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Myogenic responses of isolated adipose tissue arterioles
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Microvascular Research 66 (2003) 140 –146
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Myogenic responses of isolated adipose tissue arterioles Samir S. Undavia, Valentina Berger, Gabor Kaley, and Edward J. Messina* Department of Physiology, New York Medical College, Valhalla, NY 10595, USA Received 14 October 2002
Abstract Previous in vivo studies indicate that vascular autoregulation does take place in adipose tissue. We tested the hypothesis that adipose tissue arterioles can develop a myogenic response to increases in transmural pressure. Arterioles, isolated from the inguinal fat pad of male Wistar rats, were placed in a microvessel chamber containing a Kreb’s bicarbonate-buffered solution (pH 7.4) gassed with 10% O2 (5% CO2; 85% N2). Vessels were cannulated and pressurized to 100 mm Hg and studied under no-flow conditions. Control diameters were obtained at 100 mm Hg. Changes in arteriolar diameter were observed and measured by television microscopy and videocaliper. Diameters, in response to 20 mm Hg step increases in transmural pressure, were measured before and after removal either of extracellular calcium or of the endothelium, and administration of indomethacin (10⫺5 M) or L-NAME (3 ⫻ 10⫺4 M). Removal of calcium resulted in an increase in control diameter of 81% and completely eliminated the myogenic response. In contrast, administration of indomethacin increased control diameter by 13%. L-NAME significantly enhanced the myogenic response; however, neither endothelium removal nor indomethacin had any significant effect. These results indicate that adipose tissue arterioles are capable of eliciting a myogenic response that could contribute to the regulation of blood flow in vivo. Furthermore, it appears that calcium is essential for the myogenic response and that nitric oxide significantly contributes to the modulation of baseline myogenic tone, as well as the myogenic response. © 2003 Elsevier Inc. All rights reserved. Keywords: Microcirculation; In vitro; Endothelium; Vascular smooth muscle; Nitric oxide; Prostaglandins; Acetylcholine; Sodium nitroprusside; Phenylephrine; Rat; Inguinal fat
Introduction Previous in vivo studies have indicated that adipose tissue is capable of autoregulation (Nielsen and Secher, 1971; Henriksen et al., 1973, 1976). Autoregulation, the inherent ability of a vascular bed to preserve blood flow despite changes in arterial pressure, has been observed in a variety of vascular beds (Schubert and Mulvany, 1999). It is believed that autoregulation serves to maintain capillary hydrostatic pressure and tissue blood flow during periods of blood pressure fluctuation. Although many theories exist to explain the mechanisms of autoregulation, it is thought to be achieved mainly through the balance of two local control mechanisms: vasoactive metabolites and the myogenic response. The myogenic response is characterized as an increase in resistance * Corresponding author. Fax: ⫹1-914-594-4018. E-mail address: [email protected] (E.J. Messina). 0026-2862/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0026-2862(03)00042-6
in response to an increase in transmural pressure (Bayliss, 1902). While the degree of contribution of each mechanism in autoregulation is unknown, it is widely believed that the myogenic response, because of its similarity to the autoregulatory profile, significantly contributes to autoregulation in several vascular beds (Schubert and Mulvany, 1999). In vivo reports of autoregulation in the canine inguinal fat pad suggest that the pressure-dependent myogenic response contributes to autoregulation (Nielsen and Secher, 1971). However, the complexities of an in vivo study prevent a clear determination of the nature of this contribution and therefore an in vitro investigation of the myogenic response of adipose tissue arterioles was undertaken. The aim of our study, therefore, was to investigate the pressure-dependent behavior of adipose tissue-resistance vessels, to test their capacity to elicit a myogenic response, and to characterize this response in vitro. To accomplish this goal, second-order adipose tissue arterioles were isolated, cannulated, and subjected to step increases in pressure be-
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fore and after calcium removal, endothelium removal, and NG-nitro-L-orginine methyl ester (L-NAME) or indomethacin (IND) administration. To minimize possible interference from factors other than pressure that may influence vascular diameter, such as shear stress, our studies were performed under no-flow conditions.
Materials and methods Five to 7-week-old male Wistar rats (average weight: 180 g) were anesthetized with intraperitoneal injections of sodium pentobarbital (Nembutal, 50 mg/kg). A lower abdominal inguinal incision was made exposing the inguinal adipose tissue (2.5 cm2). This tissue was excised and placed in a petri dish containing a cold (0 – 4°C) Mops-buffered physiological salt solution (pH 7.4). This solution contained (in mM) 145 NaCl, 5.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 1.0 NaH2PO4, 5.0 dextrose, 2.0 pyruvate, 0.02 EDTA, and 3.0 3-(N-morpholino)propanesulfonic acid (Mops). The tissue was then pinned to the Silastic (transparent) bottom of the dish and allowed to equilibrate for 15 min. Using microscissors and a dissecting microscope (Olympus), sections of adipose tissue lobules as well as fascia surrounding individual second-order arterioles were removed. Following the excision of a 1- to 2-mm arteriolar segment, the vessel was transferred to a chamber containing glass micropipettes and a bicarbonate-buffered physiological salt solution (see below) at room temperature. As previously described by Sun et al. (1992), the inflow micropipette was connected with silicone tubing to a pressure-servo syringe system (Living Systems, Burlington, VT). The outflow micropipette was connected to silicone tubing to which a three-way stopcock was attached and closed. The suffusion and perfusion solution contained (in mM) 110.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 10.0 dextrose, 24.0 NaHCO3, and 0.02 EDTA and was gassed with 10% O2–5% CO2, balanced with N2 at pH 7.4. Upon mounting one end of the vessel on the inflow micropipette and securing it with a ligature, the intravascular pressure was raised slightly to flush the vessel and expel clotted blood from the free end. The free end was then mounted and secured with a ligature to the outflow micropipette, the attached stopcock was then closed, and intravascular pressure was slowly raised to 100 mm Hg. The suffusion fluid temperature was also slowly increased to 37°C at this time. Thus, in this study, arterioles were examined under no-flow conditions. Vessels were allowed to equilibrate for 1 h, during which myogenic tone developed spontaneously. The total volume of suffusion solution in the vessel chamber and reservoir was 100 ml, and the rate of flow of the suffusate was 40 ml/min. Changes in diameter of the vessels were observed and measured with television microscopy and a video caliper (Microcirculation Research Institute, Texas A & M Univer-
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sity, College Station, TX), and also recorded (Omega Engineering Inc.). To assess endothelial and arteriolar smooth muscle function, test doses of acetylcholine (ACh 10⫺7 M) and/or sodium nitroprusside (SNP 10⫺6 M), and phenylephrine (PE 10⫺7 M) were administered at a constant transmural pressure of 100 mm Hg. After the drug tests, intravascular pressure was decreased to 20 mm Hg and subsequently increased in 20 mm Hg steps to a maximum of 200 mm Hg. Each pressure step was maintained for at least 15 min, and diameters were recorded once steady-state diameters were achieved. At the conclusion of the experiment, the suffusion solution was changed to a calcium-free physiological salt solution containing ethylene glycol bis(Baminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid (EGTA) (1.0 mM). Arterioles were incubated with this solution for 10 min, and then the pressure steps from 20 to 200 mm Hg were repeated to obtain the passive diameter at each pressure value. A myogenic index was calculated at each pressure interval by a formula according to Halpern et al. (1984), 100 ⫻ 关共⌬r i/r i兲/⌬P兴, where ri signifies the internal radius of arterioles, ⌬ri signifies the change in arteriolar diameter, and ⌬P signifies the change in transmural pressure. To establish the role of arteriolar smooth muscle and the vascular endothelium in response to pressure changes, vessels were subjected to step increases in pressure as described above, from 20 to 140 mm Hg for 15 min at each pressure interval, before and after endothelial denudation. Endothelial removal and arteriolar smooth muscle relaxation were assessed with test doses of ACh 10⫺7 M and SNP 10⫺6 M (endothelium-dependent and endotheliumindependent dilators, respectively) before and after endothelial denudation. Smooth muscle constrictor activity was assessed with PE 10⫺7 M, and endothelium-independent constrictor agent, before and after endothelial denudation. All drugs were added to the reservoir connected to the vessel chamber and final concentrations are reported. As previously reported (Sun et al., 1992), endothelial denudation was achieved by perfusing the vessel with air. The outflow arteriolar end was untied from the micropipette, and 2 to 3 ml of air was slowly pushed through silicon tubing into the vessel lumen and out the free end. The vessel was then flushed with the suffusate at 20 mm Hg for 30 min, and again remounted onto the outflow micropipette. Intravascular pressure was then raised to 100 mm Hg, and vessels were allowed to equilibrate for 1 h. After a steadystate diameter was obtained, vessels were subject to step increases in transmural pressure from 20 to 140 mm Hg, following the reassessment of vessel responses to ACh 10⫺7, SNP 10⫺6, and PE 10⫺7 M. In two separate protocols, arteriolar diameters in response to step increases in intravascular pressure from 20 to 140 mm Hg were measured before and after the administration of either NG-nitro-L-arginine methyl ester (3 ⫻ 10⫺4
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Fig. 1. Effects of 20 mm Hg step increases in transmural pressure on steady state second-order arteriolar diameters (means ⫾ SE), before (䡩) and after (●) extracellular calcium removal (n ⫽ 6). Diameters of arterioles in the presence of extracellular calcium; diameters of arterioles in the absence of extracellular calcium. *Indicates significant differences between the diameters found in the presence of calcium, versus those found in the absence of calcium (*P ⬍ 0.05).
