PI3K and PKC contribute to membrane depolarization mediated by α2-adrenoceptors in the canine isolated mesenteric vein

Background Norepinephrine (NE), a classic neurotransmitter in the sympathetic nervous system, induces vasoconstriction of canine isolated mesenteric vein that is accompanied by a sustained membrane depolarization. The mechanisms underlying the NE-elicited membrane depolarization remain undefined. In the present study we hypothesized that phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) are involved in the electrical field stimulation (EFS)-induced slow membrane depolarization (SMD) in canine isolated mesenteric vein. EFS (0.1–2 Hz, 0.1 ms, 15V, 10 s)-induced changes in the membrane potential were recorded with a conventional intracellular microelectrode technique and evaluated in the absence and presence of inhibitors of neuronal activity, α-adrenoceptors, membrane ion channels, PI3K, inositol 1,4,5-triphosphate (InsP3) receptors, and PKC. Activation of PI3Kγ and PKCζ in response to exogenous NE and clonidine in the absence and presence of receptor and kinase inhibitors were also determined. Results Contractile responses to NE and clonidine (0.05 – 10 μM) were significantly diminished in the presence of yohimbine (0.1 μM). Exogenous NE (0.1 μM) and clonidine (1 μM) elicited SMD. The resting membrane potential of canine mesenteric vein smooth muscle cells was -68.8 ± 0.8 mV. EFS elicited a biphasic depolarization comprised of excitatory junction potentials and SMD that are purinergic and adrenergic in nature, respectively. The magnitude of the SMD in response to EFS at 0.5 Hz was 9.4 ± 0.7 mV. This response was reduced by 65–98% by the fast Na+ channel inhibitor tetrodotoxin (1 μM), by the inhibitor of N-type Ca2+ channels ω-conotoxin GVIA (5 nM), the non-selective α-adrenoceptor blocker phentolamine (1 μM), the selective α2-adrenoceptor blocker yohimbine (0.1 μM), the ion channel inhibitors niflumic acid (NFA, 100 μM), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 30 μM), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 200 μM), and Gd3+ (30 μM), and the PI3K inhibitors wortmannin (100 nM) and LY-294002 (10 μM). The SMD remained unchanged in the presence of the L-type Ca2+ channel blocker nicardipine (1 μM) and the InsP3 receptor blockers 2-aminoethoxydiphenylborate (2APB, 50 μM) and xestospongin C (3 μM). The inhibitor of PKC chelerythrine (1 μM), but not calphostin C (10 μM), diminished the SMD. Exogenous NE and clonidine (1 μM each) activated both PI3Kγ and PKCζ, and the activation of these kinases was abolished by preincubation of tissue with the α2-adrenoceptor blocker yohimbine. Conclusion Neuronally-released NE stimulates smooth muscle α2-adrenoceptors and activates PI3K and atypical PKC in the canine mesenteric vein. Events downstream of PKC lead to SMD and vasoconstriction. This represents a novel pathway for NE-induced membrane depolarization in a vascular smooth muscle preparation.


