INTRODUCTION

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THE LOCAL REGULATION OF DIAMETER of skeletal muscle arterioles is determined, in large part, by the interaction of responses elicited by intravascular pressure and flow/shear stress (13, 26, 27). The pressure-induced myogenic constriction of arterioles is intrinsic to the arteriolar smooth muscle (aSM) (30, 32), whereas flow-induced arteriolar dilation is mediated by factors released from the endothelium, most importantly nitric oxide (NO) (13, 26). The intracellular signal transduction of myogenic constriction depends on the pressure-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in aSM due to an influx of extracellular Ca²⁺ via voltage-operated Ca²⁺ channels (VOCs) (9, 32, 33). Furthermore, arteriolar myogenic tone can be modulated by pathways that alter the sensitivity of the contractile apparatus to Ca²⁺ (5, 10). However, the mechanisms by which endothelium-derived NO interferes with the signal transduction of pressure-induced tone are not clearly elucidated.

Previous studies in large conduit vessels and isolated smooth muscle cells demonstrated that exogenous administration of NO donors or agonist-induced endogenous release of NO activates soluble guanylate cyclase, leading to increases in cGMP levels (22) and eliciting decreases in [Ca²⁺]_i (4). It was hypothesized that NO and/or cGMP decreases [Ca²⁺]_i in smooth muscle cells by inhibiting inositol 1,4,5-trisphosphate formation or Ca²⁺ influx, enhancing Ca²⁺ uptake in the sarcoplasmic reticulum and/or eliciting membrane hyperpolarization (4, 11, 24, 29). Other studies suggested that NO, NO donors, and/or cGMP may exert a substantial part of its dilator effect via mechanisms that are independent of changes in [Ca²⁺]_i (1, 6, 20, 23, 28, 35). Furthermore, it was shown that in norepinephrine-preconstricted arterioles ACh-induced decreases of [Ca²⁺]_i were not affected by a NO synthase inhibitor (1). It is possible that NO-induced activation of Ca²⁺-sensitivity-related and [Ca²⁺]_i-related pathways significantly depends on the concentration of NO administered and/or there are significant differences between signaling mechanisms activated by NO in vessels with different sizes and functions. Because [Ca²⁺]_i and/or Ca²⁺ sensitivity of smooth muscle (25b) was likely altered by many of the agents (e.g., thromboxane A₂-receptor agonists, angiotensin II, norepinephrine, KCl) or mechanical procedures (stretching of ring preparations) used in these studies to induce vascular tone, it is difficult to ascertain the sole effect of NO/cGMP on Ca²⁺-signaling mechanisms.

Thus we aimed to elucidate the role of NO/cGMP-dependent modulation of smooth muscle Ca²⁺ signaling in arterioles in which substantial myogenic tone develops in response to intraluminal pressure that is likely to be associated with physiologically relevant levels of smooth muscle [Ca²⁺]_i and Ca²⁺ sensitivity. Also, for NO release, we utilized increases in intraluminal flow, which is a primary physiological stimulus for NO synthesis. We hypothesized that increases in intraluminal flow via the NO/cGMP pathway reduce arteriolar myogenic tone by decreasing [Ca²⁺]_i and/or Ca²⁺ sensitivity in aSM. To test this hypothesis, we characterized the effects of NO released in response to increases in intraluminal flow, NO donors, a cGMP analog, and an inhibitor of cGMP-specific phosphodiesterase known to elevate endogenous cGMP levels on aSM [Ca²⁺]_i and myogenic tone of isolated rat skeletal muscle arterioles.

	METHODS

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Simultaneous measurement of smooth muscle [Ca²+]_i and diameter of isolated arterioles. The internal diameter of isolated gracilis muscle arterioles of 12-wk-old Wistar rats (n = 30) was measured by videomicroscopy, as previously described (30, 32). In brief, arterioles were isolated and cannulated in an organ chamber containing physiological salt solution (in mmol/l: 110 NaCl, 5.0 KCl, 2.5 CaCl₂, 1.0 MgSO₄, 1.0 KH₂PO₄, 5.0 glucose, and 24.0 NaHCO₃; equilibrated with 10% O₂-5%-CO₂-85% N₂; pH 7.4). Intraluminal pressure was kept constant at 80 mmHg by a pressure servo-control system. Perfusate flow was measured with a ball flowmeter (Omega Engineering). To assess changes in [Ca²⁺]_i, the arteriolar smooth muscle was loaded with fura 2 [2 µM fura 2-acetoxymethyl ester (AM); 30 min, at 24°C] and changes in Ca²⁺ fluorescence ratio (R_Ca) (30, 32) were measured by the ratiometric fluorescence method (9, 17, 30, 32, 33) using the Ionoptix Microfluorimeter System (Ionoptix, Milton, MA).