M) or indomethacin (10⫺5 M) in the chamber solution. In both sets of experiments, responses to ACh 10⫺7 M were recorded before and after drug administration. All salts and chemicals were obtained from J.T. Baker Inc. (Phillipsburg, NJ) and Sigma Chemical Co. (St. Louis, MO). The data are presented as means ⫾ SE. Differences were considered significant at P ⬍ 0.05 and were determined by using analysis of variance (ANOVA) with the Student-Newman-Keuls method, two-way ANOVA for repeated measures with the Student-Newman-Keuls method, and paired t tests, as appropriate.
Results Pressure-diameter relationship with intact endothelium In the presence of calcium, and at a constant transmural pressure of 100 mm Hg, control diameters averaged 75.3 ⫾ 6.7 m. All second-order arterioles (n ⫽ 6), in the presence of calcium, developed active tone in response to step increases in transmural pressure (Fig. 1). In response to a transmural pressure increase from 20 to 40 mm Hg, the vessel diameter increased from 91.3 ⫾ 3.0 to 95.8 ⫾ 5.5 m. However, in response to a 20 mm Hg step increase in transmural pressure from 40 to 60 mm Hg, significant (P ⬍ 0.05) arteriolar constriction from 95.8 ⫾ 5.5 to 81.5 ⫾ 2.0 m was observed. In response to step increases in transmural pressure from 60 to 160 mm Hg, arteriolar diameters remained constant and did not change significantly (81.5 ⫾ 2.0 to 79.2 ⫾ 4.1 m). Beyond 160 mm Hg and up to 200 mm Hg, arteriolar diameter significantly (P ⬍ 0.05) increased from 79.1 ⫾ 4.1 to 126 ⫾ 6.8 m. In these same arterioles, the passive pressure-diameter relationship was obtained by the removal of calcium from
the vessel bath. After calcium removal, control diameters, at a constant transmural pressure of 100 mm Hg, increased significantly (P ⬍ 0.05) to 136 ⫾ 2.7 m from 75.3 ⫾ 6.7 m, an 81% increase in diameter. In response to a 20 mm Hg step increase in transmural pressure from 20 to 40 mm Hg, arteriolar diameter increased significantly (P ⬍ 0.05) from 96.7 ⫾ 1.0 to 120.5 ⫾ 4.6 m. At 40 mm Hg and up to 200 mm Hg, arteriolar diameter continuously increased with each 20 mm Hg increase in pressure. While arteriolar diameters increased from 120.5 ⫾ 4.6 to 140.8 ⫾ 7.4 m in response to the pressure increases, each individual diameter increase was not significantly different from the previous one. In comparison to vessel diameter changes in the presence of calcium, removal of calcium from the vessel bath did not significantly alter arteriolar diameters at 20 mm Hg. However, above 20 mm Hg, and up to 200 mm Hg, arteriolar diameters obtained in the absence of calcium were significantly (P ⬍ 0.05) higher than those obtained in the presence of calcium in the vessel bath. Arteriolar diameters, before and after removal of calcium from the vessel bath, were observed and recorded during step increases in transmural pressure from 20 to 200 mm Hg. However, in subsequent experimental protocols with endothelium removal and administration of L-NAME and INDO, arteriolar diameters were only recorded within the pressure range of 20 to 140 mm Hg. Our preliminary data indicated (not shown here) a diminished response to the agonists ACh and PE in arterioles subjected to transmural pressure exceeding 140 mm Hg. This observation is consistent with pressure-induced damage to both endothelium and vascular smooth muscle. In contrast, arteriolar responses to these agonists were not significantly different following step increases in transmural pressure to 140 mm Hg. Therefore, protocols were revised in order to avoid endothelial and vascular smooth muscle damage and experiments in this study were conducted in the pressure range of 20 to 140 mm Hg. Effect of endothelium removal on responses to drugs Control diameters were recorded at a transmural pressure of 100 mm Hg. Responses to ACh, SNP, and PE were also recorded at this pressure. Our preliminary studies indicated that, upon initial pressurization, myogenic tone, as well as the response to agonists, was more easily observed at 100 mm Hg. In additional preliminary studies, arterioles pressurized at 75 mm Hg developed poor spontaneous tone. We therefore concluded that control diameters and agonist responses must be recorded at 100 mm Hg. The presence of a functional endothelium and vascular smooth muscle was assessed by the dilation of arterioles to ACh and SNP, and their constriction in response to PE at 100 mm Hg. In nine experiments, the dilations to ACh (10⫺7 M) and SNP (10⫺6 M) before endothelium removal were 43.8 ⫾ 4.8 and 25.7 ⫾ 3.3 m, respectively. PE
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Fig. 2. Effects of 20 mm Hg step increases in transmural pressure on steady-state second-order arteriolar diameters (means ⫾ SE), before and after endothelium removal (n ⫽ 9). (䡩) Diameters of arterioles with an intact endothelium; (‘) diameters of arterioles with the endothelium removed. Arteriolar diameters measured before and after endothelium removal were not found to be significantly different.
administration resulted in a constriction of ⫺24.6 ⫾ 4.8 m. All of these responses were significantly (P ⬍ 0.05) different from zero. After removal of the endothelium in these nine experiments, ACh produced a constriction of ⫺8.8 ⫾ 1.2 m. In contrast to the change observed in the arteriolar response to ACh, responses to SNP and PE were not significantly altered as a result of endothelium removal (PE, ⫺29 ⫾ 3.7 m; SNP, 22.4 ⫾ 2.6 m). These results indicate that the endothelium was functionally removed, as demonstrated by the change in response to the endothelium-dependent dilator ACh. Furthermore, removal of the endothelium did not result in damage to the vascular smooth muscle, as was demonstrated by the preservation of arteriolar responses to the endothelium-independent agonists, SNP and PE. In our previous study (Sun et al., 1992), histological examination revealed that the endothelium was completely removed by the procedure employed in the present study.