Background
Norepinephrine (NE), a classic neurotransmitter in the sympathetic nervous system, is released from adrenergic varicosities of stimulated postganglionic nerve terminals, activates postjunctional α-adrenoceptors and gives rise to a slow membrane depolarization (SMD) and contraction [1,29]. The NE-induced SMD represents an important mechanism of excitation-contraction coupling in blood vessels however the signaling pathways underlying the NE-elicited SMD in vascular smooth muscle remain undefined.
One well-documented pathway downstream of activated G-protein coupled receptors (GPCRs) includes dissociation of G αβγ trimers and production of G α monomer and G βγ dimer, and involvement of the latter proteins in signal transduction events downstream of α-adrenoceptors. For example, G α mediates activation of phospholipase C (PLC), hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ), and generation of second messengers including inositol 1,4,5-triphosphate (InsP 3 ) and diacylglycerol, DAG [20]. These second messengers then mediate signal transduction events leading to activation of ion channels. InsP 3 has the capacity to release cytosolic Ca 2+ from intracellular stores, which then activates Ca 2+ -activated Clchannels (ClC Ca ) and membrane depolarization, required for opening of voltage-operated calcium channels (VOCC) and Ca 2+ influx. DAG, on the other hand, activates non-selective cation channels (NSCC) in rabbit portal vein [17]. In addition, it becomes increasingly clear that G βγ dimers can initiate intracellular signal transduction events as well. Phosphatidylinositol 3-kinase-γ (PI3Kγ), a member of class IB PI3Ks, was identified as a major effector of G βγ in various cell and tissue preparations [13,18]. Lipid products of the PI3Ks, phosphatidylinositol 3,4-bisphosphate (PI3,4P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P 3 ), function as second messengers and can directly affect the activity of the membrane ion channels CFTR [12] and voltage-gated potassium channels [19]. Alternatively, PI3,4P 2 and PI3,4,5P 3 can modulate membrane ion channels via activation of PKC isozymes [6,25]. For example, G βγ , PI3Kγ, and atypical PKC were shown to link activation of G-protein coupled M 2 -muscarinic receptors to metabotropic Ca 2+ and voltage-independent Clchannels in Xenopus oocytes [31]. It was also demonstrated that PI3K mediates activation of L-type Ca 2+ channels upon stimulation of M 2 -muscarinic receptors in rabbit portal vein myocytes [3] and α 2 -adrenoceptor induced vasoconstriction in porcine palmar lateral vein [27]. These studies imply that activation of GPCRs could activate membrane ion channels and SMD via PI3K-dependent mechanisms. To our knowledge, however, coupling of α-adrenoceptors to PI3Kγ and membrane depolarization in vascular smooth muscles has not yet been reported.
The α 2 -adrenoceptor blocker yohimbine reduces the con-tractile responses of canine isolated mesenteric vein to nore-pinephrine (NE) and clonidine We used canine isolated mesenteric vein to test the hypothesis that EFS-induced SMD is mediated by PI3K and PKC. Our results demonstrate both nerve stimulation and exogenous NE-mediated activation of α 2 -adrenoceptors, PI3K and PKC, and suggest a role for these kinases for the activation of membrane ion channels (e.g., ClC Ca and/ or NSCC) and development of SMD.

α 2 -Adrenoceptors mediate vasoconstriction and membrane depolarization in canine isolated mesenteric vein
Cumulative application of exogenous NE and clonidine (0.05 µM-10 µM) resulted in concentration dependent contractile responses ( Fig. 1A and 1B). In the presence of the selective α 2 -adrenoceptor antagonist yohimbine (0.1 µM) the contractile responses to 0.05-1 µM NE were virtually abolished, whereas the responses to 5 and 10 µM NE were significantly reduced (Fig. 1A). Yohimbine abolished the mechanical responses to all clonidine concentrations (Fig. 1B). These results indicate that α 2adrenoceptors are particularly important for NE-mediated vasoconstriction in this blood vessel. Moreover, exogenous application of either NE (0.1 µM, Fig. 1C) or clonidine (1 µM, Fig. 1D) elicited SMD, suggesting that α 2adrenoreceptors may be involved in the membrane potential changes in response to NE that is released upon EFS.