Experimental protocols. Arterioles were maximally dilated in a Ca²⁺-free solution, and then simultaneous changes in aSM R_Ca and development of myogenic constriction (at 80 mmHg) were assessed in response to elevation of extracellular Ca²⁺ concentration (0-2.5 mmol/l). Responses to the L-type Ca²⁺-channel inhibitor nimodipine (10¹⁰-10⁷ mol/l) (18) were obtained.

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Materials. Fura 2-AM (Molecular Probes) and ODQ (Calbiochem, San Diego, CA) were used. All other salts and chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Fura-2 AM and zaprinast were dissolved in DMSO, and nimodipine was dissolved in ethanol. The vehicle did not have vasoactive effects. All other drugs were dissolved in distilled water on the day of the experiment. All drugs were added to the organ chambers, and final concentrations are reported. After responses to each drug subsided, the system was flushed with physiological salt solution.

Data analysis. Reduction of arteriolar myogenic tone is expressed as a percentage of the maximal arterial dilation. Increases in R_Ca in response to CaCl₂ are expressed as percentage of the maximal reduction of R_Ca in Ca²⁺-free solution, whereas reductions of R_Ca in response to flow and vasoactive agents are expressed as a percentage of the baseline. All data are expressed as means ± SE. Statistical analyses were performed by analysis of variance followed by Tukey's post hoc test or Student's t-test, as appropriate. P < 0.05 was considered statistically significant.

	RESULTS

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In fura 2-loaded isolated arterioles (diameter 120 ± 4 µm) of rat gracilis muscle at an intraluminal pressure of 80 mmHg, R_Ca significantly increased and substantial myogenic tone developed in response to elevation of extracellular Ca²⁺ concentration (from 0 to 2.5 mmol/l; Fig. 1A). The VOC inhibitor nimodipine elicited significant decreases in R_Ca and simultaneous reduction of arteriolar myogenic tone in a concentration-dependent manner (Fig. 1B). Original recordings show that the change in smooth muscle [Ca²⁺]_i in response to nimodipine (10⁷ mol/l) preceded arteriolar dilation by ~3 s (Fig. 1C; representative of 6 separate experiments; diameter was measured with the automatic edge detection function of the Ionwizard software, time resolution: 0.02 s).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   A: development of myogenic tone and increases in smooth muscle Ca2+ fluorescence ratio (RCa; 0%: Ca2+ free; 100%: 2.5 mmol/l) of gracilis muscle arterioles in response to elevations of extracellular Ca2+ concentration at 80 mmHg (passive diameter: 180 ± 6 µm). Values are means ± SE; n = 5 experiments. B: dilations and decreases in RCa to nimodipine. Values are means ± SE; n = 6 experiments. C: original recordings show that the change in smooth muscle intracellular Ca2+ concentration ([Ca2+]i) in response to nimodipine (107 mol/l) precede arteriolar dilation by ~3 s (representative of 6 separate experiments). The maximal arteriolar diameter was 132 µm.