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Fig. 3. The calculated myogenic index for isolated second-order adipose tissue arterioles, before and after endothelium removal. (䡩) Myogenic index for arterioles with an intact endothelium; (‘) myogenic index for arterioles with the endothelium removed.
IND were statistically insignificant, and were found to be 69.6 ⫾ 6.7 and 78.3 ⫾ 1.0 m. Furthermore, administration of IND (n ⫽ 7) did not significantly alter arteriolar responses to increases in transmural pressure (Fig. 4). Control diameters at 100 mm Hg significantly decreased after administration of L-NAME from 70.1 ⫾ 4.6 m before L-NAME to 58.6 ⫾ 7.7 m after. Additionally, in contrast to inhibition of cyclooxygenase, inhibition of nitric oxide synthase by L-NAME (n ⫽ 7) significantly decreased arteriolar diameters at all pressure steps with the exception of 20 mm Hg, the lowest pressure used (Fig. 5). Additionally in this group of vessels, L-NAME produced a significant (P ⬍ 0.05) inhibition of the dilator response to ACh (n ⫽ 7). ACh induced arteriolar dilations before and after administration of L-NAME were 56.14 ⫾ 5.31 and 36.29 ⫾ 3.32 m, respectively.
Pressure-diameter relationship after endothelium removal While control diameters at 100 mm Hg decreased after endothelium removal, these differences in diameters were not statistically significant (63.7 ⫾ 6.7 vs 51.8 ⫾ 7.4 m). Furthermore, arteriolar diameters (n ⫽ 9) obtained at each pressure interval (from 20 to 140 mm Hg) after removal of the endothelium were also not significantly different from those obtained prior to endothelium removal (Fig. 2). The calculated myogenic index for these arterioles is presented in Fig. 3. Pressure-diameter relationship after administration of indomethacin or L-NAME Differences in control diameter at 100 mm Hg before and after inhibition of cyclooxygenase by the administration of
Fig. 4. Effects of 20 mm Hg step increases in transmural pressure on steady-state second-order arteriolar diameters (means ⫾ SE), before and after administration of indomethacin (n ⫽ 7). (䡩) Diameters of arterioles found in the absence of indomethacin; (⽧) diameters of arterioles found in the presence of indomethacin. Arteriolar diameters measured before and after indomethacin administration were not found to be significantly different.
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Fig. 5. Effects of 20 mm Hg step increases in transmural pressure on steady-state second-order arteriolar diameters (means ⫾ SE), before and after administration of L-NAME (n ⫽ 7). (䡩) Diameters of arterioles found in the absence of L-NAME; (䡲) diameters of arterioles found in the presence of L-NAME. Arteriolar diameters measured following administration of L-NAME were found to be significantly different from control (*P ⬎ 0.05) at all pressures except 20 mm Hg.
Discussion The present study has demonstrated the ability of adipose tissue arterioles to constrict in response to an increase in transmural pressure. Our data indicate that this myogenic response is present within the transmural pressure range of 40 to 200 mm Hg. At all pressure steps, except 20 mm Hg, arteriolar diameter was significantly lower than that found in the absence of extracellular calcium, revealing a dependence of the myogenic response on extracellular calcium. Control diameters at 100 mm Hg, as well as arteriolar diameters at each pressure step from 20 to 140 mm Hg, were not significantly altered after removal of the endothelium. These findings indicate that a functional endothelium is not necessary for the development of, nor does it significantly modulate, the increase in vascular smooth muscle tone in response to increases in transmural pressure. Additionally, these observations also indicate that the endothelium does not significantly modulate baseline myogenic tone at 100 mm Hg. Administration of IND, an inhibitor of cyclooxygenase, also failed to significantly alter control diameters at 100 mm Hg or arteriolar responses to step increases in transmural pressure. These results indicate that metabolites of cyclooxygenase do not significantly modulate vascular smooth muscle tone in response to step increases in transmural pressure, nor do they contribute to the modulation of baseline myogenic tone at 100 mm Hg. In contrast, inhibition of nitric oxide synthase significantly decreased control diameters at 100 mm Hg, as well as arteriolar diameters at all pressures except at 20 mm Hg. Therefore, nitric oxide (NO) appears to be not only a significant modulator of baseline myogenic tone but also of responses to changes in transmural pressure. To the best of our knowledge, this is the first
study to demonstrate and characterize, in detail, myogenic responses of arterioles isolated from adipose tissue. Studies of isolated human gluteal adipose tissue arterioles indicate the presence of a myogenic response within the range of 40 to 120 mm Hg (Coats et al., 2001). However, in contrast to our data, at a transmural pressure of 40 mm Hg, no statistically significant difference in diameters was found between human gluteal adipose tissue arterioles bathed in physiological salt solution with calcium (active vessel diameter), and those bathed in a calcium-free physiological salt solution (passive vessel diameter). Thus, these vessels did not develop myogenic tone at 40 mm Hg as did rat adipose tissue arterioles. It is also interesting to point out again that arterioles present in human adipose tissue proximal to the lateral malleolus autoregulate within the range of 70 to 150 mm Hg (Kastrup et al., 1985). Thus, at a pressure of 40 mm Hg, there is no autoregulation, and no myogenic response, suggesting the latter as a major contributor to autoregulation in human adipose tissue. In the rat, however, there may be a need to autoregulate at pressures of 40 mm Hg and above, as our data indicate a significant difference between active and passive diameters at 40 mm Hg, but not at 20 mm Hg (Fig. 1). Coats et al. (2001) also report that, for human adipose tissue arterioles, the greatest degree of constriction in response to a step increase in transmural pressure of 40 mm Hg occurred in the range of 40 to 80 mm Hg. Interestingly, our vessels also demonstrated constriction that was greatest when the transmural pressure was increased from 40 to 60 mm Hg. Coats et al. (2001) found the greatest degree of myogenic tone at a pressure of 120 mm Hg, and the largest vasoconstrictor response between the transmural pressures of 40 to 80 mm Hg. Similarly, our data indicate the largest degree of myogenic tone is present at transmural pressures of 100 –120 mm Hg, and the largest vasoconstrictor response between pressures of 40 and 60 mm Hg. Coronary vessels and adipose tissue vessels seem to behave similarly with respect to the range in which myogenic responses are elicited. Studies of rat coronary vessels show an active myogenic response within the pressure range of 20 to 200 mm Hg (Garcia et al., 1997), while our data of rat inguinal fat pad arterioles indicate an active myogenic response range between 40 and 200 mm Hg. Studies of porcine coronary arterioles indicate an active range of 20 to 140 mm Hg (Kuo et al., 1990). Human coronary arterioles, limited to being studied only within the pressure range of 20 to 100 mm Hg, indicate the presence of an active myogenic response from 40 to 100 mm Hg (Miller et al., 1997). With respect to the range of myogenic activity, skeletal muscle arterioles also behave similarly to inguinal fat pad arterioles. In rat cremaster muscle arterioles, Falcone et al. (1993) report an active myogenic response range of approximately 30 to 230 mm Hg. In adipose tissue, an arteriolar dilation of 43.8 ⫾ 4.8 m (⬃69% change from control) was observed in response to ACh (10⫺7 M). Second-order arterioles isolated from rat
