INTRODUCTION

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LITTLE INFORMATION IS AVAILABLE on Ca²⁺ handling and regulation of contractility in human resistance arteries, which are involved in the regulation of blood pressure. Studies concerning Ca²⁺ handling and response to vasoconstrictor agonists have been mainly performed in vascular smooth muscle cells in culture or in vessels taken from laboratory animals. However, there are differences in the mechanisms controlling contraction, depending on the species and vascular bed (2). Human small omental arteries have been used to investigate the influence of pathological states, such as sepsis, on arterial vasomotricity (25). However, the mechanisms regulating contraction are not entirely elucidated in these vessels. It is communally agreed that an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is a determinant for contraction in vascular smooth muscle in response to Ca²⁺-mobilizing agonists (24). In addition, recent studies (12, 29) have shown that most of these agonists are able to modulate contraction by altering myofilament Ca²⁺ sensitivity or through Ca²⁺-independent pathways. The involvement of protein kinase C (PKC) (5, 8, 14, 19), tyrosine kinase (TK) (6, 17, 22), and also Rho-associated protein kinase (ROK) (7, 10, 20) in Ca²⁺ sensitization has been reported in intact and permeabilized arteries from different species. In addition, cross talk between these different kinase pathways may be a key signaling event of Ca²⁺ sensitization of the contractile apparatus during agonist-induced contractile activation of vascular smooth muscle (22). Also, a Ca²⁺-independent kinase associated with the myofilaments may play a role in Ca²⁺ sensitization and Ca²⁺-independent contraction of smooth muscle (29).

Therefore, the aim of the present study was to investigate the relationships between [Ca²⁺]_i and contraction and the mechanisms involved in response to norepinephrine (NE) and the stable analog of thromboxane A₂, U-46619, both of which play an important role in the control of peripheral vascular resistance. The implication of PKC, TK, and ROK in the mechanisms controlling contraction was also studied.

	MATERIALS AND METHODS

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Drugs. Calphostin C and ryanodine were purchased from Calbiochem (Paris, France), GF-109203X was from Interchim (Paris, France), U-46619 was from Cayman Chemical (Ann Arbor, MI), and fura 2-acetoxymethyl ester (AM) was from Molecular Probes (Eugene, OR). Nitrendipine was a generous gift from Bayer (Wuppertal, Germany, and Paris, France), SKF-96365 was from SmithKline Beecham Pharmaceuticals (London, UK), and Y-27632 was from Yoshitomi Pharmaceutical Industries (Japan). All other products were purchased from Sigma (Grenoble, France). Table 1 shows all pharmacological agents used, the respective actions of the agents on the signaling pathway involved for vascular contraction, and the results obtained for each drug.

Arterial preparation and mounting. Omental arteries were isolated from pieces of human omentum harvested for histopathology in patients requiring a large bowel resection for cancer or inflammatory disease. The protocol used was approved by the institutional ethic committee of Strasbourg hospital (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale d'Alscae, Strasbourg, France). In addition, written consent was obtained from each patient. None of the patients (mean age 65 ± 8 yr, 22 female and 15 male) demonstrated a systemic inflammatory response syndrome nor received any cardiovascular therapy. Segments of the omental arteries were collected into cold physiological saline solution (PSS) with the following composition (in mM): 119 NaCl, 4.7 KCl, 0.4 KH₂PO₄, 14.9 NaHCO₃, 1.17 MgSO₄, 2.5 CaCl₂, and 5.5 glucose. The arteries were then cleaned of fat and connective tissue, and a 2-mm-long segment was removed and mounted in a myograph filled with PSS kept at 37°C and continuously gassed (95% O₂-5% CO₂, pH 7.4). Mechanical activity was recorded isometrically with the previously described materials, experimental conditions, and protocol (18). After the vessel was set up, it was equilibrated for 30 min before being passively stretched to an internal diameter that yields a circumference equivalent to 90% of that given by an internal pressure of 100 mmHg, which requires a load of ~400 mg. All the experiments were performed in arteries with an intact endothelium. The presence of a functional endothelium was assessed in all preparations by the ability of bradykinin (1 µM) to induce relaxation of vessels precontracted with either NE (3 µM) or U-46619 (0.3 µM). Under these experimental conditions, bradykinin (1 µM) produced 80 ± 4% (n = 21) and 78 ± 6.0% (n = 11) relaxation of vessels precontracted with NE or U-46619, respectively.

