INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A VARIETY OF Ca²⁺-permeable ion channels, including voltage-gated Ca²⁺ channels, receptor-operated channels (ROCs), and store-operated channels (SOCs), participate in the "resting" and/or agonist- or depolarization-stimulated influx of Ca²⁺ in vascular smooth muscle (VSM) (33). Recent evidence indicates that SOCs play an important role in sarcoplasmic reticulum (SR) refilling after VSM activation (2, 22, 25, 29, 43), and may even be involved in the generation and/or maintenance of myogenic tone (MT) (31, 39). Mg²⁺ is a known blocker of SOCs (2, 17, 26, 43). Thus if SOCs contribute to MT, we might predict that an elevated extracellular Mg²⁺ concentration ([Mg²⁺]_o) should interfere with the development or maintenance of tone. Indeed, blood vessel tone can be modulated by [Mg²⁺]_o (1, 23, 36). Reduction of [Mg²⁺]_o increases tone. Conversely, elevation of [Mg²⁺]_o relaxes VSM: it dilates small spontaneously constricted arteries (i.e., those with MT) and attenuates agonist-evoked vasoconstriction (1, 23). Ca²⁺ exchange in VSM is enhanced by low [Mg²⁺]_o and reduced by high [Mg²⁺]_o (36), which lowers intracellular Ca²⁺ without changing intracellular Mg²⁺ (8). Nevertheless, the mechanism(s) of action of Mg²⁺ is (are) poorly understood.

Interpretation of the effects of Mg²⁺ is problematic because various channel types might be targets of Mg²⁺ action. For example, several investigators have reported that Mg²⁺ is a blocker of voltage-gated Ca²⁺ channels (1). External Mg²⁺ has a rapid on-off rate and is a "weak blocker" of Ca²⁺ currents through L-type voltage-gated Ca²⁺ channels (LVGCCs) (21). Little is known about the effects of external Mg²⁺ on Ca²⁺ currents and Ca²⁺ entry through LVGCCs in VSM or other cell types. Dihydropyridines (DHPs) such as nifedipine (Nif) are widely viewed as selective blockers of LVGCCs (10, 34, 38) and thus are potentially useful in distinguishing the role of LVGCCs from other channels, such as SOCs, in phenomena such as MT. Recently, however, some investigators (5, 7, 35) have reported that DHPs may block other Ca²⁺ entry channels, including SOCs and/or ROCs. There also is new evidence that SOCs, which should be sensitive to Mg²⁺ (2, 17, 30, 43), may contribute to MT. These findings raise questions about the widely held view (9, 14, 18) that LVGCCs are the main source of Ca²⁺ for MT.

There is a paucity of information about the direct effects of [Mg²⁺]_o on the cytosolic Ca²⁺ concentration [Ca²⁺]_cyt in VSM. This, plus the uncertainty about the roles of LVGCCs and SOCs in the generation of MT, led us to reexamine these issues in intact, pressurized small mesenteric arteries. Small arteries were studied because MT keeps these arteries in a state of maintained (tonic) contraction that is critical for the control of blood flow and blood pressure. It is important to understand how this tone is generated by increased intralumenal pressure, and how it is maintained, because altered MT may play a role in the pathogenesis of hypertension.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Preparation of Arteries

Imaging

Diameter measurements. The arteries were continuously superfused with gassed Krebs solution and were equilibrated (45-60 min) to initial experimental conditions (37°C, 70 mmHg). Arteries with significant leaks or branches were discarded. Only vessels that exhibited stable MT were studied further under isobaric conditions.

Imaging of Ca²+. For Ca²⁺ imaging, dissected arterial segments were loaded (~3 h at room temperature) with Ca²⁺ indicator (fluo 4) in albumin-free dissection solution containing 15 µM fluo 4-AM, 1% DMSO (vol/vol), and 0.03% cremaphor EL (vol/vol). Dye-loaded arteries were mounted on the glass cannulas, pressurized to 70 mmHg, and superfused with Krebs solution for 30 min at 20-22°C to remove extracellular Ca²⁺ dye. The temperature was then raised to 37°C to permit the development of MT. Experiments were carried out at 37°C to mimic in vivo conditions and to maintain MT, although the dye was lost at a faster rate than at 20-22°C.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Diagram of the method of optically sectioning the rat small mesenteric arterial wall with confocal laser scanning microscopy. The focal plane through the center of the artery is shown. With concentric wall motion, as illustrated, the cross sections of the myocytes visualized in this plane will move horizontally and remain in focus when the artery dilates and constricts (24).

