The effects of verapamil and diltiazem on N-, P- and Q-type calcium channels mediating dopamine release in rat striatum.
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1. The putative inhibitory effects of verapamil and diltiazem on neuronal non-L-type Ca2+ channels were studied by investigating their effects on either K+- or veratridine-evoked [3H]-dopamine ([3H]-DA) release in rat striatal slices. Involvement of N-, P- and Q-type channels was identified by sensitivity of [3H]-DA release to omega-conotoxin GVIA (omega-CTx-GVIA), omega-agatoxin IVA (omega-Aga-IVA) and omega-conotoxin MVIIC (omega-CTx-MVIIC), respectively. 2. KCl (50 mM)-evoked [3H]-DA release was abolished in the absence of Ca2+, and was insensitive to dihydropyridines (up to 30 microM). It was significantly blocked by omega-CTx-GVIA (1 microM), omega-Aga-IVA (30 nM) and was confirmed to be abolished by omega-CTx-MVIIC (3 microM), indicating involvement of N-, P- and Q-type channel subtypes. 3. Verapamil and diltiazem inhibited K+-evoked [3H]-DA release in a concentration-dependent manner. The inhibitory effects of verapamil or diltiazem (each 30 microM) were fully additive to the effect of omega-CTx-GVIA (1 microM), whereas co-application with omega-Aga-IVA (30 nM) produced similar effects to those of omega-Aga-IVA alone. 4. As shown previously, veratridine-evoked [3H]-DA release in Ca2+ containing medium exclusively involves Q-type Ca2+ channels. Here, diltiazem (30 microM) did not inhibit veratridine-evoked [3H]-DA release, whereas verapamil (30 microM) partially inhibited it, indicating possible involvement of Q-type channels in verapamil-induced inhibition. However, verapamil (30 microM) inhibited this release even in the absence of extracellular Ca2+, suggesting that Na+ rather than Q-type Ca2+ channels are involved. 5. Taken together, our results suggest that verapamil can block P- and at higher concentrations possibly N- and Q-type Ca2+ channels linked to [3H]-DA release, whereas diltiazem appears to block P-type Ca2+ channels only. (+info)
Long-term effects of Ca(2+) on structure and contractility of vascular smooth muscle.
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Culture of dispersed smooth muscle cells is known to cause rapid modulation from the contractile to the synthetic cellular phenotype. However, organ culture of smooth muscle tissue, with maintained extracellular matrix and cell-cell contacts, may facilitate maintenance of the contractile phenotype. To test the influence of culture conditions, structural, functional, and biochemical properties of rat tail arterial rings were investigated after culture. Rings were cultured for 4 days in the absence and presence of 10% FCS and then mounted for physiological experiments. Intracellular Ca(2+) concentration ([Ca(2+)](i)) after stimulation with norepinephrine was similar in rings cultured with and without FCS, whereas force development after FCS was decreased by >50%. The difference persisted after permeabilization with beta-escin. These effects were associated with the presence of vasoconstrictors in FCS and were dissociated from its growth-stimulatory action. FCS treatment increased lactate production but did not affect ATP, ADP, or AMP contents. The contents of actin and myosin were decreased by culture but similar for all culture conditions. There was no effect of FCS on calponin contents or myosin SM1/SM2 isoform composition, nor was there any appearance of nonmuscle myosin. FCS-stimulated rings showed evidence of cell degeneration not found after culture without FCS or with FCS + verapamil (1 microM) to lower [Ca(2+)](i). The decreased force-generating ability after culture with FCS is thus associated with increased [Ca(2+)](i) during culture and not primarily caused by growth-associated modulation of cells from the contractile to the synthetic phenotype. (+info)
Voltage-dependent outward K(+) current in intermediate cell of stria vascularis of gerbil cochlea.
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A voltage-dependent outward K(+) (K(V)) current in the intermediate cell (melanocyte) of the cochlear stria vascularis was studied using the whole cell patch-clamp technique. The K(V) current had an activation threshold voltage of approximately -80 mV, and 50% activation was observed at -42.6 mV. The time courses of activation and inactivation were well fitted by two exponential functions: the time constants at 0 mV were 7.9 and 58.8 ms for activation and 0.6 and 4.3 s for inactivation. The half-maximal activation time was 13. 8 ms at 0 mV. Inactivation of the current was incomplete even after a prolonged depolarization of 10 s. This current was independent of intracellular Ca(2+). Quinine, verapamil, Ba(2+), and tetraethylammonium inhibited the current in a dose-dependent manner, but 4-aminopyridine was ineffective at 50 mM. We conclude that the K(V) conductance in the intermediate cell may stabilize the membrane potential, which is thought to be closely related to the endocochlear potential, and may provide an additional route for K(+) secretion into the intercellular space. (+info)
Transmural pressure inhibits prorenin processing in juxtaglomerular cell.
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Pressure control of renin secretion involves a complex integration of shear stress, stretch, and transmural pressure (TP). This study was designed to delineate TP control of renin secretion with minimal influence of shear stress or stretch and to determine its mechanism. Rat juxtaglomerular (JG) cells were applied to a TP-loading apparatus for 12 h. In cells conditioned with atmospheric pressure or atmospheric pressure + 40 mmHg, renin secretion rate (RSR) averaged 29.6 +/- 3.7 and 14.5 +/- 3.3% (P < 0.05, n = 8 cultures), respectively, and active renin content (ARC) averaged 47.3 +/- 4.6 and 38.4 +/- 3.4 ng of ANG I. h(-1). million cells(-1) (P < 0.05, n = 10 cultures), respectively. Total renin content and renin mRNA levels were unaffected by TP. The TP-induced decrease in RSR was prevented by Ca(2+)-free medium and the Ca(2+) channel blocker verapamil and was attenuated by thapsigargin and caffeine, which deplete intracellular Ca(2+) stores. Thapsigargin and caffeine, but not Ca(2+)-free medium or verapamil, prevented TP-induced decreases in ARC. The adenylate cyclase activator forskolin did not modulate TP-induced decreases in RSR or ARC. These findings suggest that TP not only stimulates Ca(2+) influx but also inhibits prorenin processing through an intracellular Ca(2+) store-dependent mechanism and thus inhibits active renin secretion by JG cells. (+info)
The effect of ketoconazole on the jejunal permeability and CYP3A metabolism of (R/S)-verapamil in humans.
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AIMS: The purpose of this human intestinal perfusion study was to investigate the effect of ketoconazole on the jejunal permeability and first-pass metabolism of (R)- and (S)-verapamil in humans. METHODS: A regional single-pass perfusion of the jejunum was performed using a Loc-I-Gut(R) perfusion tube in six healthy volunteers. Each perfusion lasted for 200 min and was divided into two periods of 100 min each. The inlet concentration of (R/S)-verapamil was 120 mg l-1 in both periods, and ketoconazole was added at 40 mg l-1 in period 2. (R/S)-verapamil was also administered as a short intravenous infusion of 5 mg, over a period of 10 min. The appearance ratios of the CYP3A formed metabolites (R)- and (S)-norverapamil were also estimated in the outlet jejunal perfusate. RESULTS: The effective jejunal permeability (Peff) of both (R)- and (S)-verapamil was unaffected by the addition of ketoconazole in period 2 suggesting that ketoconazole had no effect on the P-glycoprotein mediated efflux. However, the appearance ratio of both (R)- and (S)-norverapamil in the outlet jejunal perfusate decreased in the