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cremaster muscle, upon administration of ACh (5 ⫻ 10⫺8 M), also dilated 33.3 ⫾ 3.6 m (Huang et al., 1993). Isolated rat coronary arterioles demonstrated slight dilation in response to low concentrations of ACh (10⫺9 to 10⫺7 M). In contrast, higher concentrations of ACh (10⫺6 and 10⫺5 M) resulted in significant vasoconstriction of 16.4 ⫾ 9.3 m in rat coronary arterioles (Szekeres et al., 2001). This observation indicates a similar reactivity to ACh in skeletal muscle and adipose tissue arterioles. In adipose tissue, an arteriolar dilation of 25.7 ⫾ 3.3 m (⬃41% change from control) was observed in response to SNP (10⫺6 M). Arterioles isolated from rat cremaster muscle, upon administration of SNP (10⫺7 M), also dilated 22.4 ⫾ 2.9 m, respectively (Huang et al., 1993). Similarly in rat coronary arterioles with a control diameter of 77.3 ⫾ 6.6 m, SNP (10⫺6 M) elicited a significant dilation of ⬃30 m (Szekeres et al., 2001). Thus, rat skeletal muscle, adipose, and coronary arterioles possess a similar reactivity to SNP. Our data indicate that endothelium removal in adipose tissue arterioles completely abolished dilation to ACh, but not SNP. Furthermore, ACh administration, after endothelium removal, resulted in an arteriolar constriction of ⫺8.8 ⫾ 1.2 m. Similar results were observed in rat skeletal muscle arterioles, as removal of the endothelium abolished arteriolar dilation to ACh (but not SNP), and resulted in a vasoconstriction of ⫺1.3 ⫾ 0.4 m (Huang et al., 1993). In contrast, however, to adipose tissue and skeletal muscle arterioles, removal of the endothelium in isolated porcine coronary arterioles did not significantly change arteriolar constriction to ACh (Kuo et al., 1990). In adipose tissue arterioles, the presence of L-NAME (3 ⫻ 10⫺4 M) resulted in a partial but significant reduction of arteriolar dilation to ACh. In rat skeletal muscle, a significant partial reduction in arteriolar dilation in response to ACh was also observed in the presence of another nitric oxide synthase inhibitor, L-NNA (Schrage et al., 2000). Curiously, the administration of L-NAME (3 ⫻ 10⫺4 M) failed to achieve a complete block of arteriolar dilation to ACh. These results suggest that other mediators contribute to ACh-induced dilations. The contribution of vasodilatory mediators other than nitric oxide was not fully explored. Our preliminary results (not shown) indicate that a further reduction in ACh-induced dilations was seen in the presence of indomethacin and L-NAME, suggesting that vasodilator prostaglandins may potentially contribute to the response. Additionally, the presence of a poorly characterized endothelium-derived hyperpolarizing factor has been shown to dilate vessels in a variety of vascular beds (Busse et al., 2002). Further studies of adipose tissue arterioles are needed to resolve this matter. In response to PE (10⫺7 M), adipose tissue arterioles constricted ⫺24.6 ⫾ 4.8 m (⬃40% change from control diameter). Skeletal muscle arterioles behave similarly, as PE administration also resulted in vasoconstriction (Aaker et al., 2002). We observed significant arteriolar constriction only at concentrations greater than 10⫺7 M. Thus, ␣-adren-
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ergic receptor density and/or sensitivity differ between the two vascular beds. In contrast, in isolated canine coronary arterioles, administration of PE directly to the arteriole did not result in a significant change in diameter (Tiefenbacher et al., 1998). Our data indicate that nitric oxide contributes significantly to baseline myogenic tone in adipose tissue arterioles. Similar results were found in a study of the coronary microcirculation, as administration of L-NNA to rat coronary arterioles significantly decreased arteriolar diameters from control (Szekeres et al., 2001). In response to inhibition of the synthesis of nitric oxide in skeletal muscle, however, variations exist with regards to skeletal muscle tissue location (Aaker et al., 2002; Schrage et al., 2000). Although control diameters at 100 mm Hg, as well as arteriolar diameters at all pressure steps were increased in response to indomethacin administration, these increases were not significant. Similar results have been observed in isolated rat soleus arterioles, as indomethacin administration resulted in an insignificant increase in baseline arteriolar diameters at 80 mm Hg (Schrage et al., 2000). In rat cremaster arterioles subject to step increases in transmural pressure, administration of indomethacin also failed to alter arteriolar diameter at any of the pressure steps, except at 160 mm Hg (Huang et al., 1993). These observations indicate that in adipose tissue arterioles and some skeletal muscle arterioles, a vasoconstrictor cyclooxygenase product is contributing slightly to basal tone. Skeletal muscle and adipose tissue arterioles also present with similar myogenic indices over the pressure range studied (Sun et al., 1994) Removal of the endothelium failed to significantly alter arteriolar diameters at baseline as well as in response to increases in transmural pressure. This finding can be explained on the basis of a simultaneous removal of constrictor and dilator influences. In contrast, however, inhibition of endothelial nitric oxide production, a dilator influence, resulted in a significant decrease in arteriolar diameters. These seemingly contrasting data can be reconciled by considering that the endothelium, at baseline and in response to step changes in transmural pressure, was stimulated to release both vasodilator and vasoconstrictor agents. Thus, by inhibiting a vasodilator, we found significant decreases in arteriolar diameter. The identity of the vasoconstrictor(s) can only be speculated about at the present time. However, in response to indomethacin, arteriolar diameters, while insignificantly, did increase, indicating the production of vasoconstrictor metabolite(s) of cyclooxygenase. Schrage et al. (2000) have also reported, in isolated rat soleus arterioles, insignificant increases in baseline arteriolar diameters upon administration of indomethacin. In summary, we have shown that isolated adipose tissue arterioles have the ability to respond to increases in transmural pressure with a myogenic response. Thus, the myogenic response of adipose tissue arterioles may play an important role in autoregulation of blood flow.
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Perspective Given the 20% incidence of obesity (Mokdad et al., 2001) in the United States, it is surprising that more attention has not yet been paid to the regulation of blood flow in adipose tissue. Especially since fat can represent 18 and 24% of body weight in normal men and women, respectively, and as much as 52 and 74% of body weight in obese men and women, respectively (Leibel et al., 1995). It is our hope that this study demonstrates the feasibility of studying adipose tissue arterioles in vitro, and that more interest will develop in adipose tissue blood flow regulation to parallel the developing interest in the metabolic disturbances that are brought about by obesity.
Acknowledgments This work was supported by the National Institutes of Health Grant PO-I HL-43023.