Drugs and solutions. The dissection solution was composed of (in mM) 145 NaCl, 4.7 KCl, 1.2 MgSO₄ · 7 H₂O, 2.0 MOPS, 0.02 EDTA, 1.2 NaH₂PO₄, 2.0 CaCl₂ · 2 H₂O, 5.0 glucose, 2.0 pyruvate, and 1% albumin (pH 7.4 at 5°C). The Krebs solution was composed of (in mM) 112 NaCl, 26 NaHCO₃, 4.9 KCl, 2.5 CaCl₂, 1.2 MgSO₄ · 7H₂O, 1.2 KH₂PO₄, 11.5 glucose, and 10 HEPES (adjusted pH to 7.3-7.4 with NaOH). K⁺-Krebs contained 75 mM KCl and only 42 mM NaCl. In other cases, varying amounts of NaCl were replaced with LiCl, N-methyl-D-glucamine (NMDG)-Cl, or sufficient sucrose to maintain osmolality (see RESULTS for details). Ca²⁺-free solution (0 Ca²⁺-Krebs) was made by the omission of Ca²⁺ and addition of 0.5 mM EGTA. For solutions with a reduced [Mg²⁺]_o, the [Mg²⁺] in the Krebs solution was altered by deleting MgSO₄ and adding MgCl₂ to give the desired final concentration. For solutions with an elevated [Mg²⁺]_o, MgCl₂ was usually added to the normal Krebs. In some cases, however, 27 mM NaCl was replaced by either 8.8 mM MgCl₂ plus 6 mM sucrose (=10 mM Mg²⁺-Krebs) to maintain osmolality, or 27 mM LiCl to maintain both ionic strength and osmolality. Solution osmolalities were measured with a vapor pressure osmometer (model 5500, Wescor; Logan, UT). All osmolalities reported in RESULTS are means of multiple measurements with SE <0.5%. The normal or modified Krebs solutions were all gassed with a mixture of 90% N₂-5% CO₂-5% O₂. The tissue chamber had a volume of ~1.0 ml, and the arteries were superfused at a rate of 3-4 ml/min. See RESULTS for details of the experimental protocols and solution changes.

Data Analysis and Statistics

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effects of [Mg²+]_o on MT

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of extracellular Mg2+ concentration ([Mg2+]o) and nifedipine (Nif) on myogenic tone (MT) in rat small mesenteric arteries. In these and all subsequent experiments described in this study, the arteries were pressurized to 70 mmHg and studied at 37°C. A: representative experiment showing the effect of raising [Mg2+]o in Krebs solution from 1.2 mM (normal) to 6 mM on artery diameter. Horizontal bar at bottom indicates time of exposure to 6 mM [Mg2+]o. Horizontal dashed line indicates passive diameter (PD). MTc, control MT; MT*, residual MT during exposure to altered [Mg2+]o. B: dose-response curve showing the effect of [Mg2+]o (0.2-10 mM) on MT. "100%" indicates the MT in normal Krebs ([Mg2+]o = 1.2 mM). In B and E, ordinate (MT) = (MT*/MTc) × 100%. The IC50 for inhibition of MT was 5.0 ± 1.1 mM Mg2+ (n = 7 arteries). C: representative experiment showing the effect of raising [Mg2+]o on MT in Krebs solutions with constant (normal) osmolality (286 mosmol/kg) and ionic strength (159); see text for details. Horizontal bars at bottom indicate periods during which the indicated solute concentrations differed from those in normal Krebs. D: representative data illustrating the effect of 0.3 µM Nif on MT. E: dose-response curve showing the effect of Nif (0.01 to 0.3 µM) on MT. The IC50 was 0.03 ± 0.01 µM Nif (n = 7 arteries).

Figure 2A also illustrates the protocol used to test the effect of [Mg²⁺]_o on MT. When [Mg²⁺]_o was increased from normal (1.2 mM) to 6 mM for 2 min, the artery was partially dilated. The effect was reversible; the artery constricted back to the previous diameter when normal Krebs was restored. In Fig. 2B, the MT is presented as a percentage of MT_c, where MT = MT*/MT_c × 100%, and MT* is the residual MT at low or high [Mg²⁺]_o (Fig. 2A).

Data for seven such arteries, in which [Mg²⁺]_o between 0.2 and 10 mM were tested, are summarized in Fig. 2B. Reduction of [Mg²⁺]_o <1.2 mM increased MT. Conversely, elevation of [Mg²⁺]_o >1.2 mM reduced MT in a concentration-dependent manner. The [Mg²⁺]_o needed for a 50% reduction in MT was 5 mM.

In all of the aforementioned high-[Mg²⁺]_o experiments, MgCl₂ was simply added to the normal Krebs. Thus both the ionic strength and the osmolality were increased. To control for the high osmolality of the 10 mM Mg²⁺-Krebs (299 vs. 286 mosmol/kgH₂O for normal Krebs), we compared the effects of this solution to Krebs solution, in which 8 mM NaCl or 16 mM sucrose was added. These latter solutions, which had normal [Mg²⁺]_o and osmolalities of 300 and 301 mosmol/kgH₂O, respectively, had negligible effects on the normal MT (not shown).

