The effects of CYP2D6 and CYP3A activities on the pharmacokinetics of immediate release oxycodone

Background and purpose:

There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug–drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored.

Experimental approach:

A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg·kg⁻¹ and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session.

Key results:

CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and C_max (−0.71 < Spearman correlation coefficient ρs < −0.92). Oxymorphone C_max was 62% and 75% lower in PM than EM and UM. Noroxymorphone C_max reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone C_max by 40% and 80%, and increased noroxycodone AUC_∞ by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC_∞ and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition.

Conclusions and implications:

Drug–drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype.     

The influence of divalent cations on the analgesic effect of opioid and non-opioid drugs

It is generally accepted that divalent cations are involved in the nociceptive pathway. The effect of systemic co-administration of magnesium sulfate and calcium channel blockers (nifedipine, verapamil) on the analgesic effect of opioid (mixed mu/kappa: butorphanol) and non-opioid drugs (paracetamol) was investigated. Albino mice and rats were used as experimental animals. Magnesium sulfate and calcium channel blockers were given i.p., 30 min before the administration of butorphanol tartrate and paracetamol. Analgesia was measured using "hot-plate" ( 52.5( composite function)C), "tail-flick" (radiant heat source), "writhing" (acetic acid, 1%, i.p.) and "tail-clip" tests. The pain threshold was evaluated before and after the administration (i.p.) of the different agents. The effect of the combined administration of different agents on behavior, blood pressure and heart rate, was also determined. Nifedipine (5 mg kg(-1), i.p.) and verapamil (10 mg kg(-1), i.p.) potentiated the analgesic effect of butorphanol tartrate (0.25-2 mg kg(-1), i.p.) in all tests (synergism) and enhanced analgesic effect of paracetamol (50-125 mg kg(-1), i.p.), only in acetic acid writhing and tail-clip tests. Magensium sulfate (2.5 mg kg(-1), i.p.) potentiated the analgesic effect of butorphanol, but not that of paracetamol. Co-administration of nifedipine and verapamil with either of butorphanol (0.25-2 mg kg(-1)) or paracetamol (50-125 mg kg(-1), i.p.) produced no significant effects on motor coordination, motor performance, locomotor activity, long-term memory or on the blood pressure and heart rate of experimental animals. Co-administration of magnesium sulfate, however, significantly induced sedation, inhibition of locomotor activity, motor performance and coordination, as well as impairing of long-term memory, as compared with butorphanol and paracetamol, administered alone. We conclude that the systemic co-administration of calcium channel blockers potentiated the analgesic effect of butorphanol against thermal, mechanical and chemical pain but enhanced that of paracetamol only against mechanical and chemical pain. Magensium sulfate enhanced the analgesic effect of butorphanol, but not that of paracetamol. These findings, merit further studies in animals and humans to evaluate the potential therapeutic benefits of such interactions.     

ML-9, a myosin light chain kinase inhibitor, reduces intracellular Ca2+ concentration in guinea pig trachealis

We investigated the effects of ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], a myosin light chain kinase (MLCK) inhibitor, on intracellular Ca2+ concentration ([Ca2+]i), contraction induced by high K+ and an agonist, and capacitative Ca2+ entry in fura-2-loaded guinea pig tracheal smooth muscle. ML-9 inhibited both the increase in [Ca2+]i and the contraction induced by 60 mM K+, 1 microM methacholine or 1 microM thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. However, another MLCK inhibitor, wortmannin (3 microM), inhibited the contraction elicited by these stimuli without affecting [Ca2+]i. Under the condition that the thapsigargin-induced contraction was fully suppressed by 3 microM wortmannin, 30 microM ML-9 caused a further decrease in [Ca2+]i. The inhibitory effects of ML-9 on [Ca2+]i and the contraction elicited by methacholine were similar to those of SKF-96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), a Ca2+ channel blocker. These results indicate that ML-9 acts as a potent inhibitor of Ca2+-permeable channels independently of MLCK inhibition in tracheal smooth muscle.     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteries

Background and purpose:

Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries.

