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Muscle arteriolar and venular reactivity in two models of hypertensive rats
Microvascular Research 69 (2005) 142 – 148 www.elsevier.com/locate/ymvre
Muscle arteriolar and venular reactivity in two models of hypertensive rats Mercedes Losadaa, Sonia H. Torresb,*, Noelina Herna´ndezb, Marisela Lippoa, Amparo Sosaa a
Escuela J. M. Vargas, Facultad de Medicina, Universidad Central de Venezuela, Venezuela Instituto de Medicina Experimental, Seccio´n para el Estudio de la Adaptacio´n Muscular (SEAM), Universidad Central de Venezuela, Apdo. 50587 Sabana Grande, Caracas 1050-A, Venezuela
b
Received 16 November 2004 Available online 20 April 2005
Abstract This study was designed to test if skeletal muscle fiber composition could influence vascular response in hypertensive rats. Muscle vessels were observed by intravital microscopy in anesthetized rats and changes in diameter were measured after local administration of endothelium-dependent and -independent vasodilators. Vascular reactivity was compared in two models of hypertension deoxicorticosterone acetate and salt load (DOCA-s) hypertensive rats and spontaneously hypertensive rats (SHR). The muscles used were: the fast-twitch glycolytic muscle, extensor digitorum longus (EDL), and the slow-twitch oxidative, soleus muscle. Maximal dilation induced by vasoactive drugs was of similar magnitude in EDL and soleus arterioles. Terminal arteriole reactivity to acetylcholine and adenosine was blunted in EDL (35% and 49% reduction, respectively) and soleus muscles (42% and 34% reduction, respectively) of SHR compared with Wistar Kyoto rats. Reactivity of DOCA-s rats to acetylcholine, adenosine, and sodium nitroprusside was reduced by 38%, 50%, 39% in EDL third- and fourthorder arterioles and by 30%, 38%, 38% in soleus fourth-order arterioles, respectively. These studies show that hypertension probably induced similar vascular changes in both muscles studied. Vascular reactivity is blunted for some vasodilator drugs and is more affected in DOCA-s rats. In addition, a preferential action for bradykinin was observed on upstream arterioles but not on venules. This effect was not observed for adenosine. D 2005 Published by Elsevier Inc. Keywords: Skeletal muscle; Adenosine; Bradykinin; SHR; DOCA-salt; Arterioles; Hypertension
Introduction It is well established that increased resistance in large and small vessels is characteristic of essential hypertension and results in elevation of blood pressure. The study of diverse vascular beds and segments of the circulation has shown that morphological and physiological adaptation to hypertension may be different in macrovessels and microvessels of different vascular beds, between large and small arteries, and even within microvessels of different diameters (Henrich et al., 1978). Although it is generally assumed that the increase in skeletal muscle vascular resistance occurs to a similar
* Corresponding author. Fax: +58 212 730 7503. E-mail addresses: [email protected], [email protected] (S.H. Torres). 0026-2862/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.mvr.2005.03.004
degree in all muscles (Sexton et al., 1990), some evidence suggests that may not be uniform and varies with the fiber type composition of the muscles. Extensor digitorum longus (EDL) muscle is predominantly formed by type II fibers, and in deoxicorticosterone acetate and salt load (DOCA-s) hypertensive rats, the proportion of IIb fibers is increased; on the contrary, in soleus muscle of control and hypertensive rats, there is a higher proportion of type I and IIa fibers that are highly oxidative (Herna´ndez et al., 1996). Doppler flow probes on normotensive rats have shown impaired sympathetic vasoconstriction in fast-twitch gastrocnemius-plantaris muscles, but good contractions in vessels of the slow-twitch soleus muscle, suggesting that, under physiological conditions, the extent of sympathetic vasoconstriction may depend on the fiber type composition of the active muscle (Hansen et al., 2000). In several models of hypertensive rats, microvascular rarefaction appears to occur in skeletal muscles composed of a high
M. Losada et al. / Microvascular Research 69 (2005) 142 – 148
proportion of fast-twitch glycolytic fibers like cremaster, gracilis, and EDL muscles (Chen et al., 1981; Green et al., 1992). However, it has been reported by Herna´ndez et al. (1996) that there is a significant reduction in capillary number per fiber in soleus muscle, which is mainly formed by slow-twitch oxidative fibers. Blood flow is linearly related to skeletal muscle oxygen consumption over a wide range of metabolic rates, lending support to the idea that local control of vascular resistance in skeletal muscle is driven by metabolic processes (Aaker and Laughlin, 2002; Murrant and Sarelius, 2000). It has been shown that changes in metabolism lead to closely related changes in blood flow, via the vasodilatory actions of products of muscle contraction metabolites, like inorganic ions, purines, etc. (Gorczynski et al., 1978). Blood flow and metabolism coupling can be extremely local in nature since arteriolar dilatation is observed only in the vicinity of the contracting muscle fiber. Moreover, three generations of arterioles upstream from capillaries dilate when the muscle fibers increase their metabolism. Capillaries communicate to upstream arterioles via conducted signals, initiated at the capillary level and transmitted through gap –junction communications (Berg et al., 1997). In the present study, vascular reactivity of vessels from muscles having different oxidative capacities was tested. The effect of vasodilator drugs: acetylcholine (ACh), adenosine (AD), sodium nitroprusside (SNP), and bradykinin (BK) was evaluated on precapillary arterioles and postcapillary venules from fast-twitch glycolytic (EDL) and slow-twitch oxidative (soleus) muscles in two models of hypertension, spontaneously hypertensive rats (SHR) and DOCA-s rats. These drugs were selected in order to compare hypertension effect on different mediators.