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Halpern, W., Mongeon, S.A., Root, D.T., 1984. Stress, tension, and myogenic aspects pf small isolated extraparenchymal rat arteries, in: Stephens, N.L. (Ed.), Smooth Muscle Contraction, Dekker, New York, pp. 476 – 456. Henriksen, O., Nielsen, S.L., Paaske, W.P., 1973. Autoregulation of blood flow in human adipose tissue. Acta Physiol. Scand. 89, 531–537. Henriksen, O., Nielsen, S.L., Paaske, W., 1976. Intrinsic regulation of blood flow in adipose tissue. Acta Physiol. Scand. 98, 30 –36. Huang, A., Sun, D., Koller, A., 1993. Endothelial dysfunction augments myogenic arteriolar constriction in hypertension. Hypertension. 22, 913–921. Kastrup, J., Norgaard, T., Parving, H.H., Henriksen, O., Lassen, N.A., 1985. Impaired autoregulation of blood flow in subcutaneous tissue of long-term type 1 (insulin-dependent) diabetic patients with microangiopathy: an index of arteriolar dysfunction. Diabetologia 28, 711–717. Kuo, L., Chilian, W.M., Davis, M.J., 1990. Coronary arteriolar myogenic response is independent of endothelium. Circ. Res. 66, 860 – 866. Leibel, R.L., Rosenbaum, M., Hirsch, J., 1995. Changes in energy expenditure resulting from altered body weight. N. Engl. J. Med. 332, 621– 628. Miller Jr, F.J., Dellsperger, K.C., Gutterman, D.D., 1997. Myogenic constriction of human coronary arterioles. Am. J. Physiol. 273, H257– H264. Mokdad, A.H., Bowman, B.A., Ford, E.S., Vinicor, F., Marks, J.S., Koplan, J.P., 2001. The continuing epidemics of obesity and diabetes in the United States. J. Am. Med. Assoc. 286, 1195–1200. Nielsen, P.A., Secher, N.J., 1971. Autoregulation in adipose tissue. Pressure-flow measurements in inguinal fat pad on heparinized dogs during local hypotension, venous pressure elevation, and reactive hyperaemia. Cardiovasc. Res. 5, 572–576. Schrage, W.G., Woodman, C.R., Laughlin, M.H., 2000. Hindlimb unweighting alters endothelium-dependent vasodilation and ecNOS expression in soleus arterioles. J. Appl. Physiol. 89, 1483–1490. Schubert, R., Mulvany, M.J., 1999. The myogenic response: established facts and attractive hypotheses. Clin. Sci. (Lond.) 96, 313–326. Sun, D., Messina, E.J., Koller, A., Wolin, M.S., Kaley, G., 1992. Endothelium-dependent dilation to L-arginine in isolated rat skeletal muscle arterioles. Am. J. Physiol. 262, H1211–H1216. Sun, D., Kaley, G., Koller, A., 1994. Characteristics and origin of myogenic response in isolated gracilis muscle arterioles. Am. J. Physiol. 266, H1177–H1183. Szekeres, M., Dezsi, L., Nadasy, G.L., Kaley, G., Koller, A., 2001. Pharmacologic inhomogeneity between the reactivity of intramural coronary arteries and arterioles. J. Cardiovasc. Pharmacol. 38, 584 –592. Tiefenbacher, C.P., DeFily, D.V., Chilian, W.M., 1998. Requisite role of cardiac myocytes in coronary alpha1-adrenergic constriction. Circulation 98, 9 –12.
Microvascular Research 66 (2003) 140 –146
www.elsevier.com/locate/ymvre
Myogenic responses of isolated adipose tissue arterioles Samir S. Undavia, Valentina Berger, Gabor Kaley, and Edward J. Messina* Department of Physiology, New York Medical College, Valhalla, NY 10595, USA Received 14 October 2002
Abstract Previous in vivo studies indicate that vascular autoregulation does take place in adipose tissue. We tested the hypothesis that adipose tissue arterioles can develop a myogenic response to increases in transmural pressure. Arterioles, isolated from the inguinal fat pad of male Wistar rats, were placed in a microvessel chamber containing a Kreb’s bicarbonate-buffered solution (pH 7.4) gassed with 10% O2 (5% CO2; 85% N2). Vessels were cannulated and pressurized to 100 mm Hg and studied under no-flow conditions. Control diameters were obtained at 100 mm Hg. Changes in arteriolar diameter were observed and measured by television microscopy and videocaliper. Diameters, in response to 20 mm Hg step increases in transmural pressure, were measured before and after removal either of extracellular calcium or of the endothelium, and administration of indomethacin (10⫺5 M) or L-NAME (3 ⫻ 10⫺4 M). Removal of calcium resulted in an increase in control diameter of 81% and completely eliminated the myogenic response. In contrast, administration of indomethacin increased control diameter by 13%. L-NAME significantly enhanced the myogenic response; however, neither endothelium removal nor indomethacin had any significant effect. These results indicate that adipose tissue arterioles are capable of eliciting a myogenic response that could contribute to the regulation of blood flow in vivo. Furthermore, it appears that calcium is essential for the myogenic response and that nitric oxide significantly contributes to the modulation of baseline myogenic tone, as well as the myogenic response. © 2003 Elsevier Inc. All rights reserved. Keywords: Microcirculation; In vitro; Endothelium; Vascular smooth muscle; Nitric oxide; Prostaglandins; Acetylcholine; Sodium nitroprusside; Phenylephrine; Rat; Inguinal fat
Introduction Previous in vivo studies have indicated that adipose tissue is capable of autoregulation (Nielsen and Secher, 1971; Henriksen et al., 1973, 1976). Autoregulation, the inherent ability of a vascular bed to preserve blood flow despite changes in arterial pressure, has been observed in a variety of vascular beds (Schubert and Mulvany, 1999). It is believed that autoregulation serves to maintain capillary hydrostatic pressure and tissue blood flow during periods of blood pressure fluctuation. Although many theories exist to explain the mechanisms of autoregulation, it is thought to be achieved mainly through the balance of two local control mechanisms: vasoactive metabolites and the myogenic response. The myogenic response is characterized as an increase in resistance * Corresponding author. Fax: ⫹1-914-594-4018. E-mail address: [email protected] (E.J. Messina). 0026-2862/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0026-2862(03)00042-6
in response to an increase in transmural pressure (Bayliss, 1902). While the degree of contribution of each mechanism in autoregulation is unknown, it is widely believed that the myogenic response, because of its similarity to the autoregulatory profile, significantly contributes to autoregulation in several vascular beds (Schubert and Mulvany, 1999). In vivo reports of autoregulation in the canine inguinal fat pad suggest that the pressure-dependent myogenic response contributes to autoregulation (Nielsen and Secher, 1971). However, the complexities of an in vivo study prevent a clear determination of the nature of this contribution and therefore an in vitro investigation of the myogenic response of adipose tissue arterioles was undertaken. The aim of our study, therefore, was to investigate the pressure-dependent behavior of adipose tissue-resistance vessels, to test their capacity to elicit a myogenic response, and to characterize this response in vitro. To accomplish this goal, second-order adipose tissue arterioles were isolated, cannulated, and subjected to step increases in pressure be-
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fore and after calcium removal, endothelium removal, and NG-nitro-L-orginine methyl ester (L-NAME) or indomethacin (IND) administration. To minimize possible interference from factors other than pressure that may influence vascular diameter, such as shear stress, our studies were performed under no-flow conditions.