The solutions with high [Mg²⁺]_o also had a high ionic strength, and therefore might be expected to increase screening of membrane surface charges (15). By this mechanism, elevated [Mg²⁺]_o might reduce the voltage-dependent entry of Ca²⁺ through LVGCCs and reduce MT. To control for this, we examined the effects of elevated [Mg²⁺]_o in solutions in which the ionic strength was kept the same as in the normal Krebs. In these solutions, [Na⁺]_o was maintained constant (85 mM) by reducing the NaCl content of the Krebs; ionic strength was kept the same as in normal Krebs (ionic strength, 159; osmolality, 286 mosmol/kgH₂O) with either 27 mM LiCl, or 8.8 mM MgCl₂ + 27 mM sucrose. As shown in Fig. 2C, when the normal Krebs was replaced by modified Krebs, which contained 27 mM LiCl and only 85 mM Na⁺; there was only a small, transient decline in MT. When the solution containing 10 mM Mg²⁺ + 27 mM sucrose was introduced, however, MT was nearly completely abolished. These data indicate that high [Mg²⁺]_o inhibits MT by a mechanism that does not involve surface charge screening.

Effects of Nif on MT

Effects of Elevated [Mg²+]_o and Nif on [Ca²⁺]_cyt in Individual VSM Cells of Pressurized Arteries: Relation to MT

Data from three representative cells in each of two representative experiments are presented in Fig. 3. Figure 3A, top, shows a fluorescent image; three of the individually identified myocytes are outlined. Under control conditions, the artery generated MT, and many of the cells exhibited increases in [Ca²⁺]_cyt (detected as maintained increases in fluo 4 fluorescence; not shown). When [Mg²⁺]_o was elevated to 10 mM, the steady level of [Ca²⁺]_cyt (i.e., fluo 4 fluorescence) in each of the myocytes declined and the artery dilated, as indicated by the change in diameter. On restoration of normal Krebs (with 1.2 mM Mg²⁺), the steady [Ca²⁺]_cyt in the myocytes rose and the artery constricted. The effects of 3 and 6 mM Mg²⁺ were also tested; the effects on [Ca²⁺]_cyt were graded with [Mg²⁺]_o (not shown). A video clip of the original fluo 4 fluorescence data shown in Fig. 3A from the experiment is at http://ajpheart.physiology.org/cgi/content/full/283/6/H2691/DC1.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of 10 mM [Mg2+]o and 0.3 µM Nif on cystolic Ca2+ concentration ([Ca2+]cyt) and MT (artery diameter) in pressurized rat small mesenteric arteries. A: fluoresence image at the top shows a number of highly fluorescent myocytes (loaded with fluo 4). [Ca2+]cyt changes (measured as fluorescence in arbitrary units, a.u.) in the three myocytes within boxes numbered 1-3 are graphed (red, green, and blue, respectively) immediately below the image. Simultaneous changes in diameter ( diameter = 2 × wall displacement) are indicated by the magenta trace. See http://ajpheart.physiology.org/cgi/content/full/283/6/H2691/DC1 for a video clip. B: effect of 0.3 µM Nif on [Ca2+]cyt and artery diameter. [Ca2+]cyt (fluorescence) changes in three myocytes (red, green, and blue, respectively) and simultaneous changes in diameter (magenta) are graphed. Both [Ca2+]cyt and MT recovered after a 10-min of washout of Nif. Data are from a different artery than the data in A. Bars above graphs indicate periods of exposure to Krebs with 10 mM [Mg2+]o (A) or 0.3 µM Nif (B). PD = maximum diameter during period in 10 mM [Mg2+]o or 0.3 µM Nif; thus relaxation in 10 mM [Mg2+]o or 0.3 µM Nif indicates the magnitude of MT (see Fig. 2). A and B show representative data from one of seven and five similar experiments, respectively.

Data from a comparable experiment with Nif are shown in Fig. 3B. Nif (0.3 µM) greatly reduced the steady [Ca²⁺]_cyt in all cells and dilated the artery. When Nif was washed out, there was a delay before [Ca²⁺]_cyt started to rise again. The increase in steady [Ca²⁺]_cyt was accompanied by complete recovery of MT.