EFS gives rise to a biphasic membrane depolarization
The resting membrane potential of isolated canine mesenteric vein segments averaged -68.8 ± 0.8 mV (n = 78). EFS (0.1-2 Hz, 0.1 ms, 15 V, 10 s) gave rise to a characteristic biphasic depolarization of the cell membrane ( Fig. 2A-E), composed of a fast excitation junction potential (EJP) of presumed purinergic nature, and a SMD of adrenergic origin. The amplitude of the SMD increased with the frequency of EFS ( Fig. 2A-F). To better understand the processes that govern membrane depolarization, we exposed blood vessels to the fast Na + channel blocker tetrodotoxin (TTX, 1 µM) or to the neuronal Ntype Ca 2+ channel blocker ω-conotoxin GVIA (ω-CtX GVIA, 5 nM). Both drugs virtually abolished the electrical responses to EFS (Fig. 3A, B), suggesting that the EFS-elicited SMD requires activation of postganglionic nerve terminals and release of a neurotransmitter substance. Phentolamine (1 µM) significantly reduced the SMD in response to 0.5 Hz EFS to 2.1 ± 0.5 mV (n = 3, P < 0.05, not shown), consistent with the possibility that NE-mediated activation of α-adrenoceptors is the primary cause for the SMD in blood vessels [14,29]. Furthermore, the selective α 2 -adrenoceptor antagonist yohimbine (0.1 µM, Fig.  3C), but not the selective α 1 -adrenoceptor antagonist prazosin (1 µM; 8.9 ± 0.9, n = 3, not shown), significantly reduced the SMD in response to 0.5 Hz EFS from 9.4 ± 0.7 (n = 14) to 0.8 ± 0.5 mV (n = 3, P < 0.05), indicating that the EFS-evoked SMD is mediated primarily by α 2 -adrenoceptors. Nicardipine (1 µM) abolished the contraction elicited by 70 mM KCl (not shown), indicating that L-type Ca 2+ channels are present in this blood vessel. However, the EFS-induced SMD remained unchanged in tissues preincubated with nicardipine ( Fig. 3D), suggesting that opening of L-type Ca 2+ channels is not required for the membrane depolarization.

InsP 3 receptors are not required for the EFS-elicited slow depolarization
To determine whether EFS-induced SMD is mediated by InsP 3 receptors, veins were incubated with 2APB (50 µM), a selective inhibitor of InsP 3 receptors and hence of GPCR-PLC-InsP 3 -mediated Ca 2+ release from sarcoplasmic reticulum. Surprisingly, SMD to 0.5 Hz EFS remained unaffected by 2APB ( Fig. 5A and 5C). To test the possibility that the lack of effect of 2APB was due to poor membrane permeability, we carried out mechanical experiments with mesenteric veins, incubated with 2APB (50 µM) prior to incubation with increasing concentrations of the α 1adrenoceptor agonist methoxamine (0.1-100 µM). 2APB produced a significant rightward shift of the concentration-response curve of methoxamine (not shown) and a decrease of the EC50 from 6.2 ± 0.3 µM (n = 10) to 5.1 ± 0.2 µM (n = 6). These results suggest that activation of InsP 3 receptors may be unnecessary for the SMD. This possibility was also confirmed by the inability of another inhibitor of InsP 3 receptors, xestospongin C (3 µM), to reduce the EFS-evoked SMD ( Fig. 5B and 5C, n = 3). Therefore, the EFS-mediated SMD may not require release of Ca 2+ from InsP 3 receptor-operated stores.

PI3K blockers reduce the EFS-elicited slow depolarization
To test whether PI3Ks are involved in the EFS-induced SMD, we incubated tissue strips with the PI3K inhibitors wortmannin (100 nM) and LY-294002 (10 µM). Both inhibitors significantly reduced the SMD elicited by 0.5 Hz EFS from 9.4 ± 0.7 mV in controls to 1.0 ± 1.1 and 2.5 ± 1.6 mV, respectively (Fig. 6), indicating that activation of PI3Ks is required for the EFS-induced activation of ion channels and SMD. Incubation of veins with exogenous NE (100 nM) induced 14.1 ± 3.0 mV SMD, which was reduced to 3.0 ± 2.0 mV (n = 3) in the presence of wortmannin (100 nM, not shown), suggesting that both EFS and exogenous NE-induced SMD are mediated by PI3K.