Contraction experiments. Ca²⁺ entry blockers were used to study the Ca²⁺ entry component of the NE- and U-46619-induced contractions. Ca²⁺ entry blockers were applied at maximally active concentrations: 1 µM for the voltage-operated Ca²⁺ channel blocker nitrendipine and 30 µM for the receptor-operated Ca²⁺ channel blocker SKF-96365 (16). To study the component of the 10 µM NE- or 0.3 µM U-46619-induced contractions due to internal Ca²⁺ release, caffeine (10 mM) or ryanodine (10 µM), an activator or an inhibitor of the Ca²⁺-induced Ca²⁺ release channels, respectively, was used at maximally active concentrations. These drugs were incubated for 10 min.

Measurements of [Ca²⁺]_i. Contraction and [Ca²⁺]_i were measured simultaneously; contraction was measured as described above, and changes in [Ca²⁺]_i were determined by measuring the fluorescence of trapped fura 2 with a dual-excitation wavelength fluorometer (Fluorolog II, SPEX, Edison, NJ). The vessel segments were loaded with fura 2 by incubating the segments with PSS containing 5 µM fura 2-AM and 20% pluronic acid in the dark for 2 h. PSS was kept at 37°C and continuously gassed (95% O₂-5% CO₂, pH 7.4). For Ca²⁺ signal calibration, vessels were treated with ionomycin (20 µM), NE or U-46619, and CaCl₂ (5 mM) for the maximal fluorescence and 20 mM EGTA in Ca²⁺-free solution for the minimal fluorescence. The ratio of fluorescence (measured at 510 nm) obtained at the two excitation wavelengths (340/380 nm) was calculated after subtraction of the autofluorescence at 340 and 380 nm. The change in [Ca²⁺]_i was calculated as described previously (11) and was expressed in nanomoles per liter (nM).

Permeabilization of the small omental arteries. -Escin was used for the permeabilization of arteries using the method previously described by Kitazawa et al. (15) with minor modifications. The permeabilization process was achieved by incubating the arteries in the relaxing solution containing 50 µM -escin at room temperature for 45 min. The arteries were then mounted, passively stretched at 400 mg as described above, and equilibrated for 20 min in the relaxing solution containing 20 mM PIPES, 10 mM creatine phosphate, 5.2 mM Na₂ATP, 5.1 mM magnesium methanesulfonate, 87 mM potassium methanesulfonate, 1 µM leupeptin, 1 µM ionomycin, 0.1 µM calmodulin, and 4 mM EGTA. The experiment was carried out at room temperature. For contraction experiments, the concentration of EGTA was decreased from 4 to 2 mM. The tension developed to a high concentration of Ca²⁺ using calcium methanesulfonate (pCa 5.0) was recorded. After the arteries were reequilibrated in the relaxing solution, a submaximal Ca²⁺ concentration (pCa 6.0) was added. When the contraction reached a steady-state level, NE or U-46619 was added in the presence of GTP (10 µM). The same experimental protocol was performed on vessels that had been preincubated for 30 min with GF-109203X or genistein. In another set of experiments, Y-27632 was added cumulatively on vessels precontracted with pCa 6.0.

Western blotting. The intact arteries were incubated for 30 min at 37°C and gassed (95% O₂-5% CO₂) in the absence or in the presence of genistein or tyrphostin A-23. NE and U-46619 were added for 5 or 10 min, respectively, before homogenization. Approximately 100 µg of total protein for supernatant fractions were used for electrophoresis (10% SDS-PAGE) and transferred to a nitrocellulose membrane. Immunostaining of tyrosine phosphorylation or ROK protein was achieved using a specific anti-phosphotyrosine monoclonal antibody (UBI) or anti-ROK monoclonal antibody (Transduction Laboratories) and then reacted with peroxidase-conjugated mouse antibody (Bio-Rad). Antibody-labeled bands on the blots were detected using an Enhanced Chemiluminescence assay (Amersham). The level of tyrosine phosphorylation was measured by densitometry.