Experimental approach:

The non-hydrolyzable selective P2Y1 agonist ADPβS (3 µM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca²⁺]_i were obtained in the presence and absence of inhibitors of protein kinase C (PKC).

Key results:

ADPβS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 µM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 µM) or BIS-VIII (1 µM) tended to augment concentration-dependent dilatation to ADPβS (0.1–3 µM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 µM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca²⁺]_i in pressurized arteries confirmed the P2Y1 receptor but not M₃ muscarinic receptor desensitization.

Conclusions and implications:

These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.     

Selective determinants of inositol 1,4,5-trisphosphate and adenophostin A interactions with type 1 inositol 1,4,5-trisphosphate receptors

BACKGROUND AND PURPOSE

Adenophostin A (AdA) is a potent agonist of inositol 1,4,5-trisphosphate receptors (IP₃R). AdA shares with IP₃ the essential features of all IP₃R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP₃, but the basis of its increased affinity is unclear. Hitherto, the 2′-phosphate of AdA has been thought to provide a supra-optimal mimic of the 1-phosphate of IP₃.

EXPERIMENTAL APPROACH

We examined the structural determinants of AdA binding to type 1 IP₃R (IP₃R1). Chemical synthesis and mutational analysis of IP₃R1 were combined with ³H-IP₃ binding to full-length IP₃R1 and its N-terminal fragments, and Ca²⁺ release assays from recombinant IP₃R1 expressed in DT40 cells.

KEY RESULTS

Adenophostin A is at least 12-fold more potent than IP₃ in functional assays, and the IP₃-binding core (IBC, residues 224–604 of IP₃R1) is sufficient for this high-affinity binding of AdA. Removal of the 2′-phosphate from AdA (to give 2′-dephospho-AdA) had significantly lesser effects on its affinity for the IBC than did removal of the 1-phosphate from IP₃ (to give inositol 4,5-bisphosphate). Mutation of the only residue (R568) that interacts directly with the 1-phosphate of IP₃ decreased similarly (by ∼30-fold) the affinity for IP₃ and AdA, but mutating R504, which has been proposed to form a cation-π interaction with the adenine of AdA, more profoundly reduced the affinity of IP₃R for AdA (353-fold) than for IP₃ (13-fold).

CONCLUSIONS AND IMPLICATIONS

The 2′-phosphate of AdA is not a major determinant of its high affinity. R504 in the receptor, most likely via a cation-π interaction, contributes specifically to AdA binding.     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

Effects of C18 Fatty Acids on Intracellular Ca2+ Mobilization and Histamine Release in RBL-2H3 Cells

To investigate the underlying mechanisms of C18 fatty acids (stearic acid, oleic acid, linoleic acid and α-linolenic acid) on mast cells, we measured the effect of C18 fatty acids on intracellular Ca²⁺ mobilization and histamine release in RBL-2H3 mast cells. Stearic acid rapidly increased initial peak of intracellular Ca²⁺ mobilization, whereas linoleic acid and α-linolenic acid gradually increased this mobilization. In the absence of extracellular Ca²⁺, stearic acid (100 µM) did not cause any increase of intracellular Ca²⁺ mobilization. Both linoleic acid and α-linolenic acid increased intracellular Ca²⁺ mobilization, but the increase was smaller than that in the presence of extracellular Ca²⁺. These results suggest that C18 fatty acid-induced intracellular Ca²⁺ mobilization is mainly dependent on extracellular Ca²⁺ influx. Verapamil dose-dependently inhibited stearic acid-induced intracellular Ca²⁺ mobilization, but did not affect both linoleic acid and α-linolenic acid-induced intracellular Ca²⁺ mobilization. These data suggest that the underlying mechanism of stearic acid, linoleic acid and α-linolenic acid on intracellular Ca²⁺ mobilization may differ. Linoleic acid and α-linolenic acid significantly increased histamine release. Linoleic acid (C18:2: ω-6)-induced intracellular Ca²⁺ mobilization and histamine release were more prominent than α-linolenic acid (C18:3: ω-3). These data support the view that the intake of more α-linolenic acid than linoleic acid is useful in preventing inflammation.     