Effects of Elevated [Mg²+]_o and Nif on K⁺-Evoked Vasoconstriction

The protocol and representative experimental results are illustrated in Fig. 4, A-C. Figure 4A shows a "control" 75 mM K⁺-evoked vasoconstriction superimposed on the MT. This artery initially constricted to 67% of PD (= MT_c). Elevating the external K⁺ concentration ([K⁺]_o) to 75 mM for 2 min, which should depolarize arterial myocytes to about 20 mV, sufficient to open most LVGCCs, induced a further, reversible vasoconstriction to 46% of PD. This constriction corresponds to the sum of MT_c and the control 75 mM K⁺ (depolarization)-evoked vasoconstriction (K_c). In this and all subsequent experiments in this series, all solutions contained 1 µM phentolamine to block the -adrenoceptors and eliminate any contribution of catecholamine release from the intravascular sympathetic nerves to the observed responses. In control experiments, replacement of 70 mM NaCl by equimolar LiCl or NMDG-Cl, or sufficient sucrose to maintain osmolality (see MATERIALS AND METHODS), did not induce vasoconstriction, but relaxed arteries with MT. Thus it seems reasonable to attribute the vasoconstrictor effect of the 75 mM K⁺-Krebs to the specific depolarizing action of the high [K⁺]_o.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of [Mg2+]o and Nif on 75 mM K+-evoked vasoconstriction. A: representative recording of control 75 mM K+-evoked vasoconstriction superimposed on MTc. Kc, Peak amplitude of 75 mM K+-evoked contraction. B: the same artery was exposed first to 10 mM [Mg2+]o for 2 min, and then to 75 mM K+-Krebs containing 10 mM [Mg2+]o for another 2 min, before recovery in normal Krebs. C: 15 min later, this artery was treated with 0.3 µM Nif for 2 min, and then with 75 mM K+-Krebs containing 0.3 µM Nif for an additional 2 min, before restoration of normal Krebs. In A-C, bars at bottom indicate periods of exposure to 75 mM K+-Krebs and to 10 mM [Mg2+]o or 0.3 µM Nif. Top, dashed line shows PD. D: dose-response curves showing the effects of [Mg2+]o (1.2 mM to 10 mM) on MT (red) and 75 mM K+-induced vasoconstriction (blue). The 75 mM K+-induced vasoconstriction is significantly greater (P < 0.05) in 10 mM than in 1.2 mM Mg2+. E: dose-response curves showing the effects of Nif (0.01 µM to 0.3 µM) on MT (red) and 75 mM K+-induced vasoconstriction (blue). In D and E, %control MT = (MT*/MTc) × 100%; %control K = (K*/Kc) × 100%; MT* and K* = values at [Mg2+]o >1.2 mM or in the presence of Nif (see Fig. 2). Data points in D and E are each means from three arteries. All solutions contained 1 µM phentolamine.

After recovery for 15 min in normal Krebs, this artery was exposed first to 10 mM Mg²⁺-Krebs (for 2 min), and then to 75 mM K⁺-Krebs containing 10 mM Mg²⁺ for an additional 2 min. As expected, (Figs. 2, A and B, and 3A), elevation of [Mg²⁺]_o to 10 mM rapidly reduced MT. High K⁺ depolarization, after a very brief dilation, rapidly and markedly constricted the artery despite the continued presence of 10 mM Mg²⁺. When normal Krebs was restored, the artery rapidly dilated to its PD, and then slowly recovered MT (see beginning of the record in Fig. 4C, which shows a continuation of the experiment from Fig. 4B). With the normal (1.2 mM) [Mg²⁺]_o, raising [K⁺]_o to 75 mM evoked a smaller constriction (K_c in Fig. 3A) than in the medium with 10 mM Mg²⁺ (K* in Fig. 4B). A possible explanation is that the ability of the artery to constrict further was limited when there was already a large vasoconstriction due to the MT. The dose-response curve (Fig. 4D) shows that elevating [Mg²⁺]_o from 1.2 to 10 mM not only did not inhibit the 75 mM K⁺-induced vasoconstriction but actually increased the K⁺-induced constriction significantly (by 50 ± 18%; P < 0.05; n = 3). The final diameters reached in 75 mM K⁺ were, however, similar in both 1.2 and 10 mM Mg²⁺ (compare Fig. 4, A and B).

Treatment of the same artery with 0.3 µM Nif also markedly reduced the MT, but in the presence of Nif, subsequent elevation of [K⁺]_o to 75 mM induced very little vasoconstriction (K* in Fig. 4C). This is expected if Nif blocks the LVGCCs that are opened by the 75 mM K⁺-induced depolarization. Nif blocked the high [K⁺]_o-induced contractions with high affinity (Fig. 4E). The IC₅₀ was 0.04 ± 0.01 µM when Nif was added during the plateau of the K⁺-induced constriction (not shown). The effect of 10 µM verapamil, a member of a different class of LVGCC blocker, was identical to that of Nif on both MT and the response to 75 mM [K⁺]_o (not shown). This seems consistent with the hypothesis that MT and 75 mM K⁺-stimulated vasoconstriction are both mediated by LVGCCs. However, the fact that high [Mg²⁺]_o blocks MT, but not the K⁺-stimulated vasoconstriction, implies that the effect of Mg²⁺ is not due to block of LVGCCs.

The dose-response curves for Mg²⁺ and Nif are shown in Fig. 4, D and E, respectively. These data demonstrate clearly that, whereas Nif and high [Mg²⁺]_o both reduce MT, Nif, but not 10 mM Mg²⁺, blocks the 75 mM K⁺-induced vasoconstriction. The latter is most likely due to Ca²⁺ entry as a result of depolarization and consequent opening of LVGCCs. The implication is that most of the pressure-induced, Mg²⁺-sensitive, myogenic vasoconstriction cannot be the result of Ca²⁺ entry through LVGCCs alone.