Changes in aSM [Ca²+]_i and arteriolar diameter to increases in intraluminal flow and SNAP. Increases in intraluminal flow elicited significant reduction of arteriolar myogenic tone that could be inhibited with the NO synthase blocker L-NAME (Fig. 2A) without increases in R_Ca (Fig. 2B). SNAP and SNP (up to 10⁷ mol/l) also elicited significant arteriolar dilations without significant decrease in R_Ca, whereas arteriolar dilations to the highest concentration of NO donors (>10⁶ mol/l) were associated with slight, but significant, decreases in R_Ca (Fig. 2D). Dilations to SNAP were significantly inhibited by ODQ (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   A-D: dilations and changes of smooth muscle RCa in response to increases in intraluminal flow of indomethacin-treated gracilis muscle arterioles [in the absence and presence of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME); n = 10 experiments] or administration of the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) or sodium nitroprusside (SNP) (n = 8 experiments). Values are means ± SE. *Significant differences between groups or differences from baseline values, P < 0.05.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   A: dilation of gracilis muscle arterioles to SNAP in the absence and presence of the guanylate cyclase inhibitor 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; n = 8 experiments). B and C: dilations and changes in smooth muscle RCa to 8-bromoguanosine cGMP (8-BrcGMP) and the phosphodiesterase inhibitor zaprinast (n = 5 experiments). Values are means ± SE. *Significant differences between groups or differences from baseline values, P < 0.05.

Changes in aSM [Ca²+]_i and arteriolar diameter to 8-Br-cGMP and zaprinast. The cGMP analog 8-BrcGMP or the phosphodiesterase inhibitor zaprinast elicited significant, concentration-dependent reduction of arteriolar myogenic tone without significantly affecting R_Ca; only dilations to highest concentration of 8-BrcGMP and zaprinast were associated with slight, but significant, decreases in R_Ca (Fig. 3, B and C).

Smooth muscle [Ca²+]_i-myogenic tone relationships in arterioles. Decreases in aSM R_Ca in response to flow and agonists were plotted against the changes in diameter, yielding smooth muscle [Ca²⁺]_i-arteriolar tone relationships to which regression lines were fitted (Fig. 4A). In the presence of nimodipine, there was a linear relationship between aSM [Ca²⁺]_i and arteriolar tone (slope 2.2 ± 0.2), indicating that nimodipine-induced dilations depend on a decrease in aSM [Ca²⁺]_i.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   A: dilation of gracilis muscle arterioles as a function of changes in RCa in response to increases in intraluminal flow/shear stress or administration of SNAP, 8-BRcGMP, zaprinast, or nimodipine. B: proposed scheme for the modulation of smooth muscle Ca2+ signaling and myogenic constriction by flow-induced endothelial release of NO in indomethacin-treated arterioles. PDE, phosphodiesterase; VOC, voltage-operated Ca2+ channel; eNOS, endothelial NO synthase.

	DISCUSSION

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The main findings of the present study are that, in skeletal muscle arterioles, NO released in response to increases in intraluminal flow or administration of SNAP or SNP elicits significant decreases of myogenic tone without substantial changes in aSM [Ca²⁺]_i. Also, 8-BrcGMP and zaprinast, by increasing cGMP levels, significantly reduced myogenic tone yet elicited only minimal changes in aSM [Ca²⁺]_i.

In the presence of 80 mmHg intraluminal pressure, elevation of extracellular Ca²⁺ concentration from 0 to 2.5 mmol/l elicited development of substantial myogenic tone preceded by a significant increase in aSM [Ca²⁺]_i (Fig. 1A). Nimodipine, a known VOC inhibitor, elicited large decreases in aSM [Ca²⁺]_i and completely abolished myogenic tone (Fig. 1B). These data were obtained to confirm earlier findings in gracilis arterioles showing that arteriolar myogenic constriction is due to an influx of extracellular Ca²⁺ through voltage-gated Ca²⁺ channels (9, 17, 31, 33) and smooth muscle tone is proportional to changes in aSM [Ca²⁺]_i under control conditions.

We also confirmed, in the present conditions, our laboratory's previous findings that increases in intraluminal flow elicit significant NO-mediated reduction of arteriolar myogenic tone (Fig. 2A) (13). However, flow-induced dilations mediated by endogenous NO, unlike responses to nimodipine, were not accompanied by substantial reduction in aSM [Ca²⁺]_i (Fig. 2B). Previously, it was also shown that endogenous NO released in response to acetylcholine does not interfere with decreases in [Ca²⁺]_i mediated by a non-NO, nonprostaglandin factor (1). The NO donor SNAP and SNP (1) also elicited significant arteriolar dilations without substantial decreases in aSM [Ca²⁺]_i (Fig. 2, C and D). Collectively, these results suggest that endogenous NO released in response to intraluminal flow may exert a substantial part of its dilator effect in microvessels via mechanisms that are independent of changes in [Ca²⁺]_i.