Materials and methods Experimental animals 16 male Sprague – Dawley rats were made hypertensive by subcutaneous injections of deoxicorticosterone acetate (20 mg/kg) daily for 10 weeks, supplemented with oral salt loading by replacing drinking water with 1% NaCl solution (DOCA-s rats). 20 control rats (C) were injected with 0.20 mL 0.9% NaCl solution following the same scheme as DOCA-s rats and drank tap water. Genetic hypertensive rats: 28 male age-matched SHR, and as controls, 27 Wistar Kyoto (WK) rats were used in these studies. Hypertensive and normotensive rats’ weight was 280 –350 g. Animals were provided with regular rat chow and housed with controlled light (12-h light –dark cycle) and temperature conditions. All rats were taken from a colony maintained at the Universidad Central de Venezuela, Instituto Nacional de Higiene Rafael Rangel, Caracas, Venezuela.
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Vascular reactivity On the day of the experiment, rats were anesthetized (pentobarbital sodium 40 mg/kg, chloral hydrate 200 mg/ kg, i.p.) The trachea was cannulated to ensure a patent airway; the left carotid artery and jugular vein were cannulated for arterial pressure measurement and for the administration of supplemental anesthesia (5 mg/kg bolus, when required), respectively. Once the initial surgery was completed, the rat was placed on a circulation-heated Perspex stage and the right EDL or soleus muscle was prepared for television microscopy observations by retracting the tibialis anterior and peroneal muscles. Throughout the whole preparation and experimental time, the muscles were bathed with a de-oxygenated Krebs –Henseleit solution (NaCl 131.9 mM; KCl 4.7 mM; MgSO4I7H2O 1.17 mM; CaCl2I2H2O 2 mM; NaHCO3 22 mM) maintained at 32 – 34-C, equilibrated with 95% N2 – 5% CO2, pH 7.35. The vessels were classified according to their branching order beginning at the capillary level: terminal (4th order: A4) and preterminal (3rd order: A3 and 2d order: A2) arterioles. A similar classification was used for postcapillary venules (V4, V3, V2). The surfaces of EDL and soleus muscles were observed with a water-immersion objective lens (Zeiss 40) by an intravital microscope (Zeiss) epiilluminated with fiber optics (Schott KL1500). Vessels’ diameters were observed by a video camera (Sonny CCDIris) and recorded to a videotape (JVC). Images from the camera were also displayed in real time on a television monitor and diameter changes were measured with a calibrated reticule generator (Artek Systems Corp.) displayed on the television monitor. Molar concentrations of vasoactive drugs that produced 50% of the maximal dilation (ED50 values) were estimated in hypertensive rats; there was a shift to the right in the EC50 in A4 arterioles in SHR, but in A3 vessels, the response was the same in WK and SHR. The doses used were: ACh: 10 5 mol/L; AD: 10 5 mol/L; BK: 10 7 mol/L, and SNP: 10 5 mol/L. Drugs were obtained from Sigma. Luminal diameters (width of red cell column) were measured at rest and after randomized topical administration of 1 mL of drugs applied to the exposed EDL or soleus muscles under the objective lens by syringe, at a temperature comparable with the superfusate, without interruption of superfusate flow. Immediately after drug application, changes in diameter were measured and recorded to take in the peak response. Each vessel was given 10 min to recover to resting diameter between drug applications. The total duration of observations during an experiment did not exceed 2.5 h. Blood pressure was continuously monitored to verify that drugs applied locally did not have a systemic effect. The results were expressed as percentage of vessel change from resting diameter (D REST) to maximal dilation (D MAX) obtained with the applied drug (D MAX D REST/D REST 100). The diameter measurements were made in two vessels per rat and ‘‘n’’ represents the number of rats.
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Statistical analyses Data were analyzed by the nonpaired Student’s ‘‘t’’ test or an analysis of variance (ANOVA) with Bonferroni test corrections for multiple comparisons as appropriate. The statistical significance of differences between groups was based on a probability level of P 0.05. Figures represent mean T SEM and the table represents means T SD.
Results Mean arterial blood pressure of hypertensive rats under general anesthesia was significantly higher (167 T 4 and 177 T 7 mm Hg for SHR and DOCA-s, respectively) compared with WK (114 T 8 mm Hg) and C (113 T 7 mm Hg). Vessel resting diameters from EDL and soleus muscles in hypertensive rats were not significantly different from control rats (Table 1). EDL and soleus reactivity The possibility that vascular reactivity could depend on the fiber type composition of the muscle was tested. Dilation induced by vasoactive drugs was of similar magnitude in A3 and A4 vessels from both muscles (EDL and soleus) of normotensive rats (Control Sprague – Dawley and WK, white bars, Figs. 1 –4). Also, dilation of A3 and A4 vessels to the drugs administrated was not significantly different between EDL and soleus muscles
Fig. 1. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A4 vessels. (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. **P < 0.01. WK (n = 9) vs. SHR (n = 10).
from hypertensive rats (DOCA-s and SHR, black bars, Figs. 1– 4). Vascular reactivity of SHR vs. WK
Table 1 Mean T SD of resting arteriolar diameters (Am) in normotensive (n = 12) and hypertensive (n = 14) rats EDL (extensor digitorum longus) WK
SHR
C DOCA-s
A2 A3 A4 V2 V3 V4 A2 A3 A4 V2 V3 V4 A3 A4 A3 A4
9.37 T 7.07 T 5.42 T 13.11 T 8.42 T 5.98 T 8.64 T 6.41 T 5.18 T 13.51 T 8.77 T 5.82 T 6.20 T 5.30 T 5.83 T 4.34 T
1.27 0.80 0.36 2.29 1.84 0.82 1.32 0.69 1.01 3.53 2.12 0.55 0.35 1.05 0.56 0.78
Soleus
6.09 T 0.74 5.60 T 0.66
6.47 T 0.81 5.60 T 0.57
5.32 4.79 6.19 4.44
T T T T
WK = Wistar Kyoto. SHR = spontaneously hypertensive. DOCA-s = deoxicorticosterone acetate and salt load treated SD rats. C = Sprague – Dawley (SD) control rats.