Effects of Elevated [Mg²+]_o and Nif on [Ca²⁺]_cyt Changes Evoked by 75 mM [K⁺]_o

View larger version (93K): [in this window] [in a new window]   Fig. 5.   Effects of 10 mM [Mg2+]o on 75 mM K+-induced changes in [Ca2+]cyt and vasoconstriction. A: images (1/s) a-i obtained at the times indicated by the lower case letters in B. After a control period, [Mg2+]o was elevated from 1.2 to 10 mM; 2 min later, [K+]o was raised to 75 mM in the continued present of 10 mM [Mg2+]o, as indicated by bars at the top of the graph. B: time course of changes in arterial diameter (magenta) and fluo 4 fluorescence in three myocytes (1-3, A,a). All solution contained 1 µM phentolamine. Results are representative of data from 8 arteries. A video clip of the original data from this experiment is available at http://ajpheart.physiology.org/cgi/content/full/283/6/H2691/DC1.

Figure 6 shows a comparable experiment in which the effect of Nif was tested. As already shown (Fig. 3B), 0.3 µM Nif reduced [Ca²⁺]_cyt (i.e., fluo 4 fluorescence) in the myocytes and dilated the artery (Fig. 6, A,b and B). Elevation of [K⁺]_o to 75 mM in the continued presence of Nif had little effect on [Ca²⁺]_cyt in most cells (1 and 2 in Fig 6A,a), but induced modest elevation of [Ca²⁺]_cyt in a few cells (3 in Fig 6A,a). There was no arterial wall displacement (i.e., vasoconstriction) under these circumstances. Following washout of the Nif and high K⁺, the myocyte fluorescence slowly increased and the artery recovered MT (Fig. 6, A,i and B). When this artery was later exposed to 75 mM [K⁺]_o (in the absence of Nif), the myocytes exhibited a large increase in fluorescence and the artery constricted markedly (not shown). The transient 75 mM [K⁺]_o-induced elevation of [Ca²⁺]_cyt seen in some cells in Fig. 6 may have been due to brief opening of LVGCCs because block by Nif is use dependent (19).

View larger version (103K): [in this window] [in a new window]   Fig. 6.   Effects of 0.3 µM Nif on 75 mM K+-induced changes in [Ca2+]cyt and vasoconstriction. A: images (1/s) a-i obtained during the times indicated by the lower case letters on the graph in B. After a control period, 0.3 µM Nif was added to the Krebs; [K+]o was then raised to 75 mM as indicated by bars at the top of the graph. B: time course of changes in arterial diameter (magenta) and fluo 4 fluorescence in three myocytes (1-3, see A,a). All solution contained 1 µM phentolamine. Results are representative of data from 7 arteries. A video clip of the original data from this experiment is available at http://ajpheart.physiology.org/cgi/content/full/283/6/H2691/DC1.

Taken together, the data in Figs. 5 and 6 indicate that the 75 mM [K⁺]_o-induced rise in steady [Ca²⁺]_cyt and vasoconstriction observed in the presence of 10 mM Mg²⁺ were apparently due to opening of Nif-sensitive LVGCCs and consequent Ca²⁺ entry. Thus 10 mM Mg²⁺ does not block these channels.

Effects of Elevated [Mg²+]_o on Peak and Plateau Phases of PE-Induced Vasoconstriction

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effects of elevating [Mg2+]o on PE-induced changes in [Ca2+]cyt and vasoconstriction. A: representative "control" 5 µM PE-induced constriction. Pkc, peak amplitude of control PE-evoked constriction. B: [Mg2+]o was elevated to 10 mM before administering 5 µM PE. C: [Mg2+]o was elevated to 10 mM during the plateau of PE-induced constriction. Plc, plateau amplitude of PE-evoked constriction; Pl*, residual constriction after increasing [Mg2+]o. Data in A-C are from the same artery. D: comparison of the effects of elevated [Mg2+]o on MT (green) and PE-induced constriction (Pk: blue, protocol in B; Pl: red, protocol in C). Data are from 4 (green, blue) or 5 (red) arteries. %Control Pk = (Pk*/Pkc) × 100%; %control Pl = (Pl*/PlC) × 100%; %Control MT = (MT*/MTc) × 100%. E: effects of 10 mM [Mg2+]o on 5 µM PE-induced changes in [Ca2+]cyt (fluo 4 fluorescence) and diameter in three myocytes (red, green, and blue, respectively) from a representative artery. Comparable data were obtained in three other arteries. A video clip of the original data from this experiment is available at http://ajpheart.physiology.org/cgi/content/full/283/6/ H2691/DC1.