Exogenous NE and clonidine activate PI3Ks
To determine whether NE directly activates PI3Ks, we measured phosphorylation of Akt in control vein segments and in tissues incubated with exogenous NE (1 µM, 2APB and xestospongin C have no effect on the EFS-induced slow membrane depolarization of canine isolated mesenteric vein  Previous experiments have shown that PI3Kγ mediates signaling downstream of GPCRs [18,33]. To test whether NE activates PI3Kγ, we incubated mesenteric vein with exogenous NE, then immunoprecipitated PI3Kγ and used the immunoprecipitated kinase to phosphorylate phosphatidylinositol (PI) in vitro. As shown in Fig. 7B, stimulation with NE increased the amount of PI3P (reaction product), suggesting that NE activated PI3Kγ. NE-mediated phosphorylation of PI was reversed in strips preincubated with LY-294002, indicating that it was a PI3Kdependent event. To test whether α 2 -adrenoceptors are linked to activation of PI3Kγ, we analyzed the activation of PI3Kγ in veins incubated with clonidine (1 µM) in the absence or presence of yohimbine (0.1 µM). As shown in Fig. 7D, clonidine activated PI3Kγ to 1.8-fold the basal, while yohimbine eliminated this activation. Thus, the activation of PI3Ks, and more specifically PI3Kγ, by exogenous NE and clonidine suggests that α 2 -adrenoceptors are coupled to PI3Kγ via activation of G-proteins.

Stimulation of atypical PKC(s) contributes to the EFSelicited SMD
PI3Kγ may be linked to membrane ion channels (i.e., ClC Ca and/or NSCC) via isozymes of the multifunctional PKC family. To test this possibility, we incubated mesenteric veins with the broad-spectrum PKC blocker chelerythrine (1 µM, 1 hour). Although chelerythrine reduced the response to 0.5 Hz EFS from 9.4 ± 0.7 mV to 2.1 ± 1.5 mV, the residual SMD remained unchanged in veins, incubated simultaneously with LY-294002 and chelerythrine prior to EFS (2.6 ± 1.3). These results not only implicate the existence of a PKC-dependent component in the EFS-induced SMD ( Fig. 8A and 8C), but also suggest that PI3Kγ and PKC may signal to ion channels in a linear fashion. To narrow down the PKCs that function downstream of PI3Kγ, we used calphostin C, an inhibitor of classical and novel PKCs [32]. One-hour incubation of mesenteric veins with calphostin C (10 µM) failed to inhibit EFS-stimulated SMD (Fig. 8B and 8C). However, calphostin C significantly reduced the contractile responses to PMA (100 nM, 30 min incubation), which is mediated by classical and novel PKCs (not shown). Together, these results suggest that activation of atypical, rather than classical or novel PKC isozymes is involved in the SMD.

Exogenous NE and clonidine activate PKCζ
Since exogenous NE and clonidine activated PI3Kγ, we tested whether these agents also activate PKCζ, a specific substrate of PI3Kγ [5]. Vein segments incubated with NE (1 µM, 3 min) and clonidine (1 µM, 3 min) caused activation of for a synthetic PKCζ peptide substrate in vitro to 2.11 and 1.89-fold the basal kinase activity of non-stimulated controls (Fig. 9A and 9B). The NE-induced activation of PKCζ was significantly reduced in tissues incubated with the PI3K inhibitor LY-294002 (10 µM) and was eliminated by a specific myristoylated peptide inhibitor of PKCζ (PKCζI, 50 µM) in vitro (Fig. 9A). Similarly, the clonidine-induced activation of PKCζ was eliminated in tissues preincubated with yohimbine and LY-294002 (Fig. 9B). These results indicate that stimulation of canine mesenteric veins with NE and clonidine is associated with activation of PI3Ks and a subsequent activation of PKCζ.

Ion channel blockers and PI3K blockers do not inhibit release of NE
Reduction of the SMD could be the result of suppressed NE release from sympathetic nerve terminals in veins, incubated with protein kinase and ion channel blockers.
To test this possibility, we assayed the EFS-evoked release of NE in superfusates collected during EFS in control veins and in tissues preincubated with each of the aforementioned blockers. The average EFS -evoked overflow of NE in tissue controls (16 Hz, 0.1 ms) was 122 ± 27 fmol/mg (n = 17). In preincubated tissues, the overflow of NE changed to (fmol/mg tissue) 236 ± 40 in the presence of NFA (n = 6), 480 ± 122 by NPPB (n = 6), 142 ± 2 by DIDS (n = 6), 144 ± 5 by wortmannin (n = 5), and 214 ± 38 by LY-294002 (n = 7). Therefore, none of these agents reduced the EFS-evoked overflow of NE, indicating that their effects on the EFS-induced SMD are not due to inhibited NE release but to activation of postjunctional (i.e., smooth muscle) mechanisms.

