INTRODUCTION

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THE MICROVASCULAR ENDOTHELIUM can influence arteriolar tone and blood flow through the release of various diffusible vasoactive factors that include nitric oxide (NO), prostaglandins, and other arachidonic acid metabolites (2, 3, 6, 7, 15, 23). As in larger vessels, stimuli for the release of these endothelial factors at the microvascular level include hemodynamic shear stress (3, 7, 15) and the binding of biochemical agents to endothelial receptors (2, 5, 6, 23).

During juvenile growth, rapid increases in tissue mass are accompanied by extensive growth of the arteriolar, venular, and capillary networks (11, 19, 31, 33) and by increases in microvascular pressure (36) and tissue blood flow (33, 36). In light of these structural and hemodynamic changes, it is reasonable to expect an accompanying change in the functional relationship between endothelium and vascular smooth muscle. Growth-related changes in endothelial function have been documented in large conduit arteries (4, 5, 13, 14, 22), but little is known about how juvenile growth may affect endothelium-mediated relaxation in the microcirculation. We undertook the current study to determine how the influence of NO and prostaglandins on arteriolar tone might change during juvenile growth in the rat spinotrapezius muscle.

	METHODS

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Surgical preparation and intravital microscopy. Experiments were performed on male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) of three age groups: weanling (4-5 wk), juvenile (7-8 wk), and mature (11-12 wk). All rats were anesthetized with thiopental sodium (100 mg/kg ip), and supplemental anesthetic (20% of original dose) was given as needed. The rat was placed on a heating mat to maintain a 37°C rectal temperature. The trachea was intubated to ensure a patent airway, followed by cannulation of the right carotid artery for continuous arterial pressure measurement with a Gould P23 ID pressure transducer (Gould, Oxnard, CA). The right spinotrapezius muscle was surgically exteriorized and prepared for microscopic observation as previously described (3). With this approach, the muscle was gently drawn away from the body wall without disturbing any of its feed vessels, neural inputs, or surface fascia and secured with silk ligatures over a transparent pedestal. A three-sided superfusion chamber was then placed around the muscle and in contact with the animal's back to form an enclosed reservoir, and the chamber was sealed to the underlying pedestal with stopcock grease. Throughout the surgery and subsequent experimental period, the muscle was maintained at its in situ length and continuously superfused with a physiological electrolyte solution (119 mM NaCl, 25 mM NaHCO₃, 6 mM KCl, 3.6 mM CaCl₂) warmed to 35°C, and equilibrated with 95% N₂-5% CO₂ (pH = 7.35-7.40). The flow rate was maintained at 4-6 ml/min to minimize equilibration with atmospheric oxygen (3).

After muscle preparation, the rat was transferred to the stage of an Olympus BHMJ intravital microscope (Hyde Park, NY) that was coupled to a charged-coupled video camera (Videoscope International, Washington, DC), and the spinotrapezius muscle was transilluminated with 150-W halogen fiber optic light. Video images were displayed on a Panasonic high-resolution television monitor and stored on videotape for off-line analysis. All observations were made with a ×10 eyepiece and Nikon ×10 or ×20 water-immersion objectives (final video magnification = ×730 or ×1460). Arteriolar inner diameters were measured during videotape playback with a video caliper (Microcirculation Research Institute, Texas A & M University) that was calibrated using a stage micrometer marked in 10-µm increments. Centerline blood velocity measurements were made on-line with an optical Doppler velocimeter (Microcirculation Research Institute). The vessels chosen for study were arcade bridge arterioles that branch directly from either the brachial and thoracodorsal arteries to enter the anterior portion of the muscle or from the 11th intercostal artery to enter the posterior portion of the muscle (29).