The specific ₁-adrenoceptor agonist PE, at a concentration of 5 µM, evoked a rapid vasoconstriction. The constriction reached a maximum (Pk_c in Fig. 7A = the control peak PE response) and then relaxed slightly to a new plateau level (Pl_c). During the plateau phase there was substantial vasomotion (Fig. 7A). These rapid transient changes in diameter are superimposed on the maintained contraction (indicated by the solid line) because individual constrictions and dilations merged on this time scale. After recovery in normal Krebs, [Mg²⁺]_o was raised to 10 mM and MT was abolished (Fig. 7B). PE (5 µM) then induced a large vasoconstriction (Pk* in Fig. 7B), comparable in amplitude to that observed with normal (1.2 mM) [Mg²⁺]_o [i.e., (Pk*/Pk_c) × 100  100%]. Vasomotion was not observed during the plateau vasoconstriction in the presence of 10 mM Mg²⁺. After the peak vasoconstriction and a relatively brief plateau phase (180-240 s or less), the artery suddenly and rapidly dilated (see Fig. 7B). Alternatively, PE was applied first and [Mg²⁺]_o was then raised to 10 mM during the plateau vasoconstriction (Fig. 7C). The artery then promptly dilated to a new diameter (Pl*) that was only slightly more constricted than PD so that (Pl*/Pl_c) × 100  20%. This artery recovered its normal MT when normal Krebs was restored.

Concentration-response data (Fig. 7D) reveal that [Mg²⁺]_o between 1.2 and 10 mM did not reduce the initial peak response to 5 µM PE (or lower concentrations, not shown). However, the plateaus of both PE-evoked vasoconstriction and MT were similarly reduced by external Mg²⁺ in a concentration-dependent manner.

The effects of elevated [Mg²⁺]_o on [Ca²⁺]_cyt were investigated as shown in Fig. 7E. When 10 mM Mg²⁺ was introduced during the plateau phase of the response, [Ca²⁺]_cyt fell rapidly to low levels. The Ca²⁺ oscillations that normally accompany PE-induced vasomotion (24) also were abolished, as was the vasomotion.

Effects of Nif on Peak and Plateau Phases of PE-induced Vasoconstriction

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Effects of Nif on PE-induced changes in [Ca2+]cyt and vasoconstriction. A: control 5 µM PE-evoked vasoconstriction; this experiment is continued in B and C. B: Nif (0.3 µM) was added 2 min before administering 5 µM PE. In this artery, as in 6 others, Nif significantly attenuated the initial vasoconstriction in response to PE [Pk = (Pk*/Pkc) × 100 = 47 ± 9%; n = 7; P < 0.05]. C: Nif (0.3 µM) was added during the plateau of the PE-induced constriction, and the artery promptly dilated from Plc to Pl*. D: in this and two other experiments, when 0.3 µM Nif was added 2 min before administering 5 µM PE, the initial, peak PE-evoked vasoconstriction Pk* was greater than the control Pkc (Fig. 7A); mean = 163 ± 3% of control; n = 3. E: comparison of the effects of 0.3 µM Nif on MT (red, 7 arteries) and the plateau of the PE-induced constriction (Pl: green, 7 arteries, protocol in B); see Fig. 7 legend for methods of calculation. Because of the large variation in peak responses (Pk* in B and D), the Pk data are not graphed. F: effects of 0.3 µM Nif on 5 µM PE-induced changes in [Ca2+]cyt (fluo 4 fluorescence) and diameter in three myocytes (red, green, and blue, respectively; top) from a representative artery. Nif was added at the beginning of the plateau of the PE-evoked response to minimize bleaching (see C; note the change in time scale).

In all 10 of these experiments, the plateau vasoconstriction in response to PE was maintained during a 5- to 7-min exposure to Nif (Fig. 8, B and D) when Nif was added before PE. This contrasts with the sudden, rapid vasodilation (i.e., decline in the plateau) observed when similar experiments were performed with 10 mM Mg²⁺ (Fig. 7B). Nevertheless, when 0.3 µM Nif (like 10 mM Mg²⁺; Fig. 7C) was applied during the plateau of the PE-evoked vasoconstriction (Pl_C in Fig. 8C), most of this vasoconstriction was rapidly blocked (Pl* = residual constriction). Moreover, 0.3 µM Nif, like 10 mM Mg²⁺, prevented the vasomotion normally observed during the plateau (recorded as the thickened lines during the plateau; compare Fig. 8, A and C, with Fig. 8, B and D). Nif reduced both MT and the plateau PE-induced vasoconstriction when Nif was applied after PE (as in Fig. 8C) in a comparable, dose-dependent manner (Fig. 8E). These results are similar to those for high [Mg²⁺]_o (Fig. 7D).

The effects of Nif on the underlying PE-evoked changes in [Ca²⁺]_cyt were also determined, along with simultaneous diameter changes. Treatment with 0.3 µM Nif in the presence of 5 µM PE reduced the steady [Ca²⁺]_cyt and the [Ca²⁺]_cyt oscillations evoked by PE, and relaxed the artery. In the experiment illustrated in Fig. 8F, Nif was added only 50 s after PE, just as the vasoconstriction was reaching a maximum. Thus vasomotion was not apparent before the Nif was added. Nevertheless, abortive [Ca²⁺]_cyt oscillations and vasomotion are seen during the Nif-induced vasodilation.

