Introduction

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The pharmacology of vanadium, a Group Vb transition metal, has been extensively investigated in the last decade, particularly in terms of its insulinomimetic properties (Brichard and Henquin, 1995; Cros et al., 1992), and their possible therapeutic application in diabetes mellitus (Cam et al., 1993; Goldfine et al., 1995; Cohen et al., 1995).

Compelling evidence had accumulated, showing differences in the cellular mechanism of action of the various vanadium species. Vanadate (V⁵⁺) and vanadyl (V⁴⁺) derivatives have in vivo antidiabetic properties correlated with an increase in insulin sensitivity, whereas their cellular mechanism, though it remains controversial, is thought to be independent of insulin receptor activation (Goldfine et al., 1995). More recently developed peroxovanadium (pervanadate) derivatives were shown to be able to lower blood glucose in the insulin-dependent diabetic BB rat in vivo (Yale et al., 1995), and their cellular mechanism seems to be directly linked to insulin receptor activation (Bevan et al., 1995). The vanadate (V⁵⁺) and vanadyl (V⁴⁺) oxidation states also differ markedly in biochemical (Cantley and Aisen, 1979; Elberg et al., 1994), pharmacological (Bhanot and McNeill, 1994; Boscolo et al., 1994; Nakai et al., 1995) and toxicologic (Llobet and Domingo, 1984) properties. In particular, vanadate derivatives were shown to have prohypertensive properties (Boscolo et al., 1994), whereas vanadyl derivatives were shown to be antihypertensive agents (Bhanot and McNeill, 1994; Bhanot et al., 1994).

Since 1980 (Ozaki and Urakawa, 1980) it has been known that vanadium salts are able to induce contraction of a variety of smooth muscles tissues, including vascular smooth muscle. Vanadate salts have been studied most, and their mechanism of action remains uncertain despite extensive investigations. The structural similarity between vanadatebut not vanadyland phosphate groups, as well as the inhibitory effect of vanadate on sarcoplasmic and endoplasmic calcium-ATPase (SERCA) (Raeymakers et al., 1983), suggested the mobilization of intracellular calcium stores (Sanchez-Ferrer et al., 1988). Since the description of various isoforms of SERCA, however, it has recently been shown that vanadate has no effect on muscular isoforms (Lytton et al., 1992). The increased level of tyrosine phosphorylation, typically described in the mechanism of the insulinomimetic effects of vanadium derivatives (Tamura et al., 1984; Shechter et al., 1995), was also found to be associated with the smooth muscle contractile activities of vanadate and PV (DiSalvo et al., 1993; Laniyonu et al., 1994), which are still unknown in the case of vanadyl.

The aim of the present study was to use isolated vascular preparations to characterize further the vascular properties of VOSO₄ and to study its mechanism of action. In particular, we examined the relative contributions of calcium and tyrosine phosphorylation to its activity. We describe the concentration-dependent VOSO₄-induced contraction in aortic tissue that is regulated by, but not dependent on, the presence of endothelium and that occurs in the absence of extracellular calcium or the presence of blockers for extracellular calcium entry and intracellular calcium mobilization. Surprisingly, the activity of VOSO₄, but not that of the potent tyrosine phosphatase inhibitor PV, was amplified by the tyrosine kinase inhibitors tyrphostins, concomitantly with elevated levels of tyrosine phosphorylation.

	Materials and Methods

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Contractile effect of VOSO₄ on rat isolated aorta. Thoracic aorta rings 3 mm long were was obtained from male Wistar rats (300-350 g) and suspended at 37°C in aerated (95% O₂, 5% CO₂) Krebs-Henseleit buffer (mM): NaCl, 119; KCl, 4.7; CaCl₂, 2.5; MgSO₄, 1.0; KH2PO₄, 1.2; NaHCO₃, 25; glucose, 11.1; pH 7.4. Isometric tension recording was performed in tissues equilibrated for 60 min under 1 g tension, washed with buffer every 15 min and then exposed to 40 mM KCl to test the viability and "sensitize" preparations for subsequent exposure to contractile agents. When indicated, a second KCl (40 mM)-induced response was recorded as "internal standard." VOSO₄ was used either as single concentrations (10⁴ or 5 × 10⁴ M) or as cumulative concentrations (1 to 1000 µM). Vanadium levels were determined by atomic absorption spectrophotometry (Mongold et al., 1990).