0.49 0.47 0.59 0.43
In EDL muscle, the responses to ACh and AD were significantly reduced in A4 (Fig. 1a) and A3 (Fig. 2a) vessels in the SHR compared with WK rats. In soleus muscle of SHR, also the responses to ACh and AD were reduced in the SHR, in A4 (Fig. 1b) and A3 arterioles (Fig. 2b). However, there were no significant differences between WK and SHR in the dilation induced by BK and SNP, neither in the muscles nor in the arteriole order studied (Figs. 1 and 2). Vascular reactivity of DOCA-s vs. C A4 and A3 vessels of EDL muscle in DOCA-s rats showed reduced responses to ACh, AD, and SNP, but normal to BK (Figs. 3a and 4a). A4 arteriole responses to the drugs in soleus muscle were similar to those seen in EDL muscle (Fig. 3b). A3 soleus vessel dilations to ACh, SNP, and BK in DOCA-s were similar to normotensive rats but significantly lower to AD (Fig. 4b). Arterioles from DOCA-s rats seem to be either more affected by hypertension or to have a different mechanism underlying
M. Losada et al. / Microvascular Research 69 (2005) 142 – 148
Fig. 2. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A3 vessels. (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. *P < 0.05, **P < 0.01. WK (n = 9) vs. SHR (n = 10).
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Fig. 3. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A4 vessels (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. *P < 0.05 **P < 0.01. C (n = 11) vs. DOCA-salt (n = 8).
vasoconstriction, since SHR arterioles showed good response to two of the four drugs used, and were able to react to SNP; in contrast, DOCA-s arterioles showed reduced responses to ACh, AD, and SNP. BK effect in three arteriolar branches It is noteworthy that vascular responses to BK in both muscles and A3 and A4 arterioles were similar in hypertensive and normotensive rats (regardless of the model of hypertension). However, the dilation induced by BK in normotensive rats was lower (40%) than the dilation induced by the other drugs. To test the possibility that some drugs could exert its effect on upstream vessels, two local mediators with physiological actions were compared (BK and AD). In an additional group of WK and SHR rats, BK and AD effects were assessed in fourth-, third-, and second-order vessel branches of EDL muscle; arteriolar and venular responsiveness was also evaluated. Fig. 5a shows a preferential action of BK on A2 vessels in WK rats but this effect is not seen in venules. Dilation induced by BK on SHR was of similar magnitude in all vessel order studied; however, it was significantly reduced in A2 and all venular branches, compared with normotensive rats. AD did not show the preferential action in A2 arterioles exerted by BK in WK rats, and vascular reactivity was of similar magnitude
Fig. 4. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A3 vessels (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. *P < 0.05 **P < 0.01. C (n = 11) vs. DOCA-salt (n = 8).
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Vascular reactivity
Fig. 5. Effect of BK (a) and AD (b) on three order branches of vessels in EDL muscle of WK (n = 8) and SHR (n = 8) rats. The solid lines show that dilation induced by BK exerts a preferential effect (ANOVA) on A2 arterioles from WK (P < 0.01). Differences between dilation induced by BK and AD on arterioles (A) and venules (V) from WK vs. SHR (t test **P < 0.01) are shown. Results are expressed as mean T SEM.
in all arteriolar and venular branches (Fig. 5b). AD dilation was significantly reduced only for SHR A3 and A4, arterioles compared with WK rats.
Discussion Vessel diameters Folkow (1990) suggested that genetic hypertension involved arterial medial hypertrophy with a resulting encroachment on the lumen. In consequence, vascular resistance increases as a result of a structural change. In the present study, resting lumen diameters measured by intravital microscopy were similar to those reported by Thomas et al. (1998) in Wistar rats’ EDL muscle, and did not differ between hypertensive (SHR and DOCA-s) and normotensive rats (WK and C). Normal resting diameters in hypertensive rats compared with normotensive have been also reported for skeletal muscle microcirculation in other models of hypertension (Lombard et al., 2000; Prewitt et al., 1984). Our results suggest that vasoconstriction of the terminal portion of the arterial tree is not involved in the increase of the peripheral resistance observed in hypertension.
This study was focused on precapillary arterioles because these small vessels under physiological conditions act as metabolic sensors of muscle activity, regulating upstream blood flow to match muscle necessities (Berg et al., 1997; Gorczynski et al., 1978; Murrant and Sarelius, 2000). In addition, third- and fourth-order arterioles contribute to arterial vascular resistance in resting muscle (Dodd and Johnson, 1991). It seems reasonable that responsiveness of the skeletal muscle arterioles varies with muscle fiber type or oxidative capacity. There is evidence that supports this idea in normotensive rats, i.e., hindlimb unloading results in a diminished responsiveness of skeletal muscle arterioles from highly oxidative skeletal muscle to vasodilators and diminished vasoconstrictor capacity in low oxidative muscle (McCurdy et al., 2000). Vasodilation in response to AD and SNP is greater in gastrocnemius muscle arterioles than soleus muscle arterioles, and maximal vasodilator response to ACh is greater in soleus and red gastrocnemius muscle arterioles than in diaphragmatic and white portion of gastrocnemius arterioles (Aaker and Laughlin, 2002; Wunsch et al., 2000). Our results in two strains of normotensive (WK and Sprague – Dawley) rats did not show differences in vascular reactivity associated to muscle fiber type. In both strains, EDL muscle is predominantly formed by type II fibers, with high glycolitic capacity, and soleus muscle has a high oxidative capacity and a high proportion of type I fibers. Similar results have been found by others (Aaker and Laughlin, 2002; Jones et al., 1995), who studied vascular reactivity to AD in larger vessels (A1 and A2). In hypertension, elevated peripheral vascular resistance and arteriolar rarefaction are associated with metabolic disorders. It has been shown that total hind quarter flow is lower (76%) in SHR than WK (Sexton et al., 1990) and basal oxygen consumption, glucose uptake, and lactate production are higher in SHR (Ye and Colquhoun, 1998). Our results show that vascular reactivity to the drugs was not significantly different between EDL and soleus muscles; in consequence, neither difference in fiber type composition nor oxidative capacity of skeletal resting muscles seems to be the primary determinants of vascular reactivity in hypertensive rats. Bohlen (1986) suggested that capillary control of pressure excess in hypertensive rats involves propagated signals that elevate arteriolar resistance to compensate tissue overperfusion. This implies that vascular function might be normalized in terminal arterioles by mechanisms that are taking place in larger vessels. Our results do not support this hypothesis and suggest that attenuated vascular responses might be related to alterations in synthesis or release of intracellular mediators like nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF), which interfere to a variable degree with vasodilator mechanisms in the skeletal muscle microcirculation, and are dependent on the hypertension model but not in muscle fiber type.