Effects of Nif and Elevated [Mg²+]_o on Vasoconstriction After SR Ca²⁺ Store Depletion

As already noted, removal of external Ca²⁺ dilates the arteries to PD. Restoration of normal (2.5 mM) Ca²⁺ is then associated with prompt constriction of the arteries and recovery of MT (Fig. 9A; the beginning of the blue record was obtained 15 min after the end of the red record). The 0 Ca²⁺-Krebs (containing 0.5 mM EGTA) should slowly lower the [Ca²⁺] within the SR ([Ca²⁺]_SR) as well as lowering [Ca²⁺]_cyt. Reduction of [Ca²⁺]_SR can be accelerated and prolonged by adding the membrane-permeable, low-affinity Ca²⁺ chelator, TPEN, to the 0 Ca²⁺-Krebs (16). After TPEN treatment, restoration of normal Krebs induced an even greater vasoconstriction than normal (i.e., an "overshoot"; Fig. 9A, end of the blue record, and Fig. 9B, end of the red record) and slowed recovery to normal MT (start of the green record, ~15 min after the end of the blue record in A). This overshoot vasoconstriction after TPEN washout, and at least part of the rapid vasoconstriction induced by external Ca²⁺ restoration in the absence of TPEN, are likely due to Ca²⁺ entry through channels opened by SR Ca²⁺ store depletion (SOCs), and the consequent rise in [Ca²⁺]_cyt.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Effects of 10 mM [Mg2+]o and 0.3 µM Nif on vasoconstriction associated with SR Ca2+ refilling after store depletion and vasodilation in 0 Ca2+-Krebs ± N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) or 5 µM PE. A: exposure to 0 Ca2+-Krebs (with 0.5 mM EGTA) for 2 min dilated the representative artery to PD. The artery recovered MT when external Ca2+ (normal Krebs) was restored (control, red line). SR Ca2+ stores were then depleted in 0 Ca2+-Krebs containing 10 µM TPEN. Readmission of normal Krebs resulted in greater vasoconstriction (blue line) than in the control. The influence of TPEN was reversible, and normal MT was slowly restored (green line). B: [Mg2+]o (10 mM, blue line) or Nif (0.3 µM, green line) were added with 0 Ca2+-Krebs containing TPEN (i.e., during SR store depletion), and were included in the normal Krebs during the first 2 min of TPEN washout and Ca2+ restoration. Red line (control) shows the effects of a 2 min exposure to 0 Ca2+-Krebs + TPEN with normal [Mg2+]o and no Nif. C: artery was exposed to 0 Ca2+-Krebs for 2 min and external Ca2+ was then restored (control, red line). During a second exposure to 0 Ca2+-Krebs, 5 µM PE was added to activate the inositol 1,4,5-trisphosphate receptor-mediated SR Ca2+ release. Readmission of normal Krebs 1.5 min after PE washout resulted in greater vasoconstriction (blue line, mean = 179 ± 24% of control; n = 4; P < 0.01). D: addition of 10 mM [Mg2+]o (blue line) or 0.3 µM Nif (green line) to the normal Krebs during washout of 5 µM PE and restoration of external Ca2+ inhibited the external Ca2+-dependent vasoconstriction (control, red line). Data are representative of results from 3 (A and B) or 4 (C and D) similar experiments.

Figure 9B illustrates the effects of Nif and 10 mM [Mg²⁺]_o on the external Ca²⁺-dependent vasoconstriction after treatment with TPEN. In these experiments, 10 mM [Mg²⁺]_o or 0.3 µM Nif were added with 0 Ca²⁺-Krebs containing TPEN (i.e., during SR store depletion), and were included in the normal Krebs during the first 2 min of TPEN washout and Ca²⁺ restoration. The data demonstrate that 10 mM Mg²⁺ and 0.3 µM Nif both markedly reduce or abolish the vasoconstriction that normally occurs when TPEN is removed and external Ca²⁺ is restored. Because SOCs are blocked by high [Mg²⁺]_o (2, 43), and because DHPs may block SOCs in VSM (31, 35) the possibility is raised that the vasoconstriction observed when Ca²⁺ was restored after a period in 0 Ca²⁺-Krebs without or with TPEN was due to Ca²⁺ entry through SOCs.

