INTRODUCTION

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IT IS WELL ESTABLISHED that the contractile response to a number of agonists including norepinephrine (NE) comprises two distinct components in Ca²⁺-containing medium: an initial phasic component that results from the inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-mediated release of Ca²⁺ from intracellular Ca²⁺ stores followed by a tonic component that requires Ca²⁺ entry in the continuous presence of the agonist, due to Ca²⁺ influx (4).

Despite the existence of a large body of literature exploring the roles of Ca²⁺ influx and release from intracellular stores in the vasoconstriction induced by agonists, the relative contribution of each of these mechanisms is still under debate. In vascular smooth muscle, it seems that the mechanisms of Ca²⁺ release triggering contraction may vary between vascular beds and with the stimulus. For example, in the rat portal vein, one of the two characterized Ca²⁺ stores is sensitive to caffeine [an opener of "Ca²⁺-induced Ca²⁺ release" ryanodine-sensitive channels (CICR)], whereas both of them are sensitive to Ins(1,4,5)P₃ (33). Conversely, only one of the two Ca²⁺ stores characterized in the dog mesenteric artery is released by Ins(1,4,5)P₃, whereas the other is mobilized by Ca²⁺ (22). These observations suggest differential involvement of CICR and Ins(1,4,5)P₃-sensitive Ca²⁺ release channels and associated stores.

Ca²⁺ influx across the plasma membrane generally involves voltage-operated channels and the so-called "capacitative entry pathway" activated by store depletion (10). However, the signaling mechanism for Ca²⁺ entry via the latter pathway is complex and may also be different depending on the cell type. A number of mediators have been proposed to be implicated for this phenomenon, although no such mediator has been unequivocally demonstrated as yet. These mediators included inositol 1,3,4,5-tetrakisphosphate (17), small G proteins (5), a "Ca²⁺-influx factor," a small phosphate containing nonprotein compound (30), cytochrome P-450, or one of their products (2). Finally, a role for tyrosine kinase has been proposed in activation of the capacitative Ca²⁺ entry (16).

All of the above findings suggest that agonist stimulation may activate more than one Ca²⁺ entry pathway that may be different depending on the cell type. In resistance vessels, the presence of both ryanodine- and Ins(1,4,5)P₃-sensitive Ca²⁺ release mechanisms has been characterized in the rat small mesenteric artery (32). However, the involvement of these two mechanisms in NE-induced contraction and the relationship between Ca²⁺ store depletion and Ca²⁺ entry is unknown.

The aim of the present study is to further characterize the intracellular Ca²⁺ stores released by NE and to investigate the mechanism of Ca²⁺ entry during the tonic contraction produced by this agonist in the small mesenteric resistance artery of the rat, using different pharmacological agents active on the different pathways possibly involved.

	METHODS

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Arterial preparation and mounting. Male Wistar rats (250-350 g) bred in our institute were killed by cervical dislocation and exsanguinated by carotid artery transection. The viscera were exposed, and a proximal segment of the small bowel was removed and pinned in a dissecting dish containing physiological salt solution (PSS) of 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. In Ca²⁺-free PSS, Ca²⁺ was omitted and 1 mM EGTA was added. Branch II or III resistance arteries were cleaned of fat and connective tissue, and a segment 2 mm long was removed. The segment was then mounted in a myograph (as previously described in Ref. 3) filled with PSS continuously kept at 37°C and gassed with a mixture of 95% O₂-5% CO₂ (pH 7.4). Briefly, two tungsten wires (30 µm diam) were inserted through the lumen of the vessel. Mechanical activity was recorded isometrically by a force transducer (Kistler-Morse, DSG BE4) connected to one of the two tungsten wires, the other being attached to a support carried by a micromanipulator.

Contraction experiments. In the present study, NE was used throughout the experiments at a concentration (10 µM) giving the maximal contractile response in small mesenteric artery of the rat.