Actions of 4-chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth muscle and endothelial cells

1. Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca²⁺, apparently through ryanodine-sensitive Ca²⁺ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca²⁺, by use of Ca²⁺ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells.
2. After phenylephrine-(PE, 10 μM) sensitive Ca²⁺ stores were depleted by maximal PE stimulation in Ca²⁺-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca²⁺ was restored for a 30 min refilling period. At the end of this period, Ca²⁺ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca²⁺ store.
3. In a concentration-dependent manner (30, 100 and 300 μM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca²⁺-free media. This suggests that Ca²⁺ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca²⁺-containing or Ca²⁺-free Krebs solution.
4. Restoring Ca²⁺ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca²⁺. The contraction of tissues pretreated with 300 μM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 μM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca²⁺-free and Ca²⁺-containing medium suggesting a rapid reversal of CEP effects.
5. Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K⁺ in the absence of and after 30 min pre-incubation with 30, 100 and 300 μM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K⁺ (77.6, 41.1 and 10.8% of control).
6. Some arterial rings were pre-incubated with ryanodine (30 μM) for 30 min before the construction of PE concentration-response curves. In Ca²⁺-free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9±1.2% of 100 mM K⁺ contraction, n=11) in the presence of external Ca²⁺. EC₅₀ values for PE in ryanodine-treated tissues (1.7±0.25 μM, n=16) were not significantly different from controls (2.5±0.41 μM, n=22). Maximum contractions to PE (118.5±4.4% of 100 mM K⁺ contraction, n=16) were also unaffected by ryanodine when compared to controls (129±4.2%, n=23).
7. When fura-2 loaded smooth muscle cells (n=13) and endothelial cells (n=27) were imaged for Ca²⁺ distribution, it was observed that 100 and 300 μM CEP in Ca²⁺-free medium caused Ca²⁺ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 mM) reversed the effect of CEP on intracellular Ca²⁺ to control values.
8. These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca²⁺ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5-HT and K⁺. The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca²⁺ elevation.

     

Mutation in a protein kinase C phosphorylation site of the 5-HT1A receptor preferentially attenuates Ca2+ responses to partial as opposed to higher-efficacy 5-HT1A agonists

The Thr(149)Ala mutation in a putative protein kinase C phosphorylation site of the 5-HT(1A) receptor's second intracellular loop has been shown to affect the closing of Ca(2+) channels and Ca(2+) mobilisation without interfering with the inhibitory cAMP pathway (Mol Pharmacol 52 (1997) 164). Here, the Ca(2+) responses for a series of 5-HT(1A) agonists were compared between the wild-type (wt) and mutant Thr(149)Ala 5-HT(1A) receptor as part of a fusion protein containing a G(alpha)(15) protein. Neither the mutation nor the fusion process modified the [(3)H]WAY 100635-based ligand binding profile of the fusion proteins as compared to the wt 5-HT(1A) receptor protein. Whereas at the wt 5-HT(1A) receptor, 5-HT induced a Ca(2+) response in CHO-K1 cells via endogenous G(i/o) proteins, the Ca(2+) response to 5-HT at the mutant Thr(149)Ala 5-HT(1A) receptor was fully dependent on either the co-expression or the fusion to a recombinant G(alpha)(15) protein. Buspirone, flesinoxan and 8-OH-DPAT produced a graded partial response (26 to 62%) at the wt 5-HT(1A):G(alpha)(15) fusion protein; F 13640, 5-CT and F 14679 behaved as higher-efficacy agonists with maximal Ca(2+) responses similar to 5-HT. The maximal Ca(2+) responses at the mutant Thr(149)Ala 5-HT(1A):G(alpha)(15) fusion protein were significantly attenuated for flesinoxan and 8-OH-DPAT (-45 and -36%, respectively); the response to the other 5-HT agonists was not significantly affected. A similar effect was observed upon treatment with phorbol 12-myristate 13-acetate at the Thr(149)Ala 5-HT(1A):G(alpha)(15) fusion protein. In conclusion, the amplitude of the Ca(2+) responses induced by partial, but not that to fuller 5-HT(1A) receptor agonists, is affected by the Thr(149)Ala mutation of the 5-HT(1A):G(alpha)(15) fusion protein.     