Discussion
The NE-induced membrane depolarization is an essential requirement for opening VOCC, Ca 2+ entry and smooth muscle contraction, and hence it represents an important mechanism of autonomic neurovascular control. Previous studies [4] as well as the present work indicate that α 2adrenoceptors are involved in the SMD and the vasoconstriction of mesenteric vein. However, the downstream mechanisms that couple α 2 -adronoceptors to SMD remain undefined. For example, NE-induced activation of ClC Ca plays a key role in the associated vasoconstriction and presumed membrane depolarization in various vascular networks [11,16], but NSCC may also participate in the NE-induced vasoconstriction [17]. In the present study, we have expanded upon these previous works by directly measuring membrane potential in response to EFS of intact canine isolated mesenteric veins. We found that the SMD in response to EFS is frequency-dependent, and is sensitive to the fast Na + channel blocker TTX, to the inhibitor of neuronal N-type Ca 2+ channels ω-conotoxin GVIA [24], and to the selective antagonist of α 2 -adreno-ceptors yohimbine. Consistent with previous works, therefore, our results indicate that EFS gives rise to a SMD, which is mediated by smooth muscle α 2 -adrenoceptors and hence is primarily mediated by NE, released upon action potential. Furthermore, the SMD of the vascular smooth muscle cell membrane appears to be mediated by channels sensitive to NFA, NPPB, and DIDS. Although these inhibitors target transporters with presumed high preference for Cl - [8], they may also affect other ion channels, such as the inhibition of NSCC by DIDS [7]. Because incubation of mesenteric veins with the NSCC blocker Gd 3+ partially reduced the EFS-evoked SMD, our results suggest that activation of NSCC may indeed contribute to the EFS-elicited SMD in this blood vessel. None of the channel blockers reduced the release of NE thus demonstrating that the decrease of SMD is mediated by postjunctional smooth muscle mechanisms. The EFS-induced SMD remained unaffected by the InsP 3 receptor inhibitors 2APB and xestospongin C, ruling out a substantial role of InsP 3 receptor-mediated Ca 2+ release in this response. Therefore, smooth muscle cell chloride channels and/or NE and clonidine activate PKCζ in canine mesenteric vein Furthermore, we identified a novel signal transduction mechanism governing the slow membrane depolarization, which involves enzymes of the PKC family. First, we used two PKC inhibitors with distinct PKC isozymes selectivity. Chelerythrine, for example, acts on the conserved catalytic domain of PKC as a competitive inhibitor with respect to the phosphate acceptor and a non-competitive inhibitor with respect to ATP [32]. Therefore, chelerythrine can inhibit PKCs of all classes and effects mediated by them, including the slow depolarization in mesenteric veins. In contrast, calphostin C interacts with the regulatory domain of PKC by competing for the binding site of diacylglycerol and phorbol esters, but not Ca 2+ and phospholipids. Thus, calphostin C is more specific inhibitor for classical and novel, and less specific for atypical PKC [9,32]. In the present study, chelerythrine reduced, while calphostin C had no effect on the SMD. The lack of effect of calphostin C indicates that the EFS-induced membrane depolarization is most likely mediated by atypical PKC(s). We provided experimental support of this possibility by showing a distinct activation of the atypical PKCζ in mesenteric veins incubated with exogenous NE and clonidine. Although further studies are needed to identify the precise PKC isoform(s) contributing to the α 2 -adrenoceptor mediated SMD, the present study clearly suggests a PKC isozyme-specific regulation of the EFS-(and hence NE-) induced slow membrane depolarization in canine mesenteric vein.
The observation that atypical PKCs are involved in the SMC allowed us to speculate about the identity of some specific signaling molecules that function upstream of PKCζ, and could mediate the SMD as well. PI3Ks, and particularly PI3Kγ, was an obvious candidate for several reasons. Firstly, PI3Kγ is essential for activation of various ion transporters including L-type Ca 2+ channels [30] and metabotropic nonselective cation channels [31]. Secondly, PI3Kγ activates the phosphoinositide-dependent protein kinase 1 (PDK1), and although the latter can modulate classical (α and βI) and novel (δ and ε) PKC isoform, activation of atypical (ζ/λ/τ) PKCs, and particularly PKCζ, is a highly specific event downstream of PI3Kγ-PDK1 [28]. We experimentally supported the hypothesized role of PI3Ks by showing that the PI3K inhibitors wortmannin and LY-294002 prevent the NE and clonidine-dependent activation of PKCζ and the SMD. These data are consistent with a role of PI3K in the regulation of voltage-independent Clchannels, as well. PI3Kγ in particular is activated by G βγ dimers, which are released upon dissociation of G αβγ complexes following activation of GPCRs [18]. Our experimental data demonstrate activation of PI3Kγ in veins incubated with the G-protein cou-pled α 2 -adrenoceptor agonists NE and clonidine. Furthermore, since the NE and clonidine-mediated activation of PI3Kγ, PKCζ and of the SMD was prevented by yohimbine, our results demonstrate that activation of α 2adrenoceptors is required for activation of PI3Kγ and PKCζ, and possibly for activation of membrane Cland/or NCSS channels and SMD in canine mesenteric vein. While the molecular identity of the membrane ion channels involved in these effects is presently elusive, our electrophysiological and biochemical data provide support to the possibility that activation of α 2 -adrenoceptors, PI3K and atypical PKC (possibly PKCζ) is essential for the regulation of the autonomic nervous system and vascular smooth muscle tone.