Measurement of intracellular Ca²⁺. Simultaneous measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and contraction were performed to study the relationships between Ca²⁺ stores, Ca²⁺ entry, and the contraction induced by NE in small mesenteric arteries. 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) as described by Andriantsitohaina et al. (3). The vessel segments were loaded over a 2-h period with fura 2 by incubation in the dark in PSS containing both 5 µM fura 2-AM and 2% pluronic acid. The tissue was then returned to the chamber and washed three times with PSS (37°C) to remove excess external fura 2-AM. The experiments were performed in PSS (37°C) continuously gassed with 95% O₂-5% CO₂ (pH 7.4). At the end of each experiment, the Ca²⁺ signal was calibrated using ionomycin (20 µM), NE (10 µM), and CaCl₂ (5 mM) for the maximal fluorescence and 20 mM EGTA in Ca²⁺-free solution for the minimal fluorescence. The ratio of fluorescence of the emission of fura 2 was obtained at 510 nm and was calculated after subtraction of the autofluorescence at 340 and 380 nm. Background fluorescence and autofluorescence were measured by looking at the fluorescence of the vessel without the dye at 510 nm after its excitation at 340 and 380 nm.

Immunofluorescence labeling of SERCA2a and SERCA2b. Segments of rat small mesenteric arteries were immersed in freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 6 h. They were then soaked overnight in buffer containing 20% sucrose. The samples were then frozen in cooled isopentane (50°C) and sectioned on a cryostat microtome; 12-µm-thick sections were flow-mounted on gelatin-coated slides. The sections were then processed with the conventional indirect immunofluorescence technique. The antibodies used were the SERCA2a and SERCA2b isoform-specific polyclonal antibodies elicited in rabbits against peptides encompassing the 12 COOH-terminal amino acids (1031-1042) of SERCA2b and 9 COOH-terminal amino acids (989-997) of SERCA2a. The preparation and specificity of these antibodies have both been previously described (35). The antibodies against SERCA2a and SERCA2b were diluted 1:50 in 0.05 M phosphate-buffered 0.9% saline, pH 7.3, containing 0.5% Triton X-100. The primary antibodies were applied overnight at 4°C. The secondary antibody was the FITC-labeled anti-rabbit IgG (Biosys, France). FITC-labeled sections were coverslipped in polyvinyl alcohol mounting medium containing paraphenylene diamine. Control experiments were similarly processed omitting the primary antibodies.

Expression of results and statistical analysis. Contractions were expressed as a percentage of the maximal contractile response obtained with 10 µM NE. [Ca²⁺]_i was calculated using the equation previously described by Grynkiewicz et al. (15) and expressed in nanomolar. In the resting state, basal [Ca²⁺]_i was 108 ± 14 (n = 5).

Drugs. Acetylcholine chloride, caffeine, CHAPS, cyclopiazonic acid, DMTU, genistein, MgCl₂, NE bitartrate, NiCl₂, thapsigargin, tyrphostin 23, and staurosporine were purchased from Sigma (Grenoble, France). Calphostin C and ryanodine were purchased from Calbiochem (France). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). DCB was a generous gift from Dr. C. Frelin (Nice, France). Nitrendipine was a generous gift from Bayer (Paris, France). SKF-96365 was a generous gift from SmithKline Beecham Pharmaceuticals (London, UK). SERCA2a and SERCA2b were a generous gift from Dr. F. Wuytack (Katholieke Universiteit Leuven, Belgium). Nitrendipine was dissolved in absolute ethanol. Calphostin C, DCB, and thapsigargin were dissolved in dimethyl sulfoxide (1 mg/ml), and the final concentration of dimethyl sulfoxide in the bath was 0.1%. Genistein, tyrphostin 23, and staurosporine were dissolved in dimethyl sulfoxide, and the maximal concentration of dimethyl sulfoxide in the bath was 1.2% for 100 µM tyrphostin 23. All other drugs were diluted in deionized water (Q10, Millipore).

	RESULTS

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Acetylcholine was not able to produce relaxation in vessels precontracted with NE after endothelium denudation with CHAPS. Also, the latter procedure resulted in a 10-fold increase in sensitivity to NE, the EC₅₀ values being reduced from 1.01 ± 0.01 µM (n = 90) to 0.101 ± 0.01 µM (n = 62; P < 0.001). Maximal peak response obtained with 10 µM NE was not significantly different in vessels with and without functional endothelium [1.53 ± 0.48 g (n = 90) and 1.14 ± 0.47 g (n = 62), respectively]. These data show that the use of CHAPS to functionally denude the endothelium removed the inhibitory effect of the endothelium without significantly altering the contractile function of the vessel.