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Endothelium-dependent relaxations are achieved by a combination of endothelium-derived prostacyclin, NO, and EDHF. Relaxations induced by ACh (Shimokawa et al., 1996) and BK (Cohen and Vanhoutte, 1995) are mediated by NO and EDHF, and AD is known to cause hypotension via prostaglandin synthesis (Ray et al., 2002). NO has been proposed to play a role in the pathogenesis of hypertension (Arnal et al., 1995). Our experiments showed good reactivity of A3 and A4 arterioles of SHR to SNP in both EDL and soleus muscles, indicating that the smooth muscle pathway associated to NO effect is not altered by genetic hypertension, since these vessels are able to react to an external NO donor. This does not seem to be the case for DOCA-s rats, in which vascular reactivity to SNP is significantly blunted, demonstrating an important difference between hypertension models. ACh response is reduced in both hypertension models, including SHR arterioles that showed good response to SNP. There is evidence that endothelium-derived NO predominantly mediates AChinduced dilation, and participation of EDHF becomes apparent only after inhibition of NO synthesis (Huang et al., 2000). When one mediator is impaired by hypertension, an alternative compensatory pathway is activated; probably, this is the case in our experiments. The relative contribution of NO and EDHF is not clear, it has been suggested that the two mediators are interrelated, either by sharing common mechanisms or by reinforcing or otherwise modulating the action of the other (Cohen and Vanhoutte, 1995). Ray et al. (2002) showed that in vivo vasodilation in skeletal muscle evoked by AD is dependent on prostaglandin synthesis by endothelial cells, which then stimulates NO synthesis. Our experiments showed a reduced AD response in both hypertension models. This could be associated to insensitivity of the receptor to AD (Liu et al., 2002), and lower ability to initiate the prostaglandin synthesis or reduced prostaglandin stimulation of NO synthesis (Ray et al., 2002). In the latter situation, DOCA-s rats would be more affected than SHR, since DOCA-s showed a reduced response to an external NO donor. It is important to note that vascular response to AD was reduced only in terminal arterioles; A2 arteriolar and venular response was not affected by hypertension (Fig. 5b). BK is considered as one of the most potent relaxing agents known; however, in our experiments, the maximal dilation induced by BK in normotensive rats was lower (40%) than that induced by other drugs. Not all vessels (mainly from normotensive rats) responded to BK, some of them showed dilation without flow or inclusive reversion of flow direction. This suggested that BK action could be exerted in upstream vessels and is consistent with the results of Ye and Colquhoun (1998), showing that muscle resting oxygen consumption in perfused rat hindlimbs may be largely controlled by the ratio of nutritive to non-nutritive flow. To test this possibility, in an additional series of experiments, vascular reactivity to AD and BK was compared in three
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order branches of arterioles and venules. Dilation induced by BK in A2 arterioles of WK rats was significantly higher than in A3 and A4, while in SHR, the dilation was similar in the three branches studied. This BK effect was not seen in the three venular branches studied in WK and SHR. AD reactivity did not show differences between branches of arteries or veins. This supports the idea that some vasodilator drugs like BK exert a nonnutritive flow effect, whereas AD may direct the flow to capillary exchange functions. Jones et al. (1995) have reported similar results with AD, which produces the greatest dilatory effect in the smallest vessels of the vascular tree. In SHR, the preferential action of BK on A2 arterioles is lost or masked by a lower arteriolar dilatory capacity of A2 vessels. In conclusion, resting arteriolar diameters and vascular reactivity to vasodilator drugs were similar in EDL and soleus muscles of normotensive and hypertensive rats, suggesting that fiber type composition or oxidative capacity of the skeletal muscle has no influence on hypertension development at precapillary level. Vascular reactivity to vasoactive drugs depends on the hypertension model, i.e., SHR rats showed good response to an external NO donor SNP, while the reactivity of DOCA-s rats was reduced to endothelium-dependent drugs and SNP, probably implying that alterations in the synthesis or release of intracellular mediators interfere to a variable degree with vasodilator mechanisms. Vascular reactivity to BK depends on the arteriolar branch order, exerting a preferential action on upstream vessels, probably deviating blood flow to nonnutritive routes; this effect is lost in hypertensive rats. AD actions on terminal arterioles may ensure that blood flow satisfies local tissue necessities.
Acknowledgments We acknowledge grants No. 09-11-4571-2000 and 0933-4367-1999, from the ‘‘Consejo de Desarrollo Cientı´fico y Humanı´stico’’, Universidad Central de Venezuela.