Another protocol was also used to deplete the SR Ca²⁺ stores (Figs. 9, C and D). In these experiments, after removal of external Ca²⁺, 5 µM PE was added to the medium to release residual SR Ca²⁺ by activation of the inositol 1,4,5-trisphosphate-sensitive channels. This caused a brief, modest vasoconstriction, followed by relaxation to the PD. Washout of the PE and, 1.5 min later, restoration of the extracellular Ca²⁺ caused the arteries to constrict to a greater extent (by 79% on average) than did restoration of Ca²⁺ when the arteries were not treated with PE (Fig. 9C). In these experiments, too, the vasoconstriction induced by restoration of external Ca²⁺ was abolished by both 10 mM Mg²⁺ and 0.3 µM Nif (Fig. 9D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mechanisms Involved in Generating and Maintaining MT

In addition to the mechanisms listed above, several studies (2, 22, 25, 30, 43) suggest that SOCs play an important role in Ca²⁺ entry and the control of vascular contractility. In fact, antisense oligodeoxynucleotide knockdown of transient receptor potential isoform 6 protein, which may be a component of SOCs (3, 6), markedly attenuates MT in small cerebral arteries (37). Also, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate inhibits MT without altering [Ca²⁺]_cyt or affecting the pressure-induced increase in [Ca²⁺]_cyt (31). These findings suggest that inositol 1,4,5-trisphosphate receptor-mediated release of SR Ca²⁺ and SOCs may both also be involved in generating MT. In sum, multiple mechanisms that affect Ca²⁺ entry and Ca²⁺ sensitivity apparently all contribute to the generation and maintenance of MT.

Elevated [Mg²⁺]_o inhibits MT (1) but the mechanism of action of Mg²⁺ is unclear. Extracellular Mg²⁺ is a "weak blocker" of LVGCCs (21), and the inhibition of myogenic and hormone-induced vasoconstriction by Mg²⁺ has been attributed to this Ca²⁺ antagonist action (1). Mg²⁺ also blocks SOCs in VSM myocytes (2, 43) as well as in other types of cells (11, 30, 42).

How does Mg²+ Inhibit MT? Comparison With Action of DHPs

The final studies in this report (Fig. 9) fit the view that the SR Ca²⁺ stores in small arteries are refilled through SOCs that can be blocked by high [Mg²⁺]_o. Moreover, the SOCs in these small arteries are also blocked by low concentrations of Nif. The latter finding adds to the growing evidence that DHPs can block certain SOCs (7, 35), although this is not the case in some other vascular preparations (e.g., 2, 25, 29). Indeed, the LVGCC blockers diltiazem and D-600, as well as DHPs, were all significantly more potent in inhibiting adrenergic agonist-stimulated Ca²⁺ influx and contraction than 80 mM K⁺-stimulated Ca²⁺ influx and contraction in rabbit mesenteric arteries (5). This, too, indicates that the LVGCC blockers are also acting on another Ca²⁺ entry pathway.

The most parsimonious explanation for these findings is that the inhibitory effects of both Nif and high [Mg²⁺]_o on MT are mediated primarily by blockage of SOCs and not LVGCCs. This may also have important implications for the mechanism underlying the antihypertensive effect of DHPs. It has generally been assumed that this effect is mediated by DHP blockage of LVGCCs (34, 38). Our results, however, suggest that the antihypertensive action of DHPs may actually be the result, primarily, of blockade of SOCs in small artery myocytes.

Mg²+, DHPs, and ₁-Adrenoceptor-Mediated Vasoconstriction

In contrast, Mg²⁺ and Nif have similar inhibitory effects on the following: 1) PE-evoked, maintained [Ca²⁺]_cyt elevation; 2) superimposed [Ca²⁺]_cyt oscillations and associated vasomotion; and 3) PE-evoked tonic (plateau) vasoconstriction; these findings require a different explanation. Indeed, there is a striking similarity between the dose-response curves for inhibition of the plateau vasoconstriction and MT, respectively, by both Mg²⁺ and Nif (Figs. 7D and 8E). This may indicate that the tonic vasoconstrictor action of PE is mediated largely by Ca²⁺ entry through SOCs (2, 25) because, as noted above, both Nif and Mg²⁺ block SOCs in these small arteries.

Whereas 0.3 µM Nif and 10 mM Mg²⁺ both block PE-evoked Ca²⁺ oscillations and vasomotion, and PE-evoked plateau vasoconstriction, details of these findings may provide clues to possible underlying mechanisms. When the Nif or 10 mM Mg²⁺ was introduced before PE, the Ca²⁺ oscillations and vasomotion were blocked before any effect on the plateau constriction was observed. Thus the mechanisms that underlie the Ca²⁺ oscillations and vasomotion must differ from those responsible for the plateau vasoconstriction. It is not yet clear, however, why 10 mM Mg²⁺ or 0.3 µM Nif immediately abolished the plateau when added after PE (Figs. 7, C and E, and 8, C and F) whereas, when applied before PE, Mg²⁺ dilated the artery after a brief (180-240 s) vasoconstriction (200 s in Fig. 7C), and whereas Nif did not reduce the plateau even after 400 s (Fig. 8, B and D).

Study of "Integrated" Vascular Responses

A New Look at Generation of MT

Control of [Ca²+]_cyt and MT: Importance of Ca²⁺ Extrusion