Characterization of intracellular stores released by NE. Immunostainings using specific antibodies for SERCA2a and SERCA2b isoforms were performed to assess the presence and distribution of SERCA isoforms in different layers of the vessels (Fig. 1). SERCA2a (Fig. 1A) and SERCA2b (Fig. 1B) stainings were observed in the endothelial and in the medial layers of the vessels. No staining was observed in negative controls performed in vessels in the absence of SERCA2a and SERCA2b antibodies (Fig. 1C).

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Immunofluorescence labelings of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a and SERCA2b of small mesenteric arteries representative of 3 different experiments. A: SERCA2a. B: SERCA2b. C: negative control. Calibration bar, 50 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   A: representative traces showing effects of caffeine (Caf; 10 mM) and thapsigargin (Thaps; 1 µM) on increase in both intracellular free Ca2+ concentration ([Ca2+]i) (top traces) and tension (bottom traces) induced by norepinephrine (NE; 10 µM) in Ca2+-free medium. B: histograms showing contractions induced by NE (10 µM) in Ca2+-free medium in absence (1st bar, n = 6) and presence of 10 mM Caf (2nd bar, n = 6), 10 µM ryanodine (3rd bar, n = 5), and either 1 µM Thaps (4th bar, n = 6) or 20 µM cyclopiazonic acid (5th bar, n = 6). Contractions are percentage of maximal contraction elicited by 10 µM NE in same arteries in normal PSS. Values are means ± SE. *** P < 0.001, significantly different from control.  P < 0.05, significantly different from contraction produced by NE in presence of Caf.

Mechanisms of Ca²⁺ entry after depletion of intracellular stores with NE. None of the pharmacological agents used significantly affected the contraction produced by NE in Ca²⁺-free medium except DCB (Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   A: representative traces showing effects of nitrendipine (1 µM) and SKF-96365 (30 µM) on increase in both [Ca2+]i (top traces) and tension (bottom traces) induced by CaCl2 (2.5 mM) on NE (10 µM)-exposed vessels after depletion of intracellular stores. B: histograms showing contractions induced by addition of CaCl2 (2.5 mM) in small mesenteric resistance arteries exposed to 10 µM NE in absence (1st bar, n = 44) and presence of either 1 µM nitrendipine (2nd bar, n = 9), 30 µM SKF-96365 (3rd bar, n = 5), or 1 mM NiCl2 (4th bar, n = 5). Contractions are percentage of maximal contraction elicited by 10 µM NE in same arteries in normal PSS. Values are means ± SE. *** P < 0.001, significantly different from control.  P < 0.05, significantly different from contraction produced by NE in presence of nitrendipine.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   A: representative traces showing effects of 1,3-dimethyl-2-thiourea (DMTU; 25 mM) and amiloride (1 mM) on increase in both [Ca2+]i (top traces) and tension (bottom traces) induced by CaCl2 (2.5 mM) on NE (10 µM)-exposed vessels after depletion of intracellular stores. B: histograms showing contractions induced by addition of CaCl2 (2.5 mM) in small mesenteric resistance arteries exposed to 10 µM NE in absence (1st bar, n = 4) and in presence of either 25 mM DMTU (2nd bar, n = 5), 10 µM 3',4'-dichlorobenzamil (3rd bar, n = 5), 1 mM amiloride (4th bar, n = 5), 10 mM MgCl2 (5th bar, n = 5), or substitution of NaCl with choline (6th bar, n = 5). Contractions are percentage of maximal contraction elicited by 10 µM NE in same arteries in normal PSS. Values are means ± SE. *** P < 0.001, significantly different from control.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   A: representative traces showing effects of genistein (30 µM) on increase in both [Ca2+]i (top traces) and tension (bottom traces) induced by CaCl2 (2.5 mM) on NE (10 µM)-exposed vessels after depletion of intracellular stores. B: histograms showing contractions induced by addition of CaCl2 (2.5 mM) in small mesenteric resistance arteries exposed to 10 µM NE in absence (1st bar, n = 44) and presence of either 30 µM genistein (2nd bar, n = 6), 100 µM tyrphostin 23 (3rd bar, n = 5), genistein plus nitrendipine (4th bar, n = 5), or tyrphostin 23 plus nitrendipine (5th bar, n = 5). Contractions are percentage of maximal contraction elicited by 10 µM NE in same arteries in normal PSS. Values are means ± SE. *** P < 0.001, significantly different from control.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   A: representative traces showing effects of staurosporine (30 µM) on increase in both [Ca2+]i (top traces) and tension (bottom traces) induced by CaCl2 (2.5 mM) on NE (10 µM)-exposed vessels after depletion of intracellular stores. B: histograms showing contractions induced by addition of CaCl2 (2.5 mM) in small mesenteric resistance arteries exposed to 10 µM NE in absence (1st bar, n = 24) and presence of either 30 µM staurosporine (2nd bar, n = 7), 1 µM calphostin C (3rd bar, n = 4), genistein plus staurosporine (4th bar, n = 5), or genistein plus calphostin C (5th bar, n = 4). Contractions are percentage of maximal contraction elicited by 10 µM NE in same arteries in normal PSS. Values are means ± SE. *** P < 0.001, significantly different from control.