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Role of calcium in contractile activity of VOSO₄. The role of calcium was first assessed on rat thoracic aorta. KCl (40 mM), AVP (3 × 10⁸ M) and NEPI (6 × 10⁷ M) were used for comparison purposes. The amplitude of responses of these agonists was comparable to that of VOSO₄ (5 × 10⁴ M). Consecutive exposures to the same agent yielded similar responses (not illustrated).

Role of tyrosine phosphorylation. The role of tyrosine phosphorylation in the contractile activity of Ca⁺⁺ was assessed by using the tyrosine kinase inhibitors genistein, T₄₇ (RG 50864) and T₂₃ (RG 50810), as well as inactive tyrphostin derivatives (T₁ and T₆₃). The tyrosine kinase inhibitors were dissolved in dimethylsulfoxide (DMSO) and used at a concentration of 0.2 mM. DMSO alone had no contractile activity at the maximal concentration used (0.1%).

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Materials. VOSO₄ · 5H₂O and other reagents of analytical or sequence grade were from Prolabo (Paris, France). The PV solution was prepared according to Pumiglia et al. (1992) by incubating one part of 500 mM H₂O₂ with five parts of 10 mM sodium orthovanadate for 10 min at 37°C immediately before use. This procedure induced the formation of a mixture of peroxovanadium complexes (Campbell et al., 1989) and has been widely used in pharmacological studies showing differences between the properties of PV and those of vanadate (Bevan et al., 1995). AVP, NEPI, N, saponin, phenylmethyl-sulphonyl fluoride (PMSF), aprotinin and leupeptin were from the Sigma Chemical Co. (St Louis, MO). TMB-8, genistein and the tyrphostins were from Biomol (Plymouth Meeting, PA).

Data analysis and statistics. Results of vascular contraction were expressed as mean ± S.E.M. Half-maximal effective concentrations (EC₅₀) were calculated using a computer program for multiple iterations on the Hill equation. pD₂ values were determined as log 1/EC₅₀. Statistical comparisons were done using Newman-Keuls' test or Student's t test for paired or unpaired data, as appropriate. A probability value of less than 5% was considered significant.

	Results

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VOSO₄-induced contraction in rat aorta. Typical isolated aorta responses after the addition of a single concentration of VOSO₄ are shown in figure 1, panel A. VOSO₄ induced maximal vasoconstriction after 5 to 10 min. In most cases, a slowly developing contraction was followed by a steep increase in tension.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   VOSO4-induced contraction on rat aorta. A) Typical tracing of aorta isometric contraction obtained at 5 × 104 M VOSO4 in standard Krebs-Henseleit buffer as described in "Materials and Methods." B) Cumulative concentration-response curves of rat aorta contraction obtained with endothelium or after removal of endothelium. Contraction is expressed as percent of KCl (40 mM)-induced contraction (control). Points represent the mean (n = 6) and bars the S.E.M. Statistically significant differences are indicated by * (P  .05).

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Role of calcium. The influence of various concentrations of the calcium entry blocker N (0.01 to 1 µM) on VOSO₄-, AVP-, NEPI- and KCl-induced contractions is shown in figure 2. The inhibitory activity of N was concentration-dependent. The order of potency of N to inhibit contractile agents was AVP  KCl >> VOSO₄ > NEPI.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of nicardipine on the contractile responses of isolated rat aorta to various contractile agents. Isometric tension measurement of isolated segments of rat aorta was performed in standard Krebs-Henseleit buffer as described in "Materials and Methods." The effect of various concentrations of nicardipine on the contractile responses to VOSO4 (5 × 104 M), KCl (40 mM), AVP (3 × 108 M) and NEPI (6 × 107 M) is expressed as percent of the control responses measured on the same preparations in the absence of nicardipine. Points represent the mean ± S.E.M. (n = 8). Statistically significant differences as compared with the effect of VOSO4 are indicated by * (P < .05).