Expression of results and statistical analysis. The increase of tension was measured at the peak of contraction induced by the agonists. Contractions were expressed as a percentage of the maximal contractile response obtained with 100 mM KCl-PSS plus U-46619 (0.3 µM). The concentration-response curves to the agonist (for example, for U-46619) were not significantly different in arteries with and without functional endothelium, as previously shown (18). The EC₅₀ and maximal active tension obtained with U-46619 were 99.7 ± 21.6 nM and 92 ± 2% K⁺ 100 mM (n = 21) and 68.4 ± 33.3 nM and 89 ± 3% K⁺ 100 mM (n = 9) in the presence and absence of endothelium, respectively. All results are expressed as means ± SE of n experiments, with n representing the number of patients. A two-tailed paired Student's t-test was used to compare data obtained in the same segment before and after treatment. P < 0.05 was considered significant.

	RESULTS

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Effect of depolarization, NE, and U-46619 on [Ca²⁺]_i and contraction. Figure 1, A and B, shows increases in [Ca²⁺]_i and contraction induced by KCl, NE, and U-46619 (all at maximally active concentrations). The increases in [Ca²⁺]_i induced by NE and U-46619 were significantly lower than that produced by KCl (P < 0.05, Fig. 1A). However, the contractile responses of the vessels to KCl, NE, and U-46619 were not significantly different (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Histograms showing the increase in both intracellular Ca2+ concentration ([Ca2+]i) (A) and tension (B) induced by 100 mM KCl-physiological saline solution (PSS), 10 µM norepinephrine (NE), and 0.3 µM U-46619 in normal PSS. Values are taken at the plateau of the contraction produced by the stimulating agent. Values are means ± SE (n = 5-8 arteries). *P < 0.05 significantly different from the response produced by KCl-PSS. C and D: representative traces showing the responses in both [Ca2+]i (top) and tension (bottom) to the addition of NE (C) or U-46619 (D) in arteries exposed to KCl-PSS. In -escin-permeabilized arteries, NE- (E) and U-46619-induced (F) sensitizations were evaluated at pCa 6.0 in the presence of GTP (10 µM). Dotted line shows the effect of GTP alone. Traces are representative of 3 experiments.

Influence of extracellular Ca²⁺. In nominally Ca²⁺-free medium, the agonists elicited a transient contraction without any detectable increase in [Ca²⁺]_i (Fig. 2). Furthermore, addition of extracellular CaCl₂ in the presence of the agonists restored both [Ca²⁺]_i and tension to the same levels as previously reached in normal PSS. Nitrendipine did not significantly modify the contractile responses elicited by the agonists in Ca²⁺-free medium but greatly reduced the contraction induced by CaCl₂ in the presence of the agonist (Table 2). SKF-96365 did not affect the nitrendipine-insensitive component of the CaCl₂ responses in arteries exposed to the agonists.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Representative traces (left) showing the increase in [Ca2+]i (A and C) and contractile tension (B and D) elicited by NE (A and B) and U-46619 (C and D) in Ca2+-free PSS followed by addition of CaCl2. Histograms (right) showing the mean of the responses induced by the agonists in normal PSS, in Ca2+-free medium, and after subsequent addition of CaCl2 (1 mM) in the continuous presence of the 10 µM NE (A and B) or 0.3 µM U-46619 (C and D). Values are means ± SE (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001: significantly different from agonist responses in normal PSS. P < 0.01 and P < 0.001: significantly different from agonist responses in Ca2+-free medium.

Effects of agents interfering with Ca²⁺ storage mechanisms. In Ca²⁺-free medium, caffeine consistently elicited a moderate transient contractile response (Fig. 3), and caffeine almost abolished subsequent contraction on addition of NE. However, caffeine reduced but did not abolish contraction induced by U-46619. Exposure to ryanodine did not cause any increase in tension, but ryanodine blunted the contractile responses to both agonists to the same extent as did caffeine (Table 2).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Representative traces showing the effects of caffeine (10 mM) on contractions induced by 10 µM NE (A) and 0.3 µM U-46619 (B) in Ca2+-free medium. Traces are representative of 7 experiments.