ACh and sodium nitroprusside (SNP) (Sigma, St. Louis, MO) were applied directly to arterioles by microiontophoresis. Glass pipettes were beveled at a 23-25° angle to an outer tip diameter of 1-3 µm and filled with a 0.05 M solution of either vasoactive agent in distilled water. A current programmer (model 260, WPI, Sarasota, FL) was used to deliver currents of up to 100 nA for the ejection or retention of each ionized agent. Prostaglandin E₂ (PGE₂, Sigma) was locally applied to arterioles using a pressurized pipette to deliver 0.1-ml boluses of 10⁸ to 10⁵ M PGE₂ dissolved in superfusion solution. PGE₂ was initially dissolved at a concentration of 1 mg/ml in 0.1 ml 95% ethanol + 0.9 ml Na₂CO₃ (20 mg/100 ml). This solution was then diluted with sterile isotonic saline to a stock concentration of 1 × 10⁴ M and frozen in 3-ml aliquots. Aliquots were thawed on the day of the experiment and serially diluted to the desired experimental concentrations with superfusion solution. Aliquots remained frozen for no longer than 2 wk.

Experimental protocols. The first series of experiments was designed to assess the influence of endogenous NO on resting arteriolar tone and its contribution to agonist-stimulated, endothelium-dependent vasodilation in each age group. After a postsurgical equilibration period of 45 min, a segment of the arcade bridge was selected to evaluate arteriolar responses to the endothelium-dependent agonist ACh. A hydraulic micromanipulator (model MO-202, Narishige, Tokyo, Japan) was used to lightly position the tip of the iontophoretic micropipette on the outer arteriolar wall, and a retaining current of 40-80 nA was applied to prevent passive diffusion of ACh from the tip. Arteriolar diameter was monitored for 2 min before and 2 min after pipette placement to verify that ACh was not diffusing from the tip in vasoactive amounts. ACh was then continuously applied to the vessel for 2 min by administering a randomly selected net ejection current of 5, 10, 20, 40, or 80 nA. A subsequent recovery period of at least 2 min was allowed for the vessel to regain preapplication diameter, and the above sequence was repeated five times so that ACh was applied to the vessel at all five current doses in random order. NO production was then blocked by adding the NO synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA, 10⁴ M) to the superfusate. For the rat spinotrapezius muscle, L-NMMA at this superfusate concentration does not alter arteriolar smooth muscle responsiveness to NO (3), and NO synthase inhibition also has no effect on arteriolar smooth muscle responsiveness to prostaglandins (7). After a 15-min equilibration period, the series of ACh applications was repeated during continued exposure to the L-NMMA-containing superfusate. At the end of each experiment, passive arteriolar diameter was measured after microvascular tone had been abolished with a superfusate containing 10⁴ M adenosine (Sigma).

Previous reports from this and other laboratories (3, 23, 24) have confirmed that L-NMMA at a 10⁴ M superfusate concentration maximally inhibits NO production in the spinotrapezius muscle of rats up to an average weight of 250 g (i.e., equivalent to the juvenile group studied here). Because older rats have a thicker muscle that could limit the diffusive delivery of L-NMMA to deeper vessels, we conducted additional experiments to determine whether a higher superfusate concentration of L-NMMA is required to maximally inhibit NO synthase in mature rats. In each of three mature rats (83 ± 1 days old, 330 ± 8 g body wt), three arcade bridge segments were selected, and the responses of each segment to iontophoretically applied ACh (5- and 40-nA current doses in random order) were assessed as described above. ACh applications were then repeated under a superfusate containing 10⁴ M L-NMMA and once more under a superfusate containing 10³ M L-NMMA. At the end of each experiment, passive arteriolar diameters were measured in the presence of 10⁴ M adenosine.

L-Arginine analogs do not completely block ACh-induced arteriolar dilation (2, 3, 6, 12, 23), suggesting that some other endothelium-derived factor(s) might contribute to this response. Therefore, a second series of experiments was designed to investigate the possible contribution of prostaglandins to ACh-induced arteriolar dilation in each age group. In these experiments, ACh was iontophoretically applied to the arteriolar wall at current doses of 5, 20, and 80 nA in random order, with ACh concentration in the pipette reduced to 0.02 M for better response resolution. ACh applications were then repeated under a superfusate containing 10⁴ M L-NMMA, and once more under a superfusate containing 10⁴ M L-NMMA + 3 × 10⁵ M meclofenamate. We have previously reported that meclofenamate at this superfusate concentration completely inhibits local cyclooxygenase activity in the rat spinotrapezius muscle, as judged by a complete loss of arteriolar responses to arachidonic acid (3). Meclofenamate at this concentration also has no effect on arteriolar smooth muscle responsiveness to either prostaglandins or NO in this preparation (3, 7). At the end of each experiment, passive arteriolar diameters were measured in the presence of 10⁴ M adenosine. In additional experiments on each age group, we evaluated the effect of 3 × 10⁵ M meclofenamate alone on resting arteriolar diameter and flow.