Materials and methods Five to 7-week-old male Wistar rats (average weight: 180 g) were anesthetized with intraperitoneal injections of sodium pentobarbital (Nembutal, 50 mg/kg). A lower abdominal inguinal incision was made exposing the inguinal adipose tissue (2.5 cm2). This tissue was excised and placed in a petri dish containing a cold (0 – 4°C) Mops-buffered physiological salt solution (pH 7.4). This solution contained (in mM) 145 NaCl, 5.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 1.0 NaH2PO4, 5.0 dextrose, 2.0 pyruvate, 0.02 EDTA, and 3.0 3-(N-morpholino)propanesulfonic acid (Mops). The tissue was then pinned to the Silastic (transparent) bottom of the dish and allowed to equilibrate for 15 min. Using microscissors and a dissecting microscope (Olympus), sections of adipose tissue lobules as well as fascia surrounding individual second-order arterioles were removed. Following the excision of a 1- to 2-mm arteriolar segment, the vessel was transferred to a chamber containing glass micropipettes and a bicarbonate-buffered physiological salt solution (see below) at room temperature. As previously described by Sun et al. (1992), the inflow micropipette was connected with silicone tubing to a pressure-servo syringe system (Living Systems, Burlington, VT). The outflow micropipette was connected to silicone tubing to which a three-way stopcock was attached and closed. The suffusion and perfusion solution contained (in mM) 110.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 10.0 dextrose, 24.0 NaHCO3, and 0.02 EDTA and was gassed with 10% O2–5% CO2, balanced with N2 at pH 7.4. Upon mounting one end of the vessel on the inflow micropipette and securing it with a ligature, the intravascular pressure was raised slightly to flush the vessel and expel clotted blood from the free end. The free end was then mounted and secured with a ligature to the outflow micropipette, the attached stopcock was then closed, and intravascular pressure was slowly raised to 100 mm Hg. The suffusion fluid temperature was also slowly increased to 37°C at this time. Thus, in this study, arterioles were examined under no-flow conditions. Vessels were allowed to equilibrate for 1 h, during which myogenic tone developed spontaneously. The total volume of suffusion solution in the vessel chamber and reservoir was 100 ml, and the rate of flow of the suffusate was 40 ml/min. Changes in diameter of the vessels were observed and measured with television microscopy and a video caliper (Microcirculation Research Institute, Texas A & M Univer-
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sity, College Station, TX), and also recorded (Omega Engineering Inc.). To assess endothelial and arteriolar smooth muscle function, test doses of acetylcholine (ACh 10⫺7 M) and/or sodium nitroprusside (SNP 10⫺6 M), and phenylephrine (PE 10⫺7 M) were administered at a constant transmural pressure of 100 mm Hg. After the drug tests, intravascular pressure was decreased to 20 mm Hg and subsequently increased in 20 mm Hg steps to a maximum of 200 mm Hg. Each pressure step was maintained for at least 15 min, and diameters were recorded once steady-state diameters were achieved. At the conclusion of the experiment, the suffusion solution was changed to a calcium-free physiological salt solution containing ethylene glycol bis(Baminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid (EGTA) (1.0 mM). Arterioles were incubated with this solution for 10 min, and then the pressure steps from 20 to 200 mm Hg were repeated to obtain the passive diameter at each pressure value. A myogenic index was calculated at each pressure interval by a formula according to Halpern et al. (1984), 100 ⫻ 关共⌬r i/r i兲/⌬P兴, where ri signifies the internal radius of arterioles, ⌬ri signifies the change in arteriolar diameter, and ⌬P signifies the change in transmural pressure. To establish the role of arteriolar smooth muscle and the vascular endothelium in response to pressure changes, vessels were subjected to step increases in pressure as described above, from 20 to 140 mm Hg for 15 min at each pressure interval, before and after endothelial denudation. Endothelial removal and arteriolar smooth muscle relaxation were assessed with test doses of ACh 10⫺7 M and SNP 10⫺6 M (endothelium-dependent and endotheliumindependent dilators, respectively) before and after endothelial denudation. Smooth muscle constrictor activity was assessed with PE 10⫺7 M, and endothelium-independent constrictor agent, before and after endothelial denudation. All drugs were added to the reservoir connected to the vessel chamber and final concentrations are reported. As previously reported (Sun et al., 1992), endothelial denudation was achieved by perfusing the vessel with air. The outflow arteriolar end was untied from the micropipette, and 2 to 3 ml of air was slowly pushed through silicon tubing into the vessel lumen and out the free end. The vessel was then flushed with the suffusate at 20 mm Hg for 30 min, and again remounted onto the outflow micropipette. Intravascular pressure was then raised to 100 mm Hg, and vessels were allowed to equilibrate for 1 h. After a steadystate diameter was obtained, vessels were subject to step increases in transmural pressure from 20 to 140 mm Hg, following the reassessment of vessel responses to ACh 10⫺7, SNP 10⫺6, and PE 10⫺7 M. In two separate protocols, arteriolar diameters in response to step increases in intravascular pressure from 20 to 140 mm Hg were measured before and after the administration of either NG-nitro-L-arginine methyl ester (3 ⫻ 10⫺4
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Fig. 1. Effects of 20 mm Hg step increases in transmural pressure on steady state second-order arteriolar diameters (means ⫾ SE), before (䡩) and after (●) extracellular calcium removal (n ⫽ 6). Diameters of arterioles in the presence of extracellular calcium; diameters of arterioles in the absence of extracellular calcium. *Indicates significant differences between the diameters found in the presence of calcium, versus those found in the absence of calcium (*P ⬍ 0.05).
M) or indomethacin (10⫺5 M) in the chamber solution. In both sets of experiments, responses to ACh 10⫺7 M were recorded before and after drug administration. All salts and chemicals were obtained from J.T. Baker Inc. (Phillipsburg, NJ) and Sigma Chemical Co. (St. Louis, MO). The data are presented as means ⫾ SE. Differences were considered significant at P ⬍ 0.05 and were determined by using analysis of variance (ANOVA) with the Student-Newman-Keuls method, two-way ANOVA for repeated measures with the Student-Newman-Keuls method, and paired t tests, as appropriate.
Results Pressure-diameter relationship with intact endothelium In the presence of calcium, and at a constant transmural pressure of 100 mm Hg, control diameters averaged 75.3 ⫾ 6.7 m. All second-order arterioles (n ⫽ 6), in the presence of calcium, developed active tone in response to step increases in transmural pressure (Fig. 1). In response to a transmural pressure increase from 20 to 40 mm Hg, the vessel diameter increased from 91.3 ⫾ 3.0 to 95.8 ⫾ 5.5 m. However, in response to a 20 mm Hg step increase in transmural pressure from 40 to 60 mm Hg, significant (P ⬍ 0.05) arteriolar constriction from 95.8 ⫾ 5.5 to 81.5 ⫾ 2.0 m was observed. In response to step increases in transmural pressure from 60 to 160 mm Hg, arteriolar diameters remained constant and did not change significantly (81.5 ⫾ 2.0 to 79.2 ⫾ 4.1 m). Beyond 160 mm Hg and up to 200 mm Hg, arteriolar diameter significantly (P ⬍ 0.05) increased from 79.1 ⫾ 4.1 to 126 ⫾ 6.8 m. In these same arterioles, the passive pressure-diameter relationship was obtained by the removal of calcium from
the vessel bath. After calcium removal, control diameters, at a constant transmural pressure of 100 mm Hg, increased significantly (P ⬍ 0.05) to 136 ⫾ 2.7 m from 75.3 ⫾ 6.7 m, an 81% increase in diameter. In response to a 20 mm Hg step increase in transmural pressure from 20 to 40 mm Hg, arteriolar diameter increased significantly (P ⬍ 0.05) from 96.7 ⫾ 1.0 to 120.5 ⫾ 4.6 m. At 40 mm Hg and up to 200 mm Hg, arteriolar diameter continuously increased with each 20 mm Hg increase in pressure. While arteriolar diameters increased from 120.5 ⫾ 4.6 to 140.8 ⫾ 7.4 m in response to the pressure increases, each individual diameter increase was not significantly different from the previous one. In comparison to vessel diameter changes in the presence of calcium, removal of calcium from the vessel bath did not significantly alter arteriolar diameters at 20 mm Hg. However, above 20 mm Hg, and up to 200 mm Hg, arteriolar diameters obtained in the absence of calcium were significantly (P ⬍ 0.05) higher than those obtained in the presence of calcium in the vessel bath. Arteriolar diameters, before and after removal of calcium from the vessel bath, were observed and recorded during step increases in transmural pressure from 20 to 200 mm Hg. However, in subsequent experimental protocols with endothelium removal and administration of L-NAME and INDO, arteriolar diameters were only recorded within the pressure range of 20 to 140 mm Hg. Our preliminary data indicated (not shown here) a diminished response to the agonists ACh and PE in arterioles subjected to transmural pressure exceeding 140 mm Hg. This observation is consistent with pressure-induced damage to both endothelium and vascular smooth muscle. In contrast, arteriolar responses to these agonists were not significantly different following step increases in transmural pressure to 140 mm Hg. Therefore, protocols were revised in order to avoid endothelial and vascular smooth muscle damage and experiments in this study were conducted in the pressure range of 20 to 140 mm Hg. Effect of endothelium removal on responses to drugs Control diameters were recorded at a transmural pressure of 100 mm Hg. Responses to ACh, SNP, and PE were also recorded at this pressure. Our preliminary studies indicated that, upon initial pressurization, myogenic tone, as well as the response to agonists, was more easily observed at 100 mm Hg. In additional preliminary studies, arterioles pressurized at 75 mm Hg developed poor spontaneous tone. We therefore concluded that control diameters and agonist responses must be recorded at 100 mm Hg. The presence of a functional endothelium and vascular smooth muscle was assessed by the dilation of arterioles to ACh and SNP, and their constriction in response to PE at 100 mm Hg. In nine experiments, the dilations to ACh (10⫺7 M) and SNP (10⫺6 M) before endothelium removal were 43.8 ⫾ 4.8 and 25.7 ⫾ 3.3 m, respectively. PE
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Fig. 2. Effects of 20 mm Hg step increases in transmural pressure on steady-state second-order arteriolar diameters (means ⫾ SE), before and after endothelium removal (n ⫽ 9). (䡩) Diameters of arterioles with an intact endothelium; (‘) diameters of arterioles with the endothelium removed. Arteriolar diameters measured before and after endothelium removal were not found to be significantly different.