Effect of PKC and TK inhibitors on intact and -escin-permeabilized arteries. Figure 4, A and B, shows that staurosporine, calphostin C, and GF-109203X decreased, to the same extent, the transient contraction induced by NE or U-46619 in Ca²⁺-free medium but did not affect responses to the subsequent addition of CaCl₂ in the presence of the agonists. It should be noted that the three PKC inhibitors did not affect the contractile response to KCl (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   A and B: representative traces (left and middle) showing the effects of staurosporine (30 nM) and histograms (right) showing the effects of staurosporine (Staur, 30 nM), calphostin C (Calph, 0.1 µM), and GF-109203 (GF, 3 µM) on contractions induced by 10 µM NE (A) and 0.3 µM U-46619 (B) in Ca2+-free medium and after subsequent addition of CaCl2 (1 mM). C and D: representative traces (left and middle) showing the effects of tyrphostin A-23 (100 µM) and histograms (right) showing the effects of tyrphostin A-23 (Tyrphost, 100 µM) and genistein (Genist, 30 µM) on contractions induced by NE (C) and U-46619 (D) in the Ca2+-free medium and the subsequent addition of CaCl2. Values are means ± SE (n = 5-8). *P < 0.05: significantly different from agonist responses in Ca2+-free medium in the absence of the inhibitors. P < 0.05 and P < 0.01: different from the contraction induced by CaCl2 in the absence of the inhibitors.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   NE (10 µM)- and U-46619 (0.3 µM)-stimulated tyrosine-phosphorylated proteins. Human small mesenteric arteries were treated with or without the agonist (NE or U-46619) in the absence or in the presence of the indicated inhibitor (genistein or tyrphostin). Western blots (top) were performed, and the level of phosphorylation (histograms, bottom) was measured as described in MATERIALS AND METHODS. The data are representative of 4 experiments. Intensity of control conditions was considered as the unity. *P < 0.05 significantly different from controls.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Representative traces and histograms showing the effects of GF-109203X (5 µM) (middle) and genistein (30 µM) (bottom) on the 10 µM NE (left)- and 0.3 µM U-46619-induced (right) sensitizations evaluated at pCa 6.0 in the presence of GTP (10 µM) in -escin-permeabilized arteries (top). Traces are representative of 10 experiments.

Effect of ROK inhibitor on intact and -escin-permeabilized arteries. In intact arteries, the ROK inhibitor Y-27632 produced a concentration-dependent relaxation of vessels contracted by NE and U-46619 (Fig. 7A). However, Y-27632 had no significant effect on either contraction induced by 100 mM KCl in intact arteries (Fig. 7A) or Ca²⁺-induced contraction (pCa 6.0) in -escin-permeabilized arteries, except at the highest concentration used (0.3 mM, P < 0.05) (Fig. 7B). Finally, simultaneous measurement of [Ca²⁺]_i and contraction showed that Y-27632 (100 µM) induced relaxation of vessels precontracted with U-46619 without any decrease of [Ca²⁺]_i. The contractile response and the increase in [Ca²⁺]_i produced by U-46619 were 92 ± 2% K⁺ 100 mM and 389.3 ± 7.6 nM, respectively; U-46619-induced contraction was relaxed by Y-27632 to 3.8 ± 1.3% (P < 0.001, n = 3), but [Ca²⁺]_i was not modified [372.5 ± 9.3 nM (n = 3)]. Taken together, these results suggest that this compound is a potent inhibitor of agonist-induced smooth muscle contraction.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Cumulative concentration-response curves to Y-27632 in vessels precontracted with either KCl-PSS, NE (10 µM), or U-46619 (0.3 µM) in intact arteries (A) or with pCa 6.0 in -escin-permeabilized arteries (B). Values are means ± SE (n = 5-8).

	DISCUSSION

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The above results show the involvement of the following mechanisms in contractile responses of human omental arteries to NE and U-46619: there is a Ca²⁺ release from stores sensitive to ryanodine and caffeine, a Ca²⁺ entry via nitrendipine-sensitive mechanisms, an activation of Ca²⁺-independent signaling pathways, and an enhanced responsiveness of the contractile machinery to Ca²⁺. An important issue in this study is that these mechanisms are differentially regulated by kinases. TK appears to be a convergent pathway in regulating Ca²⁺ entry and sensitization. Interestingly, agonist-induced Ca²⁺ sensitization involves the participation of PKC, TK, and ROK inhibitor-sensitive mechanisms.