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Muscle arteriolar and venular reactivity in two models of hypertensive rats Mercedes Losadaa, Sonia H. Torresb,*, Noelina Herna´ndezb, Marisela Lippoa, Amparo Sosaa a
Escuela J. M. Vargas, Facultad de Medicina, Universidad Central de Venezuela, Venezuela Instituto de Medicina Experimental, Seccio´n para el Estudio de la Adaptacio´n Muscular (SEAM), Universidad Central de Venezuela, Apdo. 50587 Sabana Grande, Caracas 1050-A, Venezuela
b
Received 16 November 2004 Available online 20 April 2005
Abstract This study was designed to test if skeletal muscle fiber composition could influence vascular response in hypertensive rats. Muscle vessels were observed by intravital microscopy in anesthetized rats and changes in diameter were measured after local administration of endothelium-dependent and -independent vasodilators. Vascular reactivity was compared in two models of hypertension deoxicorticosterone acetate and salt load (DOCA-s) hypertensive rats and spontaneously hypertensive rats (SHR). The muscles used were: the fast-twitch glycolytic muscle, extensor digitorum longus (EDL), and the slow-twitch oxidative, soleus muscle. Maximal dilation induced by vasoactive drugs was of similar magnitude in EDL and soleus arterioles. Terminal arteriole reactivity to acetylcholine and adenosine was blunted in EDL (35% and 49% reduction, respectively) and soleus muscles (42% and 34% reduction, respectively) of SHR compared with Wistar Kyoto rats. Reactivity of DOCA-s rats to acetylcholine, adenosine, and sodium nitroprusside was reduced by 38%, 50%, 39% in EDL third- and fourthorder arterioles and by 30%, 38%, 38% in soleus fourth-order arterioles, respectively. These studies show that hypertension probably induced similar vascular changes in both muscles studied. Vascular reactivity is blunted for some vasodilator drugs and is more affected in DOCA-s rats. In addition, a preferential action for bradykinin was observed on upstream arterioles but not on venules. This effect was not observed for adenosine. D 2005 Published by Elsevier Inc. Keywords: Skeletal muscle; Adenosine; Bradykinin; SHR; DOCA-salt; Arterioles; Hypertension
Introduction It is well established that increased resistance in large and small vessels is characteristic of essential hypertension and results in elevation of blood pressure. The study of diverse vascular beds and segments of the circulation has shown that morphological and physiological adaptation to hypertension may be different in macrovessels and microvessels of different vascular beds, between large and small arteries, and even within microvessels of different diameters (Henrich et al., 1978). Although it is generally assumed that the increase in skeletal muscle vascular resistance occurs to a similar
* Corresponding author. Fax: +58 212 730 7503. E-mail addresses: [email protected], [email protected] (S.H. Torres). 0026-2862/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.mvr.2005.03.004
degree in all muscles (Sexton et al., 1990), some evidence suggests that may not be uniform and varies with the fiber type composition of the muscles. Extensor digitorum longus (EDL) muscle is predominantly formed by type II fibers, and in deoxicorticosterone acetate and salt load (DOCA-s) hypertensive rats, the proportion of IIb fibers is increased; on the contrary, in soleus muscle of control and hypertensive rats, there is a higher proportion of type I and IIa fibers that are highly oxidative (Herna´ndez et al., 1996). Doppler flow probes on normotensive rats have shown impaired sympathetic vasoconstriction in fast-twitch gastrocnemius-plantaris muscles, but good contractions in vessels of the slow-twitch soleus muscle, suggesting that, under physiological conditions, the extent of sympathetic vasoconstriction may depend on the fiber type composition of the active muscle (Hansen et al., 2000). In several models of hypertensive rats, microvascular rarefaction appears to occur in skeletal muscles composed of a high
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proportion of fast-twitch glycolytic fibers like cremaster, gracilis, and EDL muscles (Chen et al., 1981; Green et al., 1992). However, it has been reported by Herna´ndez et al. (1996) that there is a significant reduction in capillary number per fiber in soleus muscle, which is mainly formed by slow-twitch oxidative fibers. Blood flow is linearly related to skeletal muscle oxygen consumption over a wide range of metabolic rates, lending support to the idea that local control of vascular resistance in skeletal muscle is driven by metabolic processes (Aaker and Laughlin, 2002; Murrant and Sarelius, 2000). It has been shown that changes in metabolism lead to closely related changes in blood flow, via the vasodilatory actions of products of muscle contraction metabolites, like inorganic ions, purines, etc. (Gorczynski et al., 1978). Blood flow and metabolism coupling can be extremely local in nature since arteriolar dilatation is observed only in the vicinity of the contracting muscle fiber. Moreover, three generations of arterioles upstream from capillaries dilate when the muscle fibers increase their metabolism. Capillaries communicate to upstream arterioles via conducted signals, initiated at the capillary level and transmitted through gap –junction communications (Berg et al., 1997). In the present study, vascular reactivity of vessels from muscles having different oxidative capacities was tested. The effect of vasodilator drugs: acetylcholine (ACh), adenosine (AD), sodium nitroprusside (SNP), and bradykinin (BK) was evaluated on precapillary arterioles and postcapillary venules from fast-twitch glycolytic (EDL) and slow-twitch oxidative (soleus) muscles in two models of hypertension, spontaneously hypertensive rats (SHR) and DOCA-s rats. These drugs were selected in order to compare hypertension effect on different mediators.