Conclusion
In this study we provide functional and biochemical evidence that NE, released from postganglionic nerve terminals, activates postjunctional α 2 -adrenoceptors, PI3Ks and atypical PKCs in canine isolated mesenteric vein. Our results further suggest that specific isoforms of the PI3K and PKC families, i.e. PI3Kγ and PKCζ respectively, may participate in the signal transduction pathway that couples α 2 -adrenoceptors to membrane ion channels. This signal transduction pathway mediates slow membrane depolarization and vasoconstriction of canine mesenteric vein.

Tissue preparation
Seventy-four mongrel dogs of either sex (averaging 15 kg) were obtained from vendors licensed by the United States Department of Agriculture. The use of dogs was approved by the University of Nevada's Animal Care and Use Committee. The animals were sacrificed with an overdose of pentobarbitone sodium (100 mg/kg intravenously), as recommended by the Panel on Euthanasia of the American Veterinary Medical Association. Experiments were conducted with second and third order branches of the inferior mesenteric vein (0.7-1.2 mm in diameter), dissected and denuded of endothelium as outlined previously [21].

Intracellular recording of membrane potential
Ring segments (7-10 mm long; 700-800 µM external diameter) were pinned out on the sylgard bottom of a 2 ml recording chamber perfused with Krebs solution ( , as described previously [22]. EFS at supramaximal voltage with trains of square-wave pulses (0.1 ms pulse width) was applied at 0.1-2 Hz for 10 s by means of two parallel platinum electrodes on both sides of the vessel connected to a Grass S48 stimulator. Once welldefined and reproducible slow depolarizations were obtained, various drugs were applied to the superfusion solution according to the experimental protocol. The maximum depolarization was evaluated. In some experiments tissues were perfused with clonidine (1 µM) and changes in the membrane potential were monitored.