	DISCUSSION

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The above results characterize intracellular stores from which Ca²⁺ are released by NE and the mechanisms of Ca²⁺ entry triggered by the agonist in rat small mesenteric artery smooth muscle cells. One major finding is that, in NE exposed arteries, tonic contraction induced by the addition of CaCl₂ after depletion of Ca²⁺ stores involved the Na⁺/Ca²⁺ exhanger in addition to Ca²⁺ entry through both dihydropyridine- and SKF-96365-sensitive mechanisms. In addition, the data obtained with inhibitors support the involvement of both tyrosine kinase and protein kinase C in Ca²⁺ entry elicited by NE. All of the above mechanisms required the continuous presence of the agonist.

The results indicate that the release of Ca²⁺ induced by NE takes place at the level of an intracellular Ca²⁺ store that can also be mobilized by caffeine and ryanodine.

Two SERCA inhibitors, thapsigargin and cyclopiazonic acid, with different chemical structures, were used at concentrations that are able to cause a rapid increase in [Ca²⁺]_i and to empty Ca²⁺ stores in other vascular smooth muscle cells (9) and vessels (23, 27). Despite the presence in the medial layer of the two SERCA isoforms (SERCA2a and SERCA2b), which are generally found in vascular smooth muscle cells and are inhibited by both thapsigargin and cyclopiazonic acid (21), both drugs failed to increase [Ca²⁺]_i and tension in rat small mesenteric arteries. It cannot be ruled out that both thapsigargin and cyclopiazonic acid did not reach the SERCA at sufficient concentrations to entirely inhibit them, although this is unlikely because the small mesenteric arteries are much thinner than other vessels like the aorta. It is possible that the fura 2 probe could not detect local increase in [Ca²⁺]_i produced by SERCA inhibitors in the absence of any signal inducing sufficient Ca²⁺ release (31). However, the data reported above suggest that NE induced the release of Ca²⁺ from two storage compartments in small mesenteric arteries smooth muscle cells, one of them being sensitive to thapsigargin and cyclopiazonic acid, and both of them can be depleted by the CICR reagents caffeine and ryanodine. This interpretation is consistent with the recent finding that a thapsigargin-insensitive Ca²⁺ pool is present in a subcompartment of the endoplasmic reticulum in different mammalian cells (29). In addition, the presence of SERCA2a and SERCA2b in the endothelium layer of small mesenteric arteries found in the present study is consistent with previously reported endothelium-dependent relaxation elicited by SERCA inhibitors in these vessels (11).

In general, depletion of Ca²⁺ stores is believed to induce the so-called capacitative Ca²⁺ entry. This phenomenon has been described in a vascular muscle cell line (A7r5) after store depletion by thapsigargin or by an agonist, vasopressin (7). It probably results from Ca²⁺ release activated current described in various cells (10). The activation of this current relies on store depletion, but it does not require the continuous presence of the agonist (10). The ionic channels involved in the so-called capacitative Ca²⁺ entry have been studied in rat aorta (26). It was found that Ca²⁺ entry occurred through a cromakalim- and dihydropyridine-sensitive Ca²⁺ channel after store depletion by a mechanism independent of the presence of the agonist. In contrast, the continuous presence of NE was necessary, in rat small mesenteric arteries, for the increase in [Ca²⁺]_i and contraction elicited by the addition of CaCl₂ after store depletion. It has been reported that SKF-96365 is able to inhibit Ca²⁺ release activated current (10); however, because this drug also inhibits dihydropyridine-sensitive Ca²⁺ channels, as discussed below, its effect is not specific to Ca²⁺ entry through the capacitative entry route.