administration resulted in a constriction of ⫺24.6 ⫾ 4.8 m. All of these responses were significantly (P ⬍ 0.05) different from zero. After removal of the endothelium in these nine experiments, ACh produced a constriction of ⫺8.8 ⫾ 1.2 m. In contrast to the change observed in the arteriolar response to ACh, responses to SNP and PE were not significantly altered as a result of endothelium removal (PE, ⫺29 ⫾ 3.7 m; SNP, 22.4 ⫾ 2.6 m). These results indicate that the endothelium was functionally removed, as demonstrated by the change in response to the endothelium-dependent dilator ACh. Furthermore, removal of the endothelium did not result in damage to the vascular smooth muscle, as was demonstrated by the preservation of arteriolar responses to the endothelium-independent agonists, SNP and PE. In our previous study (Sun et al., 1992), histological examination revealed that the endothelium was completely removed by the procedure employed in the present study.
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Fig. 3. The calculated myogenic index for isolated second-order adipose tissue arterioles, before and after endothelium removal. (䡩) Myogenic index for arterioles with an intact endothelium; (‘) myogenic index for arterioles with the endothelium removed.
IND were statistically insignificant, and were found to be 69.6 ⫾ 6.7 and 78.3 ⫾ 1.0 m. Furthermore, administration of IND (n ⫽ 7) did not significantly alter arteriolar responses to increases in transmural pressure (Fig. 4). Control diameters at 100 mm Hg significantly decreased after administration of L-NAME from 70.1 ⫾ 4.6 m before L-NAME to 58.6 ⫾ 7.7 m after. Additionally, in contrast to inhibition of cyclooxygenase, inhibition of nitric oxide synthase by L-NAME (n ⫽ 7) significantly decreased arteriolar diameters at all pressure steps with the exception of 20 mm Hg, the lowest pressure used (Fig. 5). Additionally in this group of vessels, L-NAME produced a significant (P ⬍ 0.05) inhibition of the dilator response to ACh (n ⫽ 7). ACh induced arteriolar dilations before and after administration of L-NAME were 56.14 ⫾ 5.31 and 36.29 ⫾ 3.32 m, respectively.
Pressure-diameter relationship after endothelium removal While control diameters at 100 mm Hg decreased after endothelium removal, these differences in diameters were not statistically significant (63.7 ⫾ 6.7 vs 51.8 ⫾ 7.4 m). Furthermore, arteriolar diameters (n ⫽ 9) obtained at each pressure interval (from 20 to 140 mm Hg) after removal of the endothelium were also not significantly different from those obtained prior to endothelium removal (Fig. 2). The calculated myogenic index for these arterioles is presented in Fig. 3. Pressure-diameter relationship after administration of indomethacin or L-NAME Differences in control diameter at 100 mm Hg before and after inhibition of cyclooxygenase by the administration of
Fig. 4. Effects of 20 mm Hg step increases in transmural pressure on steady-state second-order arteriolar diameters (means ⫾ SE), before and after administration of indomethacin (n ⫽ 7). (䡩) Diameters of arterioles found in the absence of indomethacin; (⽧) diameters of arterioles found in the presence of indomethacin. Arteriolar diameters measured before and after indomethacin administration were not found to be significantly different.
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Fig. 5. Effects of 20 mm Hg step increases in transmural pressure on steady-state second-order arteriolar diameters (means ⫾ SE), before and after administration of L-NAME (n ⫽ 7). (䡩) Diameters of arterioles found in the absence of L-NAME; (䡲) diameters of arterioles found in the presence of L-NAME. Arteriolar diameters measured following administration of L-NAME were found to be significantly different from control (*P ⬎ 0.05) at all pressures except 20 mm Hg.
Discussion The present study has demonstrated the ability of adipose tissue arterioles to constrict in response to an increase in transmural pressure. Our data indicate that this myogenic response is present within the transmural pressure range of 40 to 200 mm Hg. At all pressure steps, except 20 mm Hg, arteriolar diameter was significantly lower than that found in the absence of extracellular calcium, revealing a dependence of the myogenic response on extracellular calcium. Control diameters at 100 mm Hg, as well as arteriolar diameters at each pressure step from 20 to 140 mm Hg, were not significantly altered after removal of the endothelium. These findings indicate that a functional endothelium is not necessary for the development of, nor does it significantly modulate, the increase in vascular smooth muscle tone in response to increases in transmural pressure. Additionally, these observations also indicate that the endothelium does not significantly modulate baseline myogenic tone at 100 mm Hg. Administration of IND, an inhibitor of cyclooxygenase, also failed to significantly alter control diameters at 100 mm Hg or arteriolar responses to step increases in transmural pressure. These results indicate that metabolites of cyclooxygenase do not significantly modulate vascular smooth muscle tone in response to step increases in transmural pressure, nor do they contribute to the modulation of baseline myogenic tone at 100 mm Hg. In contrast, inhibition of nitric oxide synthase significantly decreased control diameters at 100 mm Hg, as well as arteriolar diameters at all pressures except at 20 mm Hg. Therefore, nitric oxide (NO) appears to be not only a significant modulator of baseline myogenic tone but also of responses to changes in transmural pressure. To the best of our knowledge, this is the first
study to demonstrate and characterize, in detail, myogenic responses of arterioles isolated from adipose tissue. Studies of isolated human gluteal adipose tissue arterioles indicate the presence of a myogenic response within the range of 40 to 120 mm Hg (Coats et al., 2001). However, in contrast to our data, at a transmural pressure of 40 mm Hg, no statistically significant difference in diameters was found between human gluteal adipose tissue arterioles bathed in physiological salt solution with calcium (active vessel diameter), and those bathed in a calcium-free physiological salt solution (passive vessel diameter). Thus, these vessels did not develop myogenic tone at 40 mm Hg as did rat adipose tissue arterioles. It is also interesting to point out again that arterioles present in human adipose tissue proximal to the lateral malleolus autoregulate within the range of 70 to 150 mm Hg (Kastrup et al., 1985). Thus, at a pressure of 40 mm Hg, there is no autoregulation, and no myogenic response, suggesting the latter as a major contributor to autoregulation in human adipose tissue. In the rat, however, there may be a need to autoregulate at pressures of 40 mm Hg and above, as our data indicate a significant difference between active and passive diameters at 40 mm Hg, but not at 20 mm Hg (Fig. 1). Coats et al. (2001) also report that, for human adipose tissue arterioles, the greatest degree of constriction in response to a step increase in transmural pressure of 40 mm Hg occurred in the range of 40 to 80 mm Hg. Interestingly, our vessels also demonstrated constriction that was greatest when the transmural pressure was increased from 40 to 60 mm Hg. Coats et al. (2001) found the greatest degree of myogenic tone at a pressure of 120 mm Hg, and the largest vasoconstrictor response between the transmural pressures of 40 to 80 mm Hg. Similarly, our data indicate the largest degree of myogenic tone is present at transmural pressures of 100 –120 mm Hg, and the largest vasoconstrictor response between pressures of 40 and 60 mm Hg. Coronary vessels and adipose tissue vessels seem to behave similarly with respect to the range in which myogenic responses are elicited. Studies of rat coronary vessels show an active myogenic response within the pressure range of 20 to 200 mm Hg (Garcia et al., 1997), while our data of rat inguinal fat pad arterioles indicate an active myogenic response range between 40 and 200 mm Hg. Studies of porcine coronary arterioles indicate an active range of 20 to 140 mm Hg (Kuo et al., 1990). Human coronary arterioles, limited to being studied only within the pressure range of 20 to 100 mm Hg, indicate the presence of an active myogenic response from 40 to 100 mm Hg (Miller et al., 1997). With respect to the range of myogenic activity, skeletal muscle arterioles also behave similarly to inguinal fat pad arterioles. In rat cremaster muscle arterioles, Falcone et al. (1993) report an active myogenic response range of approximately 30 to 230 mm Hg. In adipose tissue, an arteriolar dilation of 43.8 ⫾ 4.8 m (⬃69% change from control) was observed in response to ACh (10⫺7 M). Second-order arterioles isolated from rat