Ca²⁺ storage and release mechanisms were studied in nominally Ca²⁺-free medium to eliminate Ca²⁺ entry (Fig. 2). Although agonist-induced contraction was not associated with any detectable increase in [Ca²⁺]_i in these conditions, experiments performed with ryanodine and caffeine suggest that the release of Ca²⁺ from a store sensitive to Ca²⁺-induced Ca²⁺ release agents was nevertheless implicated in contractile responses elicited by the agonists (Fig. 3). Also, inositol 1,4,5-trisphosphate-sensitive Ca²⁺ stores might be implicated in the contractile responses obtained in Ca²⁺-free medium. Indeed, it is generally known that contraction elicited by activators of G protein-coupled receptors such as -adrenoceptors or TP receptors induced the concomitant production of both inositol 1,4,5-trisphosphate and diacylglycerol (an activator of PKC) through the same enzymatic reaction (3). Another interpretation of these results could be that the contraction observed in Ca²⁺-free medium may result from an agonist-mediated Ca²⁺-independent pathway or sensitization of contractile proteins to Ca²⁺. However, it cannot be excluded that both agonists could induce a focal increase in [Ca²⁺]_i, probably in the vicinity of contractile proteins. Such a focal increase in [Ca²⁺]_i might not be detected by fura 2 probe, but nevertheless may be sufficient to produce contraction. Indeed, it has been reported that fura 2, in contrast to aequorin, is not able to detect a focal increase in [Ca²⁺]_i in vascular smooth muscle cells during the release and refilling of Ca²⁺ stores (21).

In the present study, CaCl₂ induced a large and sustained increase in [Ca²⁺]_i and contraction after depletion of Ca²⁺ stores by the agonists (Fig. 2). In addition, this contraction was greatly reduced by the dihydropyridine Ca²⁺-entry blocker nitrendipine but not further reduced by SKF-96365, a relative selective inhibitor of receptor-operated Ca²⁺ channels (16) (Table 2). Thus, in human small omental arteries, NE and U-46619 stimulate Ca²⁺ entry mainly through a mechanism sensitive to nitrendipine, probably via voltage-dependent Ca²⁺ channels. These data are in accordance with those reported in the literature (27) showing that U-46619 contracts human umbilical artery by promoting Ca²⁺ entry through dihydropyridine-sensitive Ca²⁺ channels.

We showed that in -escin-permeabilized arteries, NE and U-46619 increased force at a constant Ca²⁺ concentration (Fig. 1). The requirement for GTP of these responses is consistent with the activation of G protein-coupled receptors accounting for this effect of the agonists. In addition, both agonists are able to produce contractions of intact arteries in both normal and Ca²⁺-free medium without a change in [Ca²⁺]_i. Taken together, these results show the participation of either Ca²⁺-independent mechanisms, Ca²⁺ sensitization, or both in the pathway leading to contraction of human small omental arteries in response to NE and U-46619.

Ca²⁺-independent pathways might occur in the response to the two agonists. Myosin light-chain phosphorylation has not been assessed here, but the mechanism involved might be due to the activation of Ca²⁺-independent light-chain kinase, as very recently reported (29). Ca²⁺ sensitization (i.e., treatment of permeabilized smooth muscles at fixed submaximal [Ca²⁺]_i with a variety of agents leading to an increase in force concomitant with an increase in phosphorylation of the 20-kDa light chain of myosin) can be controlled by the inhibition of myosin light-chain phosphatase (MLCP) through the small GTP-binding protein RhoA or PKC. Phosphorylation of other proteins, such as caldesmon by mitogen-activating protein kinase (MAPK) or calponin by PKC, has been suggested to play a role for Ca²⁺ sensitization (12). Also, a Ca²⁺-independent light-chain kinase may play a role in Ca²⁺ sensitization (29). With regard to the first point, an inhibitor of MLCP was not directly assessed here, but the ROK inhibitor, Y-27632, which is known to selectively inhibit smooth muscle contraction by inhibiting Ca²⁺ sensitization through small GTP-binding protein (7, 28), strongly relaxed agonist- but not KCl-induced contraction (Fig. 7). In addition, the relaxation produced by Y-27632 was not associated with a decrease in [Ca²⁺]_i on vessels activated by U-46619. Thus these results suggest that a ROK-associated pathway is involved in agonist-induced Ca²⁺ sensitization of human small omental arteries. With regard to the second point, it is well known that MAPK activation can be controlled by the TK pathway. Indeed, it has been reported that, in human vascular smooth muscle cells (26), angiotensin II-stimulated contraction is regulated by MAPK, suggesting a role for this pathway in agonist-induced contraction. In the present study, the participation of TK was investigated using two TK inhibitors acting through different mechanisms (1, 9) on the contractions produced by NE and U-46619. Because KCl-mediated contraction was not affected by both inhibitors, it is unlikely that genistein and tyrphostin A-23 had a direct effect either on the voltage-dependent Ca²⁺ channels or on the contractile apparatus. However, TK inhibitors abolished agonist-induced Ca²⁺ sensitization in permeabilized arteries (Fig. 6). In addition, NE and U-46619 produced tyrosine phosphorylation of two different proteins of 42 or 58 kDa, respectively, and genistein and tyrphostin A-23 inhibited these phosphorylations (Fig. 5). The exact nature of the proteins activated by both agonists remains to be determined. In addition, whether phosphorylation of these proteins might participate in the contractile response to NE and U-46619 also needs further investigation. Nevertheless, these results suggest that TK are implicated in agonist-induced Ca²⁺ sensitization in human small omental arteries. Finally, the role of PKC was investigated using three inhibitors: staurosporine, calphostin C, and GF-109203X (Fig. 4). These inhibitors decreased contraction induced by both agonists in Ca²⁺-free medium in intact arteries in which no detectable increase of [Ca²⁺]_i was observed. In addition, GF-109203X completely abolished agonist-induced contraction in -escin-permeabilized arteries (Fig. 6). Thus it is likely that PKC is also implicated for Ca²⁺ sensitization or Ca²⁺-independent contraction in the present study.