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View larger version (50K): [in this window] [in a new window]   Fig. 3.   Effects of calcium-free buffer and TMB-8 on the contractile responses of isolated aorta induced by various agents. Isometric tension responses of isolated rat aortic segments to VOSO4 (5 × 104 M), KCl (40 mM), AVP (3 × 108 M) and NEPI (6 × 107 M) were measured in standard and in calcium-free buffer as described in "Materials and Methods," in the absence or presence of TMB-8 (30 µM). Results are expressed as percent of the control responses obtained in standard buffer on the same preparations. Values are means ± S.E.M. of 6 to 12 experiments. Results obtained with VOSO4 are significantly different (P  .05) from those obtained with the other contractile agents in all cases.

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View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of Ca++ on rabbit skinned mesenteric arteries measured in the absence or presence of various concentrations of VOSO4. Segments of rabbit mesenteric arteries were mounted for isometric tension measurement and skinned with saponin as described in "Materials and Methods." Free Ca++ concentrations were obtained by adding known amounts of CaCl2 in the presence of 4 mM EGTA. Results are expressed as grams of developed tension. Values are means ± S.E.M. of 5 to 10 experiments. * indicates a significant difference (P  .05) compared with the contraction measured in the absence of VOSO4.

Role of tyrosine phosphorylation. The influence of the tyrosine kinase inhibitors tyrphostins and genistein on the contractile activity of VOSO₄ is shown in figure 5. Genistein inhibited VOSO₄-induced contraction, but the effects of the tyrphostins varied according to the compound used (Gazit et al., 1989). According to Gazit et al. (1989), tyrphostins T₁ and T₆₃, with IC₅₀ > 1200 µM in the ability to inhibit EGF receptor tyrosine kinase, were designated as "inactive." Whereas the "inactive" tyrphostins T₁ and T₆₃ had no significant effect on contraction, T₄₇ (IC₅₀ = 2.4 µM) dramatically (14-fold) potentiated the activity of VOSO₄. T₂₃ (IC₅₀ = 35 µM), a milder tyrosine kinase inhibitor than T₄₇, moderately (4-fold) potentiated the activity of VOSO₄. In contrast to its amplification effect on VOSO₄-induced contraction, T₄₇ either had no effect on or inhibited the activity of KCl, NAD and AVP, whereas genistein was inhibitory in all cases (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Effect of various tyrosine kinase inhibitors on VOSO4-induced contraction in rat aorta. Isometric tension measurement of isolated segments of rat aorta was performed in standard Krebs-Henseleit buffer as described in "Materials and Methods." The effect of various tyrosine kinase inhibitors (0.2 mM) on the contractile responses to VOSO4 (104 M) is expressed as percent of the contraction induced by 40 mM KCl in the absence of tyrosine kinase inhibitor (control). Means values of 6 to 9 experiments are shown; vertical lines indicate S.E.M. * indicates a significant difference compared with the contraction obtained with VOSO4 in the absence of inhibitors.

View larger version (57K): [in this window] [in a new window]   Fig. 6.   Identification by Western blotting of phosphotyrosine content of aortic tissue under various conditions. Samples were prepared as described in "Materials and Methods" and resolved in SDS-PAGE. Immunoblotting was performed with RC20 antibodies and revealed by ECL detection buffers. First and second lanes correspond to A431 cell lysate and control aortic tissue without stimulation, respectively. The following lanes correspond to T47 (0.2 mM) alone, VOSO4 (0.1 mM) alone, VOSO4 (0.5 mM) alone, T47 (0.1 mM) and VOSO4 (0.1 mM), pervanadate alone (104 M) and pervanadate (0.1 mM) and T47 (0.2 mM), respectively.