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cremaster muscle, upon administration of ACh (5 ⫻ 10⫺8 M), also dilated 33.3 ⫾ 3.6 m (Huang et al., 1993). Isolated rat coronary arterioles demonstrated slight dilation in response to low concentrations of ACh (10⫺9 to 10⫺7 M). In contrast, higher concentrations of ACh (10⫺6 and 10⫺5 M) resulted in significant vasoconstriction of 16.4 ⫾ 9.3 m in rat coronary arterioles (Szekeres et al., 2001). This observation indicates a similar reactivity to ACh in skeletal muscle and adipose tissue arterioles. In adipose tissue, an arteriolar dilation of 25.7 ⫾ 3.3 m (⬃41% change from control) was observed in response to SNP (10⫺6 M). Arterioles isolated from rat cremaster muscle, upon administration of SNP (10⫺7 M), also dilated 22.4 ⫾ 2.9 m, respectively (Huang et al., 1993). Similarly in rat coronary arterioles with a control diameter of 77.3 ⫾ 6.6 m, SNP (10⫺6 M) elicited a significant dilation of ⬃30 m (Szekeres et al., 2001). Thus, rat skeletal muscle, adipose, and coronary arterioles possess a similar reactivity to SNP. Our data indicate that endothelium removal in adipose tissue arterioles completely abolished dilation to ACh, but not SNP. Furthermore, ACh administration, after endothelium removal, resulted in an arteriolar constriction of ⫺8.8 ⫾ 1.2 m. Similar results were observed in rat skeletal muscle arterioles, as removal of the endothelium abolished arteriolar dilation to ACh (but not SNP), and resulted in a vasoconstriction of ⫺1.3 ⫾ 0.4 m (Huang et al., 1993). In contrast, however, to adipose tissue and skeletal muscle arterioles, removal of the endothelium in isolated porcine coronary arterioles did not significantly change arteriolar constriction to ACh (Kuo et al., 1990). In adipose tissue arterioles, the presence of L-NAME (3 ⫻ 10⫺4 M) resulted in a partial but significant reduction of arteriolar dilation to ACh. In rat skeletal muscle, a significant partial reduction in arteriolar dilation in response to ACh was also observed in the presence of another nitric oxide synthase inhibitor, L-NNA (Schrage et al., 2000). Curiously, the administration of L-NAME (3 ⫻ 10⫺4 M) failed to achieve a complete block of arteriolar dilation to ACh. These results suggest that other mediators contribute to ACh-induced dilations. The contribution of vasodilatory mediators other than nitric oxide was not fully explored. Our preliminary results (not shown) indicate that a further reduction in ACh-induced dilations was seen in the presence of indomethacin and L-NAME, suggesting that vasodilator prostaglandins may potentially contribute to the response. Additionally, the presence of a poorly characterized endothelium-derived hyperpolarizing factor has been shown to dilate vessels in a variety of vascular beds (Busse et al., 2002). Further studies of adipose tissue arterioles are needed to resolve this matter. In response to PE (10⫺7 M), adipose tissue arterioles constricted ⫺24.6 ⫾ 4.8 m (⬃40% change from control diameter). Skeletal muscle arterioles behave similarly, as PE administration also resulted in vasoconstriction (Aaker et al., 2002). We observed significant arteriolar constriction only at concentrations greater than 10⫺7 M. Thus, ␣-adren-
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ergic receptor density and/or sensitivity differ between the two vascular beds. In contrast, in isolated canine coronary arterioles, administration of PE directly to the arteriole did not result in a significant change in diameter (Tiefenbacher et al., 1998). Our data indicate that nitric oxide contributes significantly to baseline myogenic tone in adipose tissue arterioles. Similar results were found in a study of the coronary microcirculation, as administration of L-NNA to rat coronary arterioles significantly decreased arteriolar diameters from control (Szekeres et al., 2001). In response to inhibition of the synthesis of nitric oxide in skeletal muscle, however, variations exist with regards to skeletal muscle tissue location (Aaker et al., 2002; Schrage et al., 2000). Although control diameters at 100 mm Hg, as well as arteriolar diameters at all pressure steps were increased in response to indomethacin administration, these increases were not significant. Similar results have been observed in isolated rat soleus arterioles, as indomethacin administration resulted in an insignificant increase in baseline arteriolar diameters at 80 mm Hg (Schrage et al., 2000). In rat cremaster arterioles subject to step increases in transmural pressure, administration of indomethacin also failed to alter arteriolar diameter at any of the pressure steps, except at 160 mm Hg (Huang et al., 1993). These observations indicate that in adipose tissue arterioles and some skeletal muscle arterioles, a vasoconstrictor cyclooxygenase product is contributing slightly to basal tone. Skeletal muscle and adipose tissue arterioles also present with similar myogenic indices over the pressure range studied (Sun et al., 1994) Removal of the endothelium failed to significantly alter arteriolar diameters at baseline as well as in response to increases in transmural pressure. This finding can be explained on the basis of a simultaneous removal of constrictor and dilator influences. In contrast, however, inhibition of endothelial nitric oxide production, a dilator influence, resulted in a significant decrease in arteriolar diameters. These seemingly contrasting data can be reconciled by considering that the endothelium, at baseline and in response to step changes in transmural pressure, was stimulated to release both vasodilator and vasoconstrictor agents. Thus, by inhibiting a vasodilator, we found significant decreases in arteriolar diameter. The identity of the vasoconstrictor(s) can only be speculated about at the present time. However, in response to indomethacin, arteriolar diameters, while insignificantly, did increase, indicating the production of vasoconstrictor metabolite(s) of cyclooxygenase. Schrage et al. (2000) have also reported, in isolated rat soleus arterioles, insignificant increases in baseline arteriolar diameters upon administration of indomethacin. In summary, we have shown that isolated adipose tissue arterioles have the ability to respond to increases in transmural pressure with a myogenic response. Thus, the myogenic response of adipose tissue arterioles may play an important role in autoregulation of blood flow.
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Perspective Given the 20% incidence of obesity (Mokdad et al., 2001) in the United States, it is surprising that more attention has not yet been paid to the regulation of blood flow in adipose tissue. Especially since fat can represent 18 and 24% of body weight in normal men and women, respectively, and as much as 52 and 74% of body weight in obese men and women, respectively (Leibel et al., 1995). It is our hope that this study demonstrates the feasibility of studying adipose tissue arterioles in vitro, and that more interest will develop in adipose tissue blood flow regulation to parallel the developing interest in the metabolic disturbances that are brought about by obesity.
Acknowledgments This work was supported by the National Institutes of Health Grant PO-I HL-43023.
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