A third series of experiments was designed to identify any age-related changes in the inherent responsiveness of arteriolar smooth muscle to NO or prostaglandins. Two arcade bridge segments were selected in each rat to evaluate arteriolar responses to SNP, which is enzymatically converted to NO in the vascular smooth muscle membrane (17), or PGE₂, a vasodilator prostaglandin readily produced by the microvascular endothelium (8). SNP was applied iontophoretically at current doses of 5, 20, and 80 nA (random application order) using the sequence of 2-min control, application, and recovery periods described above. The SNP applications were then repeated after the addition of L-NMMA to the superfusate (10⁴ M, 15-min equilibration period). In parallel experiments, PGE₂ was locally applied by a pressurized pipette at concentrations of 10⁸, 10⁷, 10⁶, and 10⁵ M (random application order) using 2-min control, application, and recovery periods. After all application sequences for SNP or PGE₂ were completed, the passive diameter of each selected arteriole was measured in the presence of 10⁴ M adenosine.

Flow-related shear stress is considered to be the primary stimulus for basal NO release in rat spinotrapezius muscle arterioles (7), and we found the highest levels of resting wall shear stress to be in the arterioles of 4- to 5-wk-old rats (see RESULTS). However, an analysis of the effect of L-NMMA on arteriolar diameters and blood flow suggests that there is no basal influence of NO on arteriolar tone in these rats, whereas a basal NO influence is evident in rats aged 7 wk and older (see DISCUSSION). In light of these findings, these findings, we conducted additional experiments to further investigate the relationship between shear stress and arteriolar tone in 4- to 5-wk-old rats and in 7- to 9-wk-old rats. In these experiments, polished glass micropipettes were used to gently occlude a nearby parallel arteriole, which led to an increase in flow velocity (and shear stress) in the arteriole under study. This procedure, commonly referred to as the "parallel occlusion technique," has previously been used by us and by others to study flow-dependent arteriolar dilation in this preparation (3, 7), rat cremaster muscle (15), and rat mesentery (32). In the current experiments, arteriolar diameter and flow velocity were continuously measured during a 2-min control period, a 2-min period of increased flow, and a 5-min recovery period.

Data analysis and statistics. On-line red blood cell velocity measurements and off-line diameter measurements were each recorded on a Grass polygraph. The tracings for each variable were then digitized with a Hewlett-Packard desktop scanner (HP Scanjet IIp). Measurements from these digitized images were made using integrated image analysis software (Sigma Scan/Image, Jandel Scientific, San Rafael, CA). For each vessel, the level of resting arteriolar tone was calculated as follows where D_C is control diameter and D_Max is passive diameter under adenosine. Arteriolar responses to ACh, SNP, and PGE₂ were quantified by comparing the steady-state diameter reached during application with that measured during the immediately preceding control period. Because of the difference in resting arteriolar diameters among different-aged rats (see RESULTS), arteriolar responses to locally applied agonists were normalized as a percentage of maximum dilation where D_SS is steady-state diameter during agonist application.

Arteriolar diameter and centerline red blood cell velocity values were used to calculate arteriolar volume flow (, nl/s) as follows where V_m is mean blood flow velocity, and D is vessel diameter. V_m is calculated as centerline red blood cell velocity/1.6 for vessel diameters >10 µm (1). Arteriolar wall shear rate (WSR, s¹) was calculated as follows All data are expressed as means ± SE, and statistical analysis was carried out using commercially available software (Sigma Stat, Jandel Scientific). Two-way ANOVA with one repeated factor (inhibitor treatment) was used to make simultaneous among-group comparisons for the effect of NO synthase inhibition on resting hemodynamic variables, arteriolar responses to ACh, or arteriolar responses to SNP. This test was also used for among-group comparisons on the effect of L-NMMA and then L-NMMA + meclofenamate on arteriolar responses to ACh. Post hoc analysis was carried out using the Student-Newman-Keuls method, with significance assessed at the 95% confidence level (P < 0.05) for all tests.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Microvascular variables were measured in a total of 129 rats (43 weanling, 47 juvenile and 39 mature) for all protocols combined. As shown in Table 1, mean body weight significantly increased from weanling to juvenile rats and from juvenile to mature rats. There was also a significant increase in mean arterial pressure from weanling to juvenile rats but no further increase from juvenile to mature rats. Resting and passive arteriolar diameters significantly increased from weanling to juvenile rats and from juvenile to mature rats. Calculated arteriolar tone did not change with maturation. As shown by the control hemodynamic variables listed in Tables 2 and 3, growth from the weanling to mature stages was also characterized by a significant increase in arteriolar volume flow but a decrease in resting wall shear rate. There was a larger decrease in wall shear rate from the weanling to juvenile stages than from the juvenile to mature stages.

