determining whether this represents a change in expression of these signaling molecules per se, or some other adaptation of other elements in the response pathway, is beyond the scope of the present work. Importantly, our experiments also indicate that exercise adaptation involves an upregulation of an NO-dependent pathway, which appears to contrast with our conclusion that GC-dependent pathways are downregulated but which further emphasizes that multiple mechanisms are involved in the regulation of barrier properties and have been modulated in a complex way in response to the exercise training regime.
The outcomes of these changes in microvascular exchange capacity on fluid volume distribution and hence cardiovascular homeostasis in intact exercising individuals are likely to be complex. Single bouts of intense exercise in healthy human adults have been shown to result in a retention of albumin in the vascular compartment that cannot be accounted for by changes in albumin metabolism per se (11, 12). Plasma volume is reported to decrease in mixed (male and female) groups of exercising dogs (52) and humans (39); direct measurements of interstitial colloid osmotic pressure in the Mack et al. (39) study showed that this did not change, implicating decreased barrier function per se in the response. These studies are consistent with the decrease in Ps to ADO that we report here. In other work, Haskell et al. (14) reported that ANP levels in human subjects increased under the same exercise regime as in their earlier work, and they observed an increased transcapillary escape of albumin. From our results, we speculate that the extent to which ANP release during exercise could be the driving force for albumin flux out of the vasculature will be limited by an exercise-induced diminution in the response to ANP. Although both male and female subjects were included in the study of Mack et al. (39), the data are not broken out according to sex. We suggest that at least part of the variability in the findings of these studies will likely relate to the relative numbers of males and females in the study groups. Furthermore, because our data show differences in exchange function between different regions of the microvasculature (arterioles and venules), overall fluid volume status such as relative hydration (with consequent effects on venous capacitance) will likely influence the integrative fluid volume distribution response to exercise.
In summary, we show in this study that in response to exercise training there is an overall reduction in basal permeability. In response to the metabolic product ADO, Ps is increased in arterioles, but there is a sex-dependent difference in the response of venules in that Ps in venules from males decreased and Ps in venules from females increased with ADO exposure. Regulation of permeability via both AC- and GC-dependent pathways was reduced, whereas NO-dependent regulation of permeability was upregulated. Overall, our study shows that the adaptation of microvascular exchange capacity to endurance exercise training involves multiple mechanisms that can differ in arterioles versus venules: their relative contribution to net flux is a function of sex.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-lactalbumin transport. Am J Physiol Heart Circ Physiol 252: H188–H197, 1987.
posttraining (Post)], in both F and M pigs and the sex difference in HR was maintained. B: duration of exercise stress test administered to pigs before (Pre) initiation of training regime and following 16 wk of training (Post) for the EX M and F pigs. Although the training regime resulted in a significantly increased duration (*P < 0.05), there were no discernible sex differences. C: change in HR between the stress test administered prior (HRPre) and following (HRPost) the 16-wk regime for the pigs at rest and the four stages of work (S1, 5 min at 5 km/h, 0% grade; S2, 10 min at 5 km/h, 10% grade; S3, 10 min at 6.9 km/h, 10% grade; and S4, 9.7 km/h, 10% grade until exhaustion). *P < 0.05, significant difference; **P < 0.05, EX F significantly different from EX M. SED, sendentary; bpm, beats/min.
) relative to its absence (P