After depletion of the stores, the increases in [Ca²⁺]_i and contraction induced by CaCl₂ in NE-exposed arteries were reduced by the dihydropyridine Ca²⁺ entry blocker nitrendipine and by a drug which can inhibit both voltage-operated and receptor-operated Ca²⁺ channels, SKF-96365 (19, 24). It should be noted that the inhibitory effect of SKF-96365 was greater than that of nitrendipine, whereas the latter was not able to produce further inhibition in the presence of SKF-96365. Thus, after Ca²⁺ store depletion, NE stimulates Ca²⁺ entry mainly through both voltage-dependent Ca²⁺ channels sensitive to nitrendipine. In accordance with data reported in the literature obtained in the same type of artery, voltage-dependent Ca²⁺ channels appear to play a significant role in the regulation of [Ca²⁺]_i in these vessels (25). Also, the above data showed that NE might stimulate the so-called receptor-operated channels sensitive to SKF-96365. This conclusion is consistent with the dual effect of SKF-96365 on the Ca²⁺ entry through inhibition of the opening of both receptor-operated and voltage-operated Ca²⁺ channels in rat small resistance arteries previously described by Lagaud et al. (19) in the case of ATP. It is reinforced by the finding that nonselective blockade of Ca²⁺ channels by high concentration of NiCl₂ (known to produce nonselective Ca²⁺ channels blockade) reduced the responses to CaCl₂ of NE-exposed vessels to the same extent as that observed in the presence of SKF-96365.

The hypothesis that another possible route for Ca²⁺ entry into vascular smooth muscle cells via the Na⁺/Ca²⁺ exchanger has also been tested here. The extracellular CaCl₂-induced responses on NE-exposed mesenteric arteries were abolished when extracellular Na⁺ was omitted from PSS. Also, the CaCl₂-induced increase in [Ca²⁺]_i and contraction produced by NE were greatly reduced or abolished by the Na⁺/Ca²⁺ exchange inhibitors DMTU and DCB or MgCl₂ and amiloride, respectively. Moreover, the DMTU-insensitive component of the CaCl₂-induced responses was reduced or abolished by nitrendipine or SKF-96365, respectively. The reported specific inhibitor of the Na⁺/Ca²⁺ exchanger, DCB, at a concentration below the IC₅₀ values at which it suppressed the inward Na⁺/Ca²⁺ exchange current in cardiac ventricular cell (34), also reduced the extracellular CaCl₂-induced responses on NE-exposed vessels. Taken together, these data show that extracellular Na⁺ is needed for the CaCl₂-induced responses, and they indicate that the Na⁺/Ca²⁺ exchanger may contribute to the pathway leading to Ca²⁺ entry produced by NE. Such observations are consistent with previous reports showing that NE or KCl, by depolarizing the cells, produces a rise in intracellular Na⁺ that thereby activates a reverse mode of the Na⁺/Ca²⁺ exchanger, leading to an increase of Ca²⁺ influx in vascular smooth muscle cells in addition to the opening of Ca²⁺ channels (18). Also, the present results are consistent with those reported in the literature showing the presence of the Na⁺/Ca²⁺ exchanger as a viable mechanism for Ca²⁺ transport in other cell types such as the endothelium of rabbit cardiac valve, in which the Ca²⁺ entry component is enhanced when intracellular Na⁺ concentration is elevated (20). Moreover, the present data are in agreement with those reported by Blaustein (6) showing that NE elicited a rise in intracellular Na⁺ concentration (via activation of the Na⁺/H⁺ exchanger, following its phosphorylation by protein kinase C) and a subsequent reverse mode of Na⁺/Ca²⁺ exchange operation. Indeed, we found here that the protein kinase C inhibitors staurosporine and calphostin C reduced the CaCl₂-induced responses on NE-exposed vessels. However, it is difficult to determine the exact intracellular target of protein kinase C on the CaCl₂-induced responses on NE-exposed vessels, inasmuch as its role in agonist-induced contraction is not essential in rat mesenteric small arteries (28). Nevertheless, the present results suggest that the Na⁺/Ca²⁺ exchange may participate in the increase of [Ca²⁺]_i and contraction produced by CaCl₂ in NE-exposed vessels after depletion of intracellular stores. Finally, the fact that nitrendipine reduced the DMTU-insensitive response to CaCl₂ and SKF-96365 abolished this response reinforce the hypothesis that the Ca²⁺ entry produced by NE is composed of two parts, activation of voltage-dependent Ca²⁺ channels and receptor-dependent Ca²⁺ channels sensitive to SKF-96365.