	Discussion

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Although the vasoconstricting properties of vanadium salts were known, the vascular properties of the vanadyl (V⁴⁺) form of vanadium (V), recently described as antihypertensive (Bhanot and McNeill, 1994; Bhanot et al., 1994), had not been specifically studied and characterized. In the present study, we showed that VOSO₄ produced a concentration-dependent vasoconstriction that was fully repeatable on the same preparation even in the presence of high vanadium tissue levels. Vasoconstriction 1) was not endothelium-dependent, 2) was present, although reduced in amplitude, in the absence of extracellular calcium and the presence of blockers of extracellular calcium entry or intracellular calcium mobilization and 3) was absent in skinned preparations. Unlike the response to the potent tyrosine phosphatase inhibitor PV, VOSO₄-induced vasoconstriction was amplified by tyrphostins. The amplification was associated with high levels of tyrosine phosphorylation.

VOSO₄ provoked vasoconstriction at concentrations (10-1000 µM) similar to those previously described with vanadate using the same experimental model (Shimada et al., 1986; Laniyonu et al., 1994; St-Louis et al., 1995). These concentrations appear high compared with those used for known receptor-specific agonists, such as NEPI and AVP (1 pM-1 µM), but are lower than those used for KCl (10-100 mM), a known depolarizing agent. However, we recently showed that VOSO₄ was able to contract isolated smooth muscle cells at subnanomolar concentrations (Soulié et al., 1996), while active concentrations of KCl remained within the 10 mM range (G. Cros, personal communication). These data indicate that VOSO₄ can contract smooth muscle cells at concentrations close to physiological vanadium levels. Considering the capacities of vanadium to bind to tissues, it is possible that accessibility to the reactive site is a major factor determining the active concentration of VOSO₄ in a given preparation.

In addition to yielding previously described vanadate effects, our studies also indicated that VOSO₄-induced vasoconstriction was fully repeatable on the same aortic segment, both at single and multiple cumulative concentrations. A recent study indicated that after a first exposure to PV, the aorta became unresponsive for more than 2 h not only to a second exposure to PV but also to other contractile agents (such as NEPI and KCl) (Laniyonu et al., 1994). These data suggest that VOSO₄ and PV have different mechanisms of action.

VOSO₄-induced vasoconstriction was amplified by endothelium removal. In our study, the same amplification was also obtained in the presence of methylene blue, a well-known inhibitor of guanylyl cyclase (not illustrated), which clearly indicates that VOSO₄ vascular activity was not dependent on the presence of endothelium. This effect is probably modulated by endothelium relaxing factors rather than by liberation of vasoconstrictive mediators. Although Chung et al. (1992) described an endothelium-dependent relaxation factor induced by vanadate when aorta was fully contracted with NEPI, no significant relaxing effect was obtained with VOSO₄ under the same conditions (data not shown).

On the basis of in vitro experiments showing that vanadate (analogous to phosphate) was able to inhibit calcium ATPases (Raeymaekers et al., 1983; Aureliano and Madeira, 1994), it has been widely accepted that inhibition of Ca⁺⁺-ATPase and the consequent intracellular calcium release are responsible for vanadate-induced contraction of smooth muscle (Sanchez-Ferrer et al., 1988; Ozaki and Urakawa, 1980).

Because compelling studies have noted differences in pharmacological properties between vanadyl and vanadate salts, the role of calcium in the mechanism of action of VOSO₄ should be re-assessed.

Our results indicate that extracellular calcium influx plays a significant role in determining the amplitude of VOSO₄-induced contraction. Indeed, in the presence of the voltage-dependent slow Ca⁺⁺ channel blocker N and in the absence of extracellular calcium, the VOSO₄-induced response was reduced to 70% and 35% of control, respectively. Thus, as is true of vanadate, VOSO₄ contractile activity in vascular tissue is not totally dependent on extracellular calcium, which suggests that calcium influx is not the initiating event of contraction.