The effect of L-NMMA on resting arteriolar diameter, blood flow, and wall shear rate is also shown in Table 2. Whereas L-NMMA had no effect on any of these variables in weanling rats, exposure to L-NMMA in juvenile and mature rats caused a significant reduction in resting arteriolar diameters (by 14 ± 4% and 13 ± 4%, respectively) and volume flow (by 22 ± 9% and 24 ± 7%, respectively). Wall shear rate was not significantly affected by L-NMMA in any age group. Pilot experiments for this and other studies in our laboratory have confirmed that the effect of L-NMMA on arteriolar tone is directly attributable to the interruption of NO synthase activity rather than some other effect on arteriolar smooth muscle. In those experiments, the effect of 10⁴ M L-NMMA on arterioles in rat spinotrapezius muscle (a 12 ± 3% constriction) and arterioles in rat small intestine (a 7 ± 2% constriction) was completely reversed when 5 × 10³ M L-arginine was also added to the superfusate, indicating specificity for substrate inhibition (unpublished observations).

The effect of meclofenamate on resting arteriolar diameter, blood flow, and wall shear rate is shown in Table 3. Exposure to meclofenamate significantly reduced resting arteriolar diameters in all three age groups, with diameter reductions averaging 15 ± 2%, 18 ± 3%, and 13 ± 4% from control in weanling, juvenile, and mature rats, respectively. Arteriolar blood flow was also significantly reduced by meclofenamate in all groups, whereas wall shear rate was not significantly affected by meclofenamate in any group.

Arteriolar responsiveness to iontophoretically applied ACh in each age group is illustrated in Fig. 1. In each group, the magnitude of dilation was dose dependent, ranging from an average of 37-52% of maximum dilation at the 5-nA current dose to 87-92% of maximum dilation at the 80-nA current dose. There were no significant differences among the age groups in the mean response to any current dose of ACh. In contrast, there were age-dependent differences in the effect of L-NMMA on ACh-induced dilation. As shown in Fig. 2, exposure to L-NMMA significantly reduced the mean dilator response to ACh at every current dose in weanling, juvenile, and mature rats. However, this reduction was not of equal magnitude in all age groups. Figure 3 illustrates this comparison more clearly by showing the portion of responses to ACh inhibited by L-NMMA in each group. The effect of L-NMMA on ACh-induced dilation was significantly less in mature rats than in weanling rats. Addition of meclofenamate to the L-NMMA superfusate caused an additional reduction in ACh responses only in mature rats (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Arteriolar responses to iontophoretically applied ACh in each age group. N, number of rats studied per group.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Arteriolar responses to ACh before and during superfusion with 104 M NG-monomethyl-L-arginine (L-NMMA) in weanling rats (A), juvenile rats (B), and mature rats (C). * P < 0.05 vs. control. Number of rats per group same as in Fig. 1.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Percentage of control responses to ACh inhibited by L-NMMA. * P < 0.05 vs. weanling group. Number of rats per group same as in Fig. 1.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Arteriolar responses to ACh before and during superfusion with 104 M L-NMMA, then during superfusion with 104 M L-NMMA + 3 × 105 M meclofenamate in weanling rats (A; N = 6), juvenile rats (B; N = 5), and mature rats (C; N = 5). * P < 0.05 vs. Control.  P < 0.05 vs. L-NMMA value. N, number of rats per group.