There is evidence that tyrosine kinases participate in the regulation of Ca²⁺ entry associated with agonist-induced contraction in smooth muscle cells (16). In the present study, we used two groups of tyrosine kinase inhibitors, a compound interacting with the ATP binding site of the enzyme, genistein, and a compound that interacts with the substrate binding site of the enzyme, tyrphostin 23. Both tyrosine kinase inhibitors reduced the CaCl₂-induced increase in [Ca²⁺]_i and the resultant contraction in NE-exposed vessels. Although the inhibitory effect of genistein and tyrphostin 23 on tyrosine phosphorylation was not directly assessed in the present study, the properties of these agents at the concentrations used have been previously well documented (1, 13). Multiple effects of tyrosine kinase inhibitors on vascular smooth muscle contraction have been reported in the literature. These effects include blockade of a step involved in Ca²⁺ entry and Ca²⁺ store refilling by the agonist and blockade of the effect of Ca²⁺ on the contractile apparatus. In the present study, neither genistein nor tyrphostin 23 affected the increase in [Ca²⁺]_i and contraction produced by NE in Ca²⁺-free medium, suggesting that tyrosine kinase did not play a role in mediating the responses linked to the release of intracellular Ca²⁺ produced by NE. Furthermore, the inhibitory effects of genistein and staurosporine or genistein and calphostin C on the CaCl₂-induced responses in NE-exposed vessels were additive, suggesting that tyrosine kinase did not affect the protein kinase C-sensitive component of the contraction associated with the entry of Ca²⁺ produced by NE. Because nitrendipine did not produce an additional inhibitory effect on the Ca²⁺-induced responses on NE-exposed vessels in the presence of either genistein or tyrphostin 23, it is most likely that tyrosine kinase activation by NE modulates the entry of Ca²⁺ linked to the opening of voltage-dependent Ca²⁺ channels sensitive to nitrendipine. Tyrosine kinase inhibitors might reduce the Ca²⁺-induced responses in vessels exposed to NE at the level of the contractile apparatus. However, neither genistein nor tyrphostin 23 modified significantly responses to KCl depolarization (KCl, 100 mM) in the same arteries. Also, genistein did not significantly affect the response to CaCl₂ (3 µM) on vessels permeabilized with -escin (data not shown). Taken together, these data suggest that tyrosine kinase inhibitors might not mediate their effects through the inhibition of contractile machinery. Also, it should be noted that at the concentrations used, both genistein and tyrphostin 23 have been reported to inhibit tyrosine kinase activity but not other kinases linked to the signal transduction leading to vascular contraction such as myosin light-chain kinase (1, 13). Therefore, the present study provides pharmacological evidence supporting the involvement of tyrosine kinase in the Ca²⁺ influx activated by NE-mediated store depletion at the level of dihydropyridine-sensitive Ca²⁺ entry in rat small resistance arteries.

We cannot exclude that the complexity of the signal transduction involved in the responses to NE might be due to the activation of multiple ₁-adrenoceptor populations. Indeed, the characterization of ₁-adrenoceptor population in small resistance arteries shows that NE mainly activates ₁-adrenoceptor population, which belongs to _1A-, _1B-, and the pharmacologically defined _IL-subtypes (8, 12, 33). Further studies are needed to better understand the nature of the receptor subtypes implicated in the observed different pathways in these arteries.

In summary, the present study shows that NE releases Ca²⁺ from intracellular stores that are sensitive to caffeine and partially sensitive to SERCA inhibitors. They also suggest that, in addition to the participation of nitrendipine-sensitive and SKF-96365-sensitive channels, the Na⁺/Ca²⁺ exchange may contribute to the Ca²⁺ influx produced by NE after depletion of intracellular stores. Finally, this Ca²⁺ influx requires ongoing receptor activation and probably involves tyrosine kinase and protein kinase C.