Intracellular calcium release is unlikely to play a role for the following reasons: 1) In calcium-free buffer, a higher proportion of the contraction was conserved for VOSO₄ (35%) than for any other agent, including NEPI (17% of control). Because NEPI activity is dependent on calcium mobilization from intracellular stores followed by calcium entry from extracellular space (Kowarski et al., 1985), comparison of the inhibiting effects of N and calcium-free buffer on NEPI-induced contraction (16% and 83% of control, respectively) indicates that a major proportion of intracellular mobilizable calcium is absent in our "calcium-free" experimental conditions. 2) TMB-8, a known intracellular calcium mobilization inhibitor, did not modify VOSO₄-induced contraction, either in the presence or in the absence of extracellular calcium, whereas it did inhibit NEPI-induced contraction. 3) Recent data (Lytton et al., 1992) have shown that though all sarcoplasmic or endoplasmic Ca⁺⁺-ATPases (SERCA) isoforms are inhibited by the Ca⁺⁺-ATPase inhibitor thapsigargin, only the SERCA3 isoform is inhibited by vanadate. However, SERCA3 is not expressed in muscle, and other hypotheses must be considered.

It is also possible that the vanadyl ion directly activates the contractile machinery or sensitizes it to the effect of intracellular calcium. Experiments using skinned rabbit mesenteric arteriesthe procedure is unsuccessful in rat aortashowed that, much like vanadate (Nayler and Sparrow, 1983; Sunano et al., 1988), VOSO₄ not only had no effect in the absence of calcium but also inhibited calcium-induced contractions at concentrations higher than 10 µM. Therefore, direct activation or sensitizing is unlikely.

Another hypothesis compatible with our results is that VOSO₄ initiates at the cellular membrane level a cascade of events leading to sensitization to the effects of intracellular calcium and, secondarily, to the entry of extracellular calcium. Such indirect sensitization would be impaired by saponin in our experimental conditions in which agonist-induced contraction is abolished (Itoh et al., 1983). In addition, although the aortic tissue accumulated high amounts of vanadium (4 mM) after a first exposure to VOSO₄, it was still able to respond to a low extracellular concentration of VOSO₄ (0.1 mM), which further suggests that the "initiating event" of contraction is located at the membrane level. A possible candidate for that "initiating event" is phospholipase D (PLD) activation. Indeed, PLD can be activated by an increase in tyrosine phosphorylation (Bourgoin and Grinstein, 1992), and an increase in tyrosine phosphorylation is involved in the mechanism of the insulinomimetic activity of vanadium salts (Brichard and Henquin, 1995). Activation of PLD induces the formation of phosphatidic acid and diacylglycerol, which could sensitize the contractile machinery to calcium without the elevation of intracellular calcium levels. In our study, the response to VOSO₄ was partially blocked by the PLD inhibitor butanol in the presence or absence of calcium (not illustrated), which suggests the possible participation of PLD in the VOSO₄-induced response.

Although the initiating event of contraction was not determined in the present study, the possible role of a tyrosine phosphorylation cascade in the regulation of VOSO₄-induced contraction was explored. Indeed, various studies have shown that tyrosine kinase inhibitors are able to antagonize the smooth muscle contractile activities of vanadate (Di Salvo et al., 1994), PV (Laniyonu et al., 1994) and even various agonists such as angiotensin II (Marrero et al., 1994) and NEPI (Toma et al., 1995). These results indicate that tyrosine phosphorylation plays a major role in the regulation of smooth muscle contraction (Hollenberg, 1994). Although the nonspecific tyrosine kinase inhibitor genistein, which blocks ATP binding to the enzyme, partially inhibited VOSO₄-, KCl-, AVP- and NEPI-induced contractions, the tyrphostins, which are analogous to phosphorylated tyrosine, dramatically amplified VOSO₄-induced vasoconstriction. Their potency was correlated with the ability to antagonize EGF (or PDGF)-induced tyrosine phosphorylation (Gazit et al., 1989). These events occurred both in the presence and in the absence of extracellular calcium and were not dependent on the presence of endothelium. Furthermore, though no change in the degree of tyrosine phosphorylation was apparent with VOSO₄ or T₄₇, a major phosphorylation occurred in the presence of both of them. Unlike VOSO₄, PV, a potent tyrosine phosphatase inhibitor, induced an increase in tyrosine phosphorylation that was prevented by T₄₇, whereas T₄₇ had no influence on PV-induced contraction, as previously shown by Laniyonu et al. (1994).