In the additional experiments to determine whether the 10⁴ M superfusate concentration of L-NMMA was sufficient to maximally inhibit NO synthase in mature rats, we found that a 10-fold greater superfusate concentration had no further effect on either resting arteriolar diameter or on arteriolar responses to intermediate and high current doses of ACh (data not shown).

Arteriolar responses to locally applied SNP and PGE₂ are shown in Fig. 5. In each age group, the magnitude of SNP-induced dilation was dose dependent, ranging from 22-39% of maximum dilation at 5 nA to 87-94% of maximum dilation at 80 nA. Dilation in response to PGE₂ was also dose dependent, ranging from 5 to 23% of maximum dilation with 10⁸ M PGE₂ to 86-91% of maximum dilation with 10⁵ M PGE₂. There were no differences among age groups in the mean dilator response to SNP or PGE₂ at any level, with the exception of a significantly smaller response to the lowest concentration of PGE₂ in mature rats. The presence of L-NMMA had no effect on arteriolar responses to SNP in any age group (Fig. 6), verifying that NO synthase inhibition had no effect on arteriolar smooth muscle responsiveness to NO in these experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Arteriolar responses to locally applied sodium nitroprusside (SNP, A) and prostaglandin E2 (PGE2, B) in each age group. N, number of rats per group. * P < 0.05 vs. weanling and juvenile groups.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Arteriolar responses to SNP before and during superfusion with 104 M L-NMMA in each age group. N, number of rats per group.

As shown in Fig. 7, there was a clear difference between weanling and juvenile rats in arteriolar responsiveness to an acute elevation in luminal shear stress. For these experiments, control arteriolar diameter averaged 26 ± 2 µm in weanling rats and 35 ± 2 µm in juvenile rats. Occlusion of a nearby vessel produced a rapid and significant increase in arteriolar wall shear rate that was of similar magnitude in the two age groups (from 936 ± 62 to 1,313 ± 120 s¹ in weanling rats and from 855 ± 78 to 1,330 ± 121 s¹ in juvenile rats). In juvenile rats, the arteriole under study began to dilate 5-7 s after this shear rate increase, reaching a significantly greater steady-state diameter (49 ± 4 µm) within 30 s. In contrast, arterioles in weanling rats did not dilate after the shear rate increase, with diameter averaging 27 ± 2 µm after 2 min of a sustained increase in wall shear rate.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Steady-state arteriolar diameters in weanling and juvenile rats at normal resting wall shear rate (Control) and after wall shear rate is increased by occlusion of a nearby parallel vessel (Occlusion). See text for details. * P < 0.05 vs. control diameter in same age group. Data represent means of 6 vessels in 3 rats for weanling group and 7 vessels in 4 rats for juvenile group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

From the weanling to mature stages, we found approximately a 60% increase in both the resting and passive diameters of arcade bridge arterioles in the rat spinotrapezius muscle (Table 1). Despite this pronounced arteriolar wall growth, the resting tone of these vessels does not change over this period. This is consistent with the findings of Wang and Prewitt (33), who reported a stability of arteriolar tone in the maturing rat cremaster muscle. In contrast, arteriolar tone progressively increases during maturation in the hamster cremaster muscle (27). This discrepancy may reflect an interspecies difference in the relative importance of structural versus functional adjustments at the microvascular level during maturational changes in tissue metabolism. With the assumption that changes in arcade bridge volume flow accurately reflect changes in total network blood flow, the 25% volume flow increase from weanling to juvenile rats (Table 2) is consistent with previous reports of growth-related increases in total blood flow to rat spinotrapezius muscle (36) and rat cremaster muscle (33). However, from our recent finding that spinotrapezius muscle mass increases by almost 2.5-fold over this same period (19), blood flow per gram of tissue must actually decrease with growth. This trend continues during progression from the juvenile to mature stages, in which muscle mass undergoes an additional 50% increase (19) with no further increase in arcade bridge volume flow (Table 2). This finding is consistent with various studies documenting a progressive decrease in tissue metabolic demand as growth continues (28, 30).