Materials and methods Experimental animals 16 male Sprague – Dawley rats were made hypertensive by subcutaneous injections of deoxicorticosterone acetate (20 mg/kg) daily for 10 weeks, supplemented with oral salt loading by replacing drinking water with 1% NaCl solution (DOCA-s rats). 20 control rats (C) were injected with 0.20 mL 0.9% NaCl solution following the same scheme as DOCA-s rats and drank tap water. Genetic hypertensive rats: 28 male age-matched SHR, and as controls, 27 Wistar Kyoto (WK) rats were used in these studies. Hypertensive and normotensive rats’ weight was 280 –350 g. Animals were provided with regular rat chow and housed with controlled light (12-h light –dark cycle) and temperature conditions. All rats were taken from a colony maintained at the Universidad Central de Venezuela, Instituto Nacional de Higiene Rafael Rangel, Caracas, Venezuela.
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Vascular reactivity On the day of the experiment, rats were anesthetized (pentobarbital sodium 40 mg/kg, chloral hydrate 200 mg/ kg, i.p.) The trachea was cannulated to ensure a patent airway; the left carotid artery and jugular vein were cannulated for arterial pressure measurement and for the administration of supplemental anesthesia (5 mg/kg bolus, when required), respectively. Once the initial surgery was completed, the rat was placed on a circulation-heated Perspex stage and the right EDL or soleus muscle was prepared for television microscopy observations by retracting the tibialis anterior and peroneal muscles. Throughout the whole preparation and experimental time, the muscles were bathed with a de-oxygenated Krebs –Henseleit solution (NaCl 131.9 mM; KCl 4.7 mM; MgSO4I7H2O 1.17 mM; CaCl2I2H2O 2 mM; NaHCO3 22 mM) maintained at 32 – 34-C, equilibrated with 95% N2 – 5% CO2, pH 7.35. The vessels were classified according to their branching order beginning at the capillary level: terminal (4th order: A4) and preterminal (3rd order: A3 and 2d order: A2) arterioles. A similar classification was used for postcapillary venules (V4, V3, V2). The surfaces of EDL and soleus muscles were observed with a water-immersion objective lens (Zeiss 40) by an intravital microscope (Zeiss) epiilluminated with fiber optics (Schott KL1500). Vessels’ diameters were observed by a video camera (Sonny CCDIris) and recorded to a videotape (JVC). Images from the camera were also displayed in real time on a television monitor and diameter changes were measured with a calibrated reticule generator (Artek Systems Corp.) displayed on the television monitor. Molar concentrations of vasoactive drugs that produced 50% of the maximal dilation (ED50 values) were estimated in hypertensive rats; there was a shift to the right in the EC50 in A4 arterioles in SHR, but in A3 vessels, the response was the same in WK and SHR. The doses used were: ACh: 10 5 mol/L; AD: 10 5 mol/L; BK: 10 7 mol/L, and SNP: 10 5 mol/L. Drugs were obtained from Sigma. Luminal diameters (width of red cell column) were measured at rest and after randomized topical administration of 1 mL of drugs applied to the exposed EDL or soleus muscles under the objective lens by syringe, at a temperature comparable with the superfusate, without interruption of superfusate flow. Immediately after drug application, changes in diameter were measured and recorded to take in the peak response. Each vessel was given 10 min to recover to resting diameter between drug applications. The total duration of observations during an experiment did not exceed 2.5 h. Blood pressure was continuously monitored to verify that drugs applied locally did not have a systemic effect. The results were expressed as percentage of vessel change from resting diameter (D REST) to maximal dilation (D MAX) obtained with the applied drug (D MAX D REST/D REST 100). The diameter measurements were made in two vessels per rat and ‘‘n’’ represents the number of rats.
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Statistical analyses Data were analyzed by the nonpaired Student’s ‘‘t’’ test or an analysis of variance (ANOVA) with Bonferroni test corrections for multiple comparisons as appropriate. The statistical significance of differences between groups was based on a probability level of P 0.05. Figures represent mean T SEM and the table represents means T SD.
Results Mean arterial blood pressure of hypertensive rats under general anesthesia was significantly higher (167 T 4 and 177 T 7 mm Hg for SHR and DOCA-s, respectively) compared with WK (114 T 8 mm Hg) and C (113 T 7 mm Hg). Vessel resting diameters from EDL and soleus muscles in hypertensive rats were not significantly different from control rats (Table 1). EDL and soleus reactivity The possibility that vascular reactivity could depend on the fiber type composition of the muscle was tested. Dilation induced by vasoactive drugs was of similar magnitude in A3 and A4 vessels from both muscles (EDL and soleus) of normotensive rats (Control Sprague – Dawley and WK, white bars, Figs. 1 –4). Also, dilation of A3 and A4 vessels to the drugs administrated was not significantly different between EDL and soleus muscles
Fig. 1. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A4 vessels. (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. **P < 0.01. WK (n = 9) vs. SHR (n = 10).
from hypertensive rats (DOCA-s and SHR, black bars, Figs. 1– 4). Vascular reactivity of SHR vs. WK
Table 1 Mean T SD of resting arteriolar diameters (Am) in normotensive (n = 12) and hypertensive (n = 14) rats EDL (extensor digitorum longus) WK
SHR
C DOCA-s
A2 A3 A4 V2 V3 V4 A2 A3 A4 V2 V3 V4 A3 A4 A3 A4
9.37 T 7.07 T 5.42 T 13.11 T 8.42 T 5.98 T 8.64 T 6.41 T 5.18 T 13.51 T 8.77 T 5.82 T 6.20 T 5.30 T 5.83 T 4.34 T
1.27 0.80 0.36 2.29 1.84 0.82 1.32 0.69 1.01 3.53 2.12 0.55 0.35 1.05 0.56 0.78
Soleus
6.09 T 0.74 5.60 T 0.66
6.47 T 0.81 5.60 T 0.57
5.32 4.79 6.19 4.44
T T T T
WK = Wistar Kyoto. SHR = spontaneously hypertensive. DOCA-s = deoxicorticosterone acetate and salt load treated SD rats. C = Sprague – Dawley (SD) control rats.