Transmitter release experiments and HPLC assay of NE
Segments of endothelium-denuded mesenteric veins (52.5 ± 3.2 mg wet weight, n = 30) were placed in 200-µl BRANDEL superfusion chambers as previously described [23]. After 45 min equilibration, the tissues were subjected to a 30-seconds "conditioning" stimulation with a train of square wave pulses of 0.3 ms duration and a frequency of 4 Hz. Thirty minutes later the blood vessels were subjected to EFS for 60 s with a train of suprathreshold pulses of 0.1 ms duration at 16 Hz. Samples of the superfusion solution were collected before the electrical stimulation (resting overflow) and during the electrical stimulation (electrically evoked overflow) in ice-cold test tubes. Samples were analyzed for NE content by high performance liquid chromatography (HPLC) technique in conjunction with electrochemical detection [23].

Mechanical responses
Ring preparations (3 mm long) were mounted in 3 ml organ baths by inserting two stainless steel triangle mounts into the lumen, and force displacements were further investigated as described previously [21]. The baths contained Krebs solution, which routinely contained indomethacin (1 µM) and Nω-nitro-L-arginine (l-NNA, 100 µM) to block potential residual effects of the endothelium and to eliminate possible time dependent effects due to activation of inducible nitric oxide synthase (iNOS) and/or cyclooxygenase. Thus, the contractile responses to KCl and nerve stimulation were reproducible over many hours when these blockers were included in the bathing solution. A resting force of 0.5 g was applied to the vein segments. This was found to stretch vessels to near the optimum length for tension development.

Preparation of tissue homogenates and cytosolic fractions
Total protein extracts were prepared by glass-glass homogenization of mesenteric veins, pulverized under liquid nitrogen, with a buffer composed of (mM): 10 Tris-HCl (pH 7.4), 5 EDTA, 5 EGTA, 10 sodium pyrophosphate, 10 NaF, 1 sodium orthovanadate, 0.1 AEBSF and 0.001 leupeptin. Insoluble material was pelleted by centrifugation at 3,000 g (S3 supernatants) for 5 min at 4°C. S3 supernatants were transferred into clean tubes and centrifuged at 120,000 g for 60 min at 4°C to obtain S120 supernatants, which were used for assay of PI3K and PKC activity.

PI3K activation assay
Activation of PI3K was assayed by the phosphorylation of the downstream protein kinase Akt. Equal amounts of total supernatant protein (30 µg) were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and total and phospho-Ser473-Akt were assayed by immunoblotting, using a rabbit polyclonal or mouse monoclonal antibodies, respectively. Blots were scanned to obtain images and the immunoreactive bands were analyzed by densitometry, using the Quantity One software (BioRad). Changes in protein phosphorylation were calculated by normalizing the band density phospho-Akt to total Akt, and then were presented relative to the untreated group controls.

PI3Kγ activation assay
PI3Kγ was immunoprecipitated from S120 supernatants with a mouse monoclonal antibody, immobilized on Protein A/G agarose plus beads (Santa Cruz Biotechnology, Inc.). Kinase activity was assayed by phosphorylation of phosphatidylinositol (PI) in vitro, as described previously [34].

PKCζ activation assay
PKCζ activation was assayed by in vitro phosphorylation of a synthetic peptide substrate (PKCζPS), ERMRPRKRQGSVRRRV [15]. S120 fractions from control and treated tissues were used as enzyme sources. The phosphorylation reactions were stopped by cooling on ice, and 10 µl reactions were spotted on P81 filter strips. Excess radioactivity was removed from the filters by 3 washes with 75 mM ortho-phosphoric acid and a final wash with ethanol. The filters were air-dried prior to radiography and spots were quantified by densitometry, using a BioRad Model 525 Molecular Imager. In order to verify PKCζ-specific phosphorylation of the substrate, in some phosphorylation reactions we added a specific myristoylated PKCζ peptide inhibitor (PKCζI, 50 µM, Calbiochem).

Statistical analysis
Data are presented as means ± SEM. Means were compared by analysis of variance (one-way ANOVA) (Graph-PadPrism v. 3, GraphPad Software, Inc.). A probability value of less than 0.05 was considered significant. In the intracellular recording and mechanical response experiments, n refers to the number of rings, and hence dogs, included in each experimental group.