Microvascular growth is undoubtedly the result of a complex interaction among biochemical signals (the activity of circulating factors as well as local metabolic and growth factors) and local mechanical forces such as circumferential wall stress and wall shear stress (31). Some investigators have reported that arterial or arteriolar growth and remodeling occurs in such a way as to maintain a constant wall shear stress (9, 18, 21, 33, 34), although adaptation to shear stress alone may lead to an unstable network structure (10). As a result of both the increase in arteriolar diameter and a decrease in mean blood flow velocity (data not shown), wall shear rate in the arterioles that we studied decreased by 44% from the weanling to juvenile stages and by an additional 30% from the juvenile to mature stages (Table 2). Although these calculations reveal that wall shear stress is not maintained within some narrow range during arteriolar enlargement, they do not necessarily rule out a role for shear stress as an important stimulus for arteriolar wall growth. Given the changes in endothelial function that can occur during growth (see below), it is possible that there could also be a change in the set point around which luminal shear stress is regulated (21).

Studies on isolated large artery segments indicate that juvenile growth is characterized by marked changes in the endothelium-dependent control of vascular tone. With juvenile development in the rabbit, there is a progressive decline in the responsiveness of mesenteric, hepatic, saphenous, and ear arteries to the endothelium-dependent agonists ACh, ATP, and substance P (4). In the rat, endothelial P₂ purinergic receptors coupled to the L-arginine/NO pathway are nonfunctional in the aorta at 4-6 wk of age but become fully functional by 13 wk of age (13). Over this same time period, there is also a progressive decrease in aortic relaxation to ACh due to the offsetting actions of an endothelium-derived vasoconstrictor prostanoid (14). The endothelium-dependent relaxation of rat mesenteric arteries to histamine also declines after 8 wk of age (22), and bradykinin causes an endothelium-dependent constriction of these vessels until 4 wk of age but an endothelium-dependent relaxation of these vessels in older animals (5). In contrast to the large arteries, there have been relatively few studies on possible growth-related changes in endothelium-dependent control at the arteriolar level.

Local prostanoids and arteriolar tone during rapid growth. As an extension of previous reports that the cyclooxygenase inhibitor indomethacin reduces arteriolar diameters in the spinotrapezius muscle of 6- to 8-wk-old rats (7, 12), we found that meclofenamate at a concentration that completely inhibits cyclooxygenase activity (3) reduces resting arteriolar diameters by approximately the same amount in all age groups we studied (Table 3). This observation suggests that locally produced vasodilator prostanoids exert a continuous influence on resting arteriolar tone in young rats and that this influence is well maintained during subsequent juvenile growth. Our finding that the inherent responsiveness of arteriolar smooth muscle to PGE₂ does not change over this growth period (Fig. 5) reinforces this conclusion.

The stimulus for basal prostanoid production in spinotrapezius muscle arterioles has not been identified, but a report by Friebel and colleagues (7) suggests that it is not the shear stress associated with resting blood flow. In that study, cyclooxygenase inhibition had no effect on the increase in arteriolar tone that follows a reduction in luminal blood flow below resting levels, suggesting that basal prostanoid activity is unrelated to the continuous influence of shear stress on arteriolar tone. The dilation of spinotrapezius muscle arterioles following an increase in luminal blood flow above resting levels is also not dependent on an increase in prostanoid activity (3, 7). These reports clearly contrast with other findings that cyclooxygenase inhibition greatly reduces the shear-dependent dilation of arterioles in rat cremaster muscle and in rat gracilis muscle (15, 16), suggesting that there is some degree of heterogeneity among different vascular beds in the specific endothelium-derived mediators that directly couple luminal blood flow to arteriolar tone.

Growth-related changes in influence of NO on arteriolar tone. A growing body of evidence suggests that NO may be more important than prostanoids in the local endothelium-dependent control of arterioles in rat spinotrapezius muscle (3, 7, 23, 24, 26). In juvenile and adult rats, inhibition of NO synthase reduces the resting diameter of spinotrapezius muscle arterioles by an amount similar to that seen with cyclooxygenase inhibition (Table 2) (3, 7, 23, 26), suggesting that basally released NO also exerts a continuous influence on resting arteriolar tone in this vascular bed. However, changes in NO activity alone are almost entirely responsible for acute adjustments in the tone of these arterioles following an increase in local oxygen availability (26) or changes in luminal blood flow (3, 7).