0.49 0.47 0.59 0.43
In EDL muscle, the responses to ACh and AD were significantly reduced in A4 (Fig. 1a) and A3 (Fig. 2a) vessels in the SHR compared with WK rats. In soleus muscle of SHR, also the responses to ACh and AD were reduced in the SHR, in A4 (Fig. 1b) and A3 arterioles (Fig. 2b). However, there were no significant differences between WK and SHR in the dilation induced by BK and SNP, neither in the muscles nor in the arteriole order studied (Figs. 1 and 2). Vascular reactivity of DOCA-s vs. C A4 and A3 vessels of EDL muscle in DOCA-s rats showed reduced responses to ACh, AD, and SNP, but normal to BK (Figs. 3a and 4a). A4 arteriole responses to the drugs in soleus muscle were similar to those seen in EDL muscle (Fig. 3b). A3 soleus vessel dilations to ACh, SNP, and BK in DOCA-s were similar to normotensive rats but significantly lower to AD (Fig. 4b). Arterioles from DOCA-s rats seem to be either more affected by hypertension or to have a different mechanism underlying
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Fig. 2. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A3 vessels. (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. *P < 0.05, **P < 0.01. WK (n = 9) vs. SHR (n = 10).
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Fig. 3. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A4 vessels (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. *P < 0.05 **P < 0.01. C (n = 11) vs. DOCA-salt (n = 8).
vasoconstriction, since SHR arterioles showed good response to two of the four drugs used, and were able to react to SNP; in contrast, DOCA-s arterioles showed reduced responses to ACh, AD, and SNP. BK effect in three arteriolar branches It is noteworthy that vascular responses to BK in both muscles and A3 and A4 arterioles were similar in hypertensive and normotensive rats (regardless of the model of hypertension). However, the dilation induced by BK in normotensive rats was lower (40%) than the dilation induced by the other drugs. To test the possibility that some drugs could exert its effect on upstream vessels, two local mediators with physiological actions were compared (BK and AD). In an additional group of WK and SHR rats, BK and AD effects were assessed in fourth-, third-, and second-order vessel branches of EDL muscle; arteriolar and venular responsiveness was also evaluated. Fig. 5a shows a preferential action of BK on A2 vessels in WK rats but this effect is not seen in venules. Dilation induced by BK on SHR was of similar magnitude in all vessel order studied; however, it was significantly reduced in A2 and all venular branches, compared with normotensive rats. AD did not show the preferential action in A2 arterioles exerted by BK in WK rats, and vascular reactivity was of similar magnitude
Fig. 4. Bar graphs show increase (%) of arteriolar diameters induced by topical vasodilator drugs observed in A3 vessels (a) EDL and (b) soleus muscles. Results are expressed as mean T SEM. *P < 0.05 **P < 0.01. C (n = 11) vs. DOCA-salt (n = 8).
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Vascular reactivity
Fig. 5. Effect of BK (a) and AD (b) on three order branches of vessels in EDL muscle of WK (n = 8) and SHR (n = 8) rats. The solid lines show that dilation induced by BK exerts a preferential effect (ANOVA) on A2 arterioles from WK (P < 0.01). Differences between dilation induced by BK and AD on arterioles (A) and venules (V) from WK vs. SHR (t test **P < 0.01) are shown. Results are expressed as mean T SEM.
in all arteriolar and venular branches (Fig. 5b). AD dilation was significantly reduced only for SHR A3 and A4, arterioles compared with WK rats.
Discussion Vessel diameters Folkow (1990) suggested that genetic hypertension involved arterial medial hypertrophy with a resulting encroachment on the lumen. In consequence, vascular resistance increases as a result of a structural change. In the present study, resting lumen diameters measured by intravital microscopy were similar to those reported by Thomas et al. (1998) in Wistar rats’ EDL muscle, and did not differ between hypertensive (SHR and DOCA-s) and normotensive rats (WK and C). Normal resting diameters in hypertensive rats compared with normotensive have been also reported for skeletal muscle microcirculation in other models of hypertension (Lombard et al., 2000; Prewitt et al., 1984). Our results suggest that vasoconstriction of the terminal portion of the arterial tree is not involved in the increase of the peripheral resistance observed in hypertension.
This study was focused on precapillary arterioles because these small vessels under physiological conditions act as metabolic sensors of muscle activity, regulating upstream blood flow to match muscle necessities (Berg et al., 1997; Gorczynski et al., 1978; Murrant and Sarelius, 2000). In addition, third- and fourth-order arterioles contribute to arterial vascular resistance in resting muscle (Dodd and Johnson, 1991). It seems reasonable that responsiveness of the skeletal muscle arterioles varies with muscle fiber type or oxidative capacity. There is evidence that supports this idea in normotensive rats, i.e., hindlimb unloading results in a diminished responsiveness of skeletal muscle arterioles from highly oxidative skeletal muscle to vasodilators and diminished vasoconstrictor capacity in low oxidative muscle (McCurdy et al., 2000). Vasodilation in response to AD and SNP is greater in gastrocnemius muscle arterioles than soleus muscle arterioles, and maximal vasodilator response to ACh is greater in soleus and red gastrocnemius muscle arterioles than in diaphragmatic and white portion of gastrocnemius arterioles (Aaker and Laughlin, 2002; Wunsch et al., 2000). Our results in two strains of normotensive (WK and Sprague – Dawley) rats did not show differences in vascular reactivity associated to muscle fiber type. In both strains, EDL muscle is predominantly formed by type II fibers, with high glycolitic capacity, and soleus muscle has a high oxidative capacity and a high proportion of type I fibers. Similar results have been found by others (Aaker and Laughlin, 2002; Jones et al., 1995), who studied vascular reactivity to AD in larger vessels (A1 and A2). In hypertension, elevated peripheral vascular resistance and arteriolar rarefaction are associated with metabolic disorders. It has been shown that total hind quarter flow is lower (76%) in SHR than WK (Sexton et al., 1990) and basal oxygen consumption, glucose uptake, and lactate production are higher in SHR (Ye and Colquhoun, 1998). Our results show that vascular reactivity to the drugs was not significantly different between EDL and soleus muscles; in consequence, neither difference in fiber type composition nor oxidative capacity of skeletal resting muscles seems to be the primary determinants of vascular reactivity in hypertensive rats. Bohlen (1986) suggested that capillary control of pressure excess in hypertensive rats involves propagated signals that elevate arteriolar resistance to compensate tissue overperfusion. This implies that vascular function might be normalized in terminal arterioles by mechanisms that are taking place in larger vessels. Our results do not support this hypothesis and suggest that attenuated vascular responses might be related to alterations in synthesis or release of intracellular mediators like nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF), which interfere to a variable degree with vasodilator mechanisms in the skeletal muscle microcirculation, and are dependent on the hypertension model but not in muscle fiber type.