Role of Ca2+-independent phospholipase A2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts

Store-operated calcium (SOC) channels and capacitative Ca2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [3H] arachidonic acid (AA) release, 45Ca2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca2+]i. Thapsigargin-induced elevation of [Ca2+]i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca2+ influx was sensible to receptor-operated Ca2+ channel blockers (NiCl2, LaCl3) but not to voltage-dependent Ca2+ channel blocker as verapamil. Thus, AA released by Ca2+-independent phospholipase A2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).     

Cell adhesion modulates 5-HT(1D) and P2Y receptor signal trafficking differentially in LTK-8 cells

In this study, we investigated adhesion-induced changes in cellular responses to serotonin 5-HT(1D) and purinergic P2Y receptor stimulation. We demonstrated that detachment of LTK-8 cells increased 5-HT(1D) receptor-mediated intracellular Ca(2+) and extracellular signal regulated kinase (ERK) phosphorylation responses without affecting the adenylate cyclase response. Additionally, detachment enabled 5-HT(1D) receptor stimulation to inhibit P2Y receptor-induced [Ca(2+)](i) mobilization. Such a cross talk between the two receptor systems was not observed in attached cells. P2Y receptor-induced Ca(2+) response was insensitive to adhesion state of the cells, while ERK phosphorylation response was enhanced upon detachment. Integrity of the actin cytoskeleton did not appear to play a role in adhesion sensitivity of 5-HT(1D)-mediated responses, as treatment of attached cells with cytochalasin D did not mimic detachment-induced effects. Effects of detachment were reversed immediately after re-attachment of the suspended cells on poly-l-lysine coated cover slips, suggesting that the involvement of integrins or focal adhesion complexes is unlikely. Taken collectively, our results demonstrate that not only cellular responses induced by different G protein-coupled receptors, but also different responses induced by a particular G protein-coupled receptor, can be affected differentially by the adhesion status of cells. This suggests an important role for cell adhesion in controlling the coupling of a single G protein-coupled receptor to different intracellular responses.     

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca²⁺ concentration [Ca²⁺]_i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca²⁺]_i with EC₅₀ values of about 2 M and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP₃) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca²⁺]_i were 300–350 nM and mainly, almost 90%, due to influx of extracellular Ca²⁺. The efficacy of pilocarpine for stimulating InsP andCa²⁺ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca²⁺]_i with EC₅₀ values of about 20 M and 7 M, respectively. Maximal InsP formation was only 10–15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca²⁺]_i were similar in both cell types. Formation of InsP₃ was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca²⁺]_i increases induced by m2 mAChR activation were exclusively due to Ca²⁺ mobilization from intracellular stores.The efficacy of pilocarpine for stimulating InsP and Ca²⁺ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca²⁺]_i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca²⁺ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.     

Agonist potency differentiates G protein activation and Ca2+ signalling by the orexin receptor type 1

The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Galphas protein backbone with various Galpha donor sequences (Galphas/x) at the N and C termini, and measured cAMP as functional output. The Galphas/x chimeric proteins with Galpha11(Galphaq) and Galpha16 structure in the C terminus were stimulated by the OX1R. Concentration-response curves with Galphas/16 revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+]i via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M3 receptor. However, in contrast to the M3 receptor, the OX1R elevated [Ca2+]i via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of Gq/G11 protein activation.     

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.     