Consistent with previous reports (3, 7, 23, 26), L-NMMA reduced resting arteriolar diameters in the juvenile and mature rats studied here. In contrast, L-NMMA had no effect on arteriolar diameters in weanling rats (Table 2). We found no differences among groups in arteriolar responsiveness to the NO donor SNP (Fig. 5A), suggesting that vascular smooth muscle responsiveness to NO does not change over the growth period we studied. Therefore, the lack of effect of L-NMMA in weanling rats is most likely due to either the absence of basal NO production or a rapid inactivation of basally released NO before it can reach vascular smooth muscle. The latter possibility seems less likely because any condition promoting the rapid inactivation of NO should also reduce the contribution of NO to ACh-induced dilation, and this is clearly not the case in weanling rats (Fig. 3). Therefore, by the process of elimination, it appears that an absence of basal NO production is the most likely explanation for the inability of L-NMMA to reduce arteriolar diameters in weanling rats.

In the spinotrapezius muscle of juvenile rats, the shear stress associated with resting blood flow serves as the main stimulus for arteriolar NO release under basal conditions (7). We and others (3, 7) also reported that increases in shear stress above this resting level produce a rapid arteriolar dilation that is largely due to increased NO production. Our current findings suggest that in rats only a few weeks younger than those previously studied, there is a lack of basal NO production despite the arterioles normally being subjected to relatively high wall shear stresses (Table 2). One explanation for these observations is that the mechanism by which endothelial shear stress is sensed may not yet be developed in the youngest animals. This possibility is supported by our additional finding that acute increases in shear stress promote the dilation of arterioles in juvenile rats but not in weanling rats (Fig. 7). However, we cannot rule out the possibility that the arteriolar endothelium in weanling rats is capable of responding to shear stress at some level but simply senses shear stress relative to a different set point than the endothelium in older rats. Further studies involving a wider range of shear stress changes would be required to distinguish between these two possibilities.

Depending on the vascular bed, ACh-induced arteriolar dilation can be mediated by different combinations of endothelial factors (2, 6, 12, 24). We and others (3, 12, 25) found that L-NMMA significantly reduces the arteriolar response to ACh in all age groups (Fig. 2), confirming a major contribution of NO to this response in rat spinotrapezius muscle. Consistent with earlier findings in this preparation (12, 24), we also found no evidence for a contribution of prostaglandins to ACh-induced dilation in weanling or juvenile rats (Fig. 4). The smaller effect of L-NMMA on ACh responses in mature rats (Fig. 4) is probably not the result of insufficient L-NMMA delivery to the arteriolar wall, because L-NMMA at a 10-fold higher concentration was found to have no additional effect on ACh responses in these animals. It is also unlikely that arteriolar smooth muscle is less responsive to NO in mature rats, because as noted above, arteriolar responses to SNP were not different among groups (Fig. 5). We conclude that the decreased inhibitory effect of L-NMMA on ACh responses in mature rats is most likely due to an increased contribution of some other endothelial factor(s) to these responses. Our observations in the presence of L-NMMA + meclofenamate (Fig. 4) suggest that this factor may be a vasodilator prostaglandin, which would indicate an increasing role of prostaglandins in some aspects of microvascular control in the growing spinotrapezius muscle. Interestingly, the reverse of this process may occur in other vascular beds. For example, ACh-induced dilation of pial arterioles is mediated primarily by the release of a vasodilator prostaglandin in newborn pigs but becomes progressively more dependent on NO release during juvenile growth (35).

In summary, growth of the arteriolar network in rat spinotrapezius muscle is accompanied by an increase in total blood flow and a decrease in proximal arteriole wall shear stress. Despite the relatively high shear stresses in weanling rats, there is no evidence of a basal NO influence on arteriolar tone in these animals. In contrast, ACh-stimulated NO release may be greater in weanling and juvenile rats than in mature rats, with vasodilator prostaglandins contributing to this response only in mature rats. Although NO is apparently more important than prostaglandins in the regulation of arteriolar tone in rat spinotrapezius muscle, this is clearly not the case in some other vascular beds. For example, arteriolar responses to oxygen or increased luminal blood flow are mediated entirely by the altered production of endothelium-derived prostaglandins in rat cremaster muscle (15, 20). Such differences highlight the need to study the effect of growth on endothelial function in additional vascular beds to gain a more complete understanding of how blood flow control mechanisms can evolve to meet changing tissue demands during maturation.