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Endothelium-dependent relaxations are achieved by a combination of endothelium-derived prostacyclin, NO, and EDHF. Relaxations induced by ACh (Shimokawa et al., 1996) and BK (Cohen and Vanhoutte, 1995) are mediated by NO and EDHF, and AD is known to cause hypotension via prostaglandin synthesis (Ray et al., 2002). NO has been proposed to play a role in the pathogenesis of hypertension (Arnal et al., 1995). Our experiments showed good reactivity of A3 and A4 arterioles of SHR to SNP in both EDL and soleus muscles, indicating that the smooth muscle pathway associated to NO effect is not altered by genetic hypertension, since these vessels are able to react to an external NO donor. This does not seem to be the case for DOCA-s rats, in which vascular reactivity to SNP is significantly blunted, demonstrating an important difference between hypertension models. ACh response is reduced in both hypertension models, including SHR arterioles that showed good response to SNP. There is evidence that endothelium-derived NO predominantly mediates AChinduced dilation, and participation of EDHF becomes apparent only after inhibition of NO synthesis (Huang et al., 2000). When one mediator is impaired by hypertension, an alternative compensatory pathway is activated; probably, this is the case in our experiments. The relative contribution of NO and EDHF is not clear, it has been suggested that the two mediators are interrelated, either by sharing common mechanisms or by reinforcing or otherwise modulating the action of the other (Cohen and Vanhoutte, 1995). Ray et al. (2002) showed that in vivo vasodilation in skeletal muscle evoked by AD is dependent on prostaglandin synthesis by endothelial cells, which then stimulates NO synthesis. Our experiments showed a reduced AD response in both hypertension models. This could be associated to insensitivity of the receptor to AD (Liu et al., 2002), and lower ability to initiate the prostaglandin synthesis or reduced prostaglandin stimulation of NO synthesis (Ray et al., 2002). In the latter situation, DOCA-s rats would be more affected than SHR, since DOCA-s showed a reduced response to an external NO donor. It is important to note that vascular response to AD was reduced only in terminal arterioles; A2 arteriolar and venular response was not affected by hypertension (Fig. 5b). BK is considered as one of the most potent relaxing agents known; however, in our experiments, the maximal dilation induced by BK in normotensive rats was lower (40%) than that induced by other drugs. Not all vessels (mainly from normotensive rats) responded to BK, some of them showed dilation without flow or inclusive reversion of flow direction. This suggested that BK action could be exerted in upstream vessels and is consistent with the results of Ye and Colquhoun (1998), showing that muscle resting oxygen consumption in perfused rat hindlimbs may be largely controlled by the ratio of nutritive to non-nutritive flow. To test this possibility, in an additional series of experiments, vascular reactivity to AD and BK was compared in three
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order branches of arterioles and venules. Dilation induced by BK in A2 arterioles of WK rats was significantly higher than in A3 and A4, while in SHR, the dilation was similar in the three branches studied. This BK effect was not seen in the three venular branches studied in WK and SHR. AD reactivity did not show differences between branches of arteries or veins. This supports the idea that some vasodilator drugs like BK exert a nonnutritive flow effect, whereas AD may direct the flow to capillary exchange functions. Jones et al. (1995) have reported similar results with AD, which produces the greatest dilatory effect in the smallest vessels of the vascular tree. In SHR, the preferential action of BK on A2 arterioles is lost or masked by a lower arteriolar dilatory capacity of A2 vessels. In conclusion, resting arteriolar diameters and vascular reactivity to vasodilator drugs were similar in EDL and soleus muscles of normotensive and hypertensive rats, suggesting that fiber type composition or oxidative capacity of the skeletal muscle has no influence on hypertension development at precapillary level. Vascular reactivity to vasoactive drugs depends on the hypertension model, i.e., SHR rats showed good response to an external NO donor SNP, while the reactivity of DOCA-s rats was reduced to endothelium-dependent drugs and SNP, probably implying that alterations in the synthesis or release of intracellular mediators interfere to a variable degree with vasodilator mechanisms. Vascular reactivity to BK depends on the arteriolar branch order, exerting a preferential action on upstream vessels, probably deviating blood flow to nonnutritive routes; this effect is lost in hypertensive rats. AD actions on terminal arterioles may ensure that blood flow satisfies local tissue necessities.
Acknowledgments We acknowledge grants No. 09-11-4571-2000 and 0933-4367-1999, from the ‘‘Consejo de Desarrollo Cientı´fico y Humanı´stico’’, Universidad Central de Venezuela.
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