Cross-talk between kinase pathways (PKC/ROK or TK/ROK) in the regulation of Ca²⁺ sensitization has been previously described. It has been suggested that Rho p21, which activates ROK, mediates tyrosine phosphorylation of multiple substrates (13, 23), and the Rho/ROK pathway could be activated by PKC (5). In the present study, inhibition of agonist-induced Ca²⁺ sensitization by PKC, TK, and ROK inhibitors (also, these inhibitors had differential effects on Ca²⁺ handling, as discussed below) supports the view that these kinases interact with each other in the Ca²⁺ sensitization of myofilaments in human small omental arteries.

The role of PKC and TK on agonist-stimulated Ca²⁺ entry through the plasma membrane was also investigated. With regard to PKC, neither staurosporine, calphostin C, nor GF-109203X had any effect on contraction produced by CaCl₂ in the presence of agonists after Ca²⁺ store depletion (Fig. 4). At the concentrations used in the present study, PKC inhibitors did not affect the contractile response produced by KCl depolarization. These data contrast with those found in rat vas deferens, in which NE induces contraction via the activation of PKC and a subsequent influx of Ca²⁺ through voltage-dependent Ca²⁺ channels (4). Possible reasons for differential results might be due to differences in the species, in the tissues studied, or both. Nevertheless, activation of PKC appears not to be essential in agonist-induced Ca²⁺ entry in human small omental arteries. With regard to the participation of TK in the regulation of Ca²⁺ entry (Fig. 4), both genistein and tyrphostin A-23 reduced the CaCl₂-induced responses in NE- or U-46619-exposed vessels without affecting the contractile response to KCl depolarization. Results obtained with KCl did not support the view that TK inhibitors directly affect the activity of voltage-dependent Ca²⁺ channels. These results suggest that TK affects a step involved in the activation of Ca²⁺ entry linked to nitrendipine-sensitive channels after stimulation by both agonists. Interestingly, NE and U-46619 produced tyrosine phosphorylation and Ca²⁺ sensitization through a TK inhibitor-sensitive mechanism (as discussed above). Thus tyrosine phosphorylation may be a convergent pathway for agonist-mediated Ca²⁺ entry and sensitization of smooth muscle of human small omental arteries.

Finally, the use of TK inhibitors in agonist-induced responses in Ca²⁺-free medium did not support the view that these kinases play an important role in mediating the responses linked to the tonic but not phasic component of contraction produced by NE and U-46619. In contrast, the PKC pathway modulates the phasic but not the tonic component of agonist-induced contraction.

In conclusion, NE- and U-46619-induced contractions are associated with an increase in [Ca²⁺]_i, an increase in Ca²⁺-independent signaling pathways, or an enhancement of the sensitivity of the myofilaments to Ca²⁺ in human small omental arteries. Ca²⁺ influx through nitrendipine-sensitive channels and Ca²⁺ release from caffeine- and ryanodine-sensitive stores are involved in the mechanisms leading to contraction. A dual role of the TK pathway in regulating the Ca²⁺ influx and Ca²⁺-independent signaling pathways or Ca²⁺ sensitization elicited by both agonists is suggested, whereas PKC and ROK are not implicated in Ca²⁺ influx.

This study provides new information on the mechanisms regulating contractility in resistance arteries in humans. Here we described the involvement of multiple regulatory kinase pathways in regulating Ca²⁺ handling in human small omental arteries.