These data indicate that the correlation between the contractile process and the degree of tyrosine phosphorylation is still unclear, even with compounds known to act on the level of tyrosine phosphorylation, such as PV. It is surprising that a tyrosine kinase inhibitor, which was able to inhibit the increase of tyrosine phosphorylation induced by PV in our experimental conditions, amplifies both VOSO₄-induced contraction and tyrosine phosphorylation. These data indicate either some additional unknown effect of tyrphostins or a complex regulatory process in the tyrosine phosphorylation cascade.

In addition, our data confirmed the differences previously noted among vanadium salts in their cellular mechanisms of action. Indeed, the insulinomimetic activity of PV has been related to the phosphosphorylation of insulin receptor (IR) by inhibition of the IR tyrosine phosphatase (Shisheva and Shechter, 1993a), whereas vanadyl and vanadate are thought to act on the postreceptor cascade (Shisheva and Shechter, 1993b). Contrary to what was recently shown in stretched or pharmacologically stimulated coronary arteries (Adam et al., 1995), the activity of mitogen-activated protein kinase does not seem to be associated with VOSO₄-induced contraction, because no protein band was detected at 45 kDa. It is interesting to note, however, that among the three substrates (205, 116 and 86 kDa) shown by Di Salvo et al. (1993) to be tyrosine-phosphorylated during guinea pig taenia coli exposure to vanadate, two of them were also detected in our experimental conditions after exposure to PV (90 and 200 kDa) or to VOSO₄ and T₄₇ (200 kDa). Further characterization and identification of these bands may yield new insights into the role of tyrosine phosphorylation in the regulation of smooth muscle contraction.

It was recently shown in rat adipocytes that vanadate was able to stimulate in vitro a staurosporine-sensitive cytosolic tyrosine kinase that might be involved in its insulinomimetic activity (Shisheva and Shechter 1993b; Shechter et al., 1995). Preliminary results obtained in our laboratory on vascular tissue indicated that staurosporine had no effect on VOSO₄ in the absence of calcium, while blocking the amplification by T₄₇ (data not shown). Further studies are necessary to establish whether such a tyrosine kinase is involved in the regulation of vascular smooth muscle contractility.

Finally, the question of why a vasoconstricting compound has antihypertensive properties should be asked. When used in vitro, vanadium may have different properties or even properties apparently opposite to those it exhibits during chronic in vivo studies, as we recently showed for insulin secretion (Cadène et al., 1996). Another possibility is that although VOSO₄ induces vasoconstriction in vitro, chronic VOSO₄ treatment lowers insulin resistancea known prohypertensive factorand subsequently corrects hypertension (Bhanot et al., 1995).

In summary, our results indicate that the vascular activity of VOSO₄, 1) is regulated by, but not dependent on, the presence of endothelium, 2) occurs in the absence of extracellular calcium and in the presence of an intracellular calcium mobilization blocker and 3) is amplified by the tyrosine kinase inhibitors tyrphostins (amplification is associated with high levels of tyrosine phosphorylation). As is true of its insulinomimetic activity, the cellular mechanism of the vascular activity of vanadyl appears different from that of PV. Further studies will determine the initiating event of VOSO₄-induced contraction. The surprising amplification of both VOSO₄-induced vascular response and tyrosine phosphorylation by a relatively specific growth factor tyrosine kinase inhibitor may be of interest for future studies on the regulation of tyrosine phosphorylation cascade and its role in the regulation of vascular contractility.