Previous studies proposed that dilation to NO can be mediated by both cGMP-dependent and/or independent pathways, activation of which can result in a decrease in smooth muscle [Ca²⁺]_i and/or sensitivity to Ca²⁺ (3, 4, 12). Thus the role of cGMP in NO-induced [Ca²⁺]_i-independent dilation in arterioles needed to be clarified. We found that the cGMP pathway plays a key role in the signal transduction of NO-mediated relaxation of aSM because arteriolar dilations both to SNAP (Fig. 3A) and to increases in flow (12) were significantly inhibited by the guanylate cyclase blocker ODQ (8).

To establish further the role of cGMP in the [Ca²⁺]_i-independent pathways in gracilis arterioles involved in NO-mediated dilation, we administrated exogenously 8-BrcGMP, a cell-permeable analog of cGMP, and zaprinast, a cGMP-specific phosphodiesterase inhibitor that is known to elevate endogenous cGMP levels (7). We found that both 8-BrcGMP and zaprinast mimicked the effects of flow-induced NO release and the NO donor by eliciting significant dilations without substantial decreases in aSM [Ca²⁺]_i (Fig. 3, B and C).

Further analysis of the compiled data obtained for the arteriolar smooth muscle [Ca²⁺]_i-dilation relationships (32) revealed that increases in intraluminal flow, a NO donor, or increases in intracellular cGMP levels elicit significantly greater dilations for a given decrease in aSM [Ca²⁺]_i than nimodipine (Fig. 3A). We interpret these data to mean that dilation of skeletal muscle arterioles by the NO/cGMP pathway primarily depends on a decrease in Ca²⁺ sensitivity of the contractile apparatus rather than a decrease in [Ca²⁺]_i in aSM.

The mechanisms responsible for cGMP-induced decrease in Ca²⁺ sensitivity may include activation of the myosin light chain phosphatase pathway (20, 34), inhibition of protein kinase C activity (19), phosphorylation of heat shock protein 20 (16), or inhibition of RhoA (25, 25b) or mitogen-activated protein kinase (5, 21) pathways, which are thought to modulate the sensitivity of the contractile apparatus to Ca²⁺ in vascular smooth muscle cells(2, 25b).

On the basis of the present and previous findings (30, 31) and data from the literature, we propose a model for describing the regulation of pressure-induced arteriolar tone by flow/shear stress-induced release of NO via modulation of smooth muscle Ca²⁺ signaling (Fig. 3B). Accordingly, 1) increases in intraluminal pressure elicit an increase in aSM [Ca²⁺]_i because of an influx of extracellular Ca²⁺ (9, 30, 33) that activates the contractile apparatus' resulting in arteriolar myogenic constriction; 2) NO released to intraluminal flow/shear stress elevates cGMP levels in aSM that decreases Ca²⁺ sensitivity (2, 16, 19, 20, 25) of the contractile apparatus, thereby reducing myogenic constriction; and 3) thus the magnitude of myogenic tone depends both on pressure-induced increases in [Ca²⁺]_i and changes in the sensitivity of the contractile apparatus to Ca²⁺ (5, 9, 10, 32, 33). Our data suggest that NO released to flow regulates arteriolar tone in vivo primarily by modulation of Ca²⁺ sensitivity.

The importance of the present findings is underscored by recent studies showing that the increased tone of resistance arterioles in hypertension is associated with alterations in NO- mediated responses in microvessels (13) and an increased, endothelium-dependent aSM Ca²⁺ sensitivity (30). Furthermore, it is likely that a decreased arteriolar Ca²⁺ sensitivity due to an increased flow-induced microvascular NO synthesis may underlie the beneficial effects of estrogen (13, 15) and regular daily exercise (26). These results also suggest that therapeutical reduction of aSM Ca²⁺ sensitivity, rather than [Ca²⁺]_i, might be a more relevant approach to decreasing peripheral vascular tone and hence blood pressure.

In summary, our findings suggest that endothelial release of NO and elevation of aSM cGMP levels in response to increases in intraluminal flow decrease the Ca²⁺ sensitivity of aSM contractile apparatus (rather then altering [Ca²⁺]_i), which is likely to be the primary in vivo mechanism of NO to regulate skeletal muscle arteriolar tone.