Effects of a myosin light chain kinase inhibitor, wortmannin, on cytoplasmic Ca2+ levels, myosin light chain phosphorylation and force in vascular smooth muscle

Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 2157–2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 M) decreased MLC phosphorylation and the amplitude of contractions induced by high K⁺ (72.7 mM) to a level seen at rest. This occurred without a change in cytosolic Ca²⁺ levels ([Ca²⁺]_i). In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 M) and prostaglandin F₂ (PGF₂, 10 M) without a change in the [Ca²⁺]_i. On the other hand, wortmannin (1 or 10 M) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF₂ to a level seen at rest. In the absence of external Ca²⁺, caffeine (20 mM) induced a transient increase in [Ca²⁺]_i and force with an increase in MLC phosphorylation. Wortmanmn completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [Ca²⁺]_i. In the absence of external Ca²⁺, phenylephrine induced a small transient increase in [Ca²⁺]_i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca²⁺]_i and MLC phosphorylation. Wortmannin (1 M) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca²⁺]_i nor the sustained contraction. Wortmannin inhibited the Ca²⁺-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the Ca²⁺-independent, ATP-induced contraction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entry of Ca²⁺ or through the release of Ca²⁺ from the sarcoplasmic reticulum. The results also suggest that the activation of receptors by norepinephrine and PGF₂. induces a contraction via a MLC phosphorylation-independent pathway or through a pathway which is dependent on the resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.     

Polymorphisms of drug-metabolizing enzymes CYP2C9, CYP2C19, CYP2D6, CYP1A1, NAT2 and of P-glycoprotein in a Russian population

Objective The frequency of functionally important mutations and alleles of genes coding for xenobiotic metabolizing enzymes shows a wide ethnic variation. However, little is known of the frequency distribution of the major allelic variants in the Russian population.Methods Using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) genotyping assays and the real-time PCR with fluorescent probes, the frequencies of functionally important variants of the cytochromes P₄₅₀ (CYP) 2C9, 2C19, 2D6, 1A1 as well as arylamine N-acetyltransferase 2 (NAT2) and P-glycoprotein (MDR1) were determined in a sample of 290 Russian volunteers derived from Voronezh area.Results CYP2C9*2 and *3 alleles were found with allelic frequencies of 10.5% and 6.7%, respectively. The novel intron-2 T>C mutation at exon 2 +73 bp occurred in 24.8% of alleles. CYP2C19*2 and *3 alleles occurred in 11.4% and 0.3%, respectively. Six persons (2.1%) carried two of these CYP2C19 alleles responsible for poor metabolizing activity. Of all subjects, 5.9% were CYP2D6 poor metabolizers, whereas 3.4% were addressed to ultra-rapid metabolizers (CYP2D6*1×2/*1). The CYP1A1*2A allele was found in 4.7%, *2B in 5.0%, *4 in 2.6%, and the 5-mutations –3219C>T, –3229G>A, and the novel –4335G>A in 6.0%, 2.9% and 26.0% of alleles, respectively. Genotyping of eight different single nucleotide polymorphisms in the NAT2 gene provided in 58.0% a genotype associated with slow acetylation. The MDR1 triple variants G2677T and G2677A in exon 21 had an allelic frequency of 41.9% and 3.3%, respectively, and the variant C3435T in exon 26 one of 54.3%. Frequencies of functionally important haplotypes were calculated.Conclusion The overview of allele distribution of important xenobiotic-metabolizing enzymes among a Russian population shows similarity to other Caucasians. The data will be useful for clinical pharmacokinetic investigations and for drug dosage recommendations in the Russian population.     

Analysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b,A1, and A3 adenosine receptors in lymphoid organs

Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice demonstrated that the cyclic AMP-elevating A2a receptor plays a non-redundant role in protection from excessive acute inflammatory tissue damage and in the down-regulation of proinflammatory cytokine production, it remained to be established whether genetic deficiency of the A2a receptor is accompanied by a compensatory up-regulation of the cAMP-elevating A2b receptor and/or other adenosine receptors. Here, we show that most of the cAMP response to adenosine is abolished in lymphoid tissues of A2a receptor-deficient mice, although some response remains in splenocytes. No significant changes were observed in A2b, A1, and A3 mRNA levels in the thymus or lymph nodes of A2a receptor-deficient mice, but small increases in mRNA expression of these receptors were detected in the spleen. These data suggest that regulation of the expression of A2b, A1, and A3 receptors is not affected significantly by the absence of A2a receptors and may provide further explanation of earlier in vivo observations of increased tissue damage and of longer persistence of proinflammatory cytokines in animals with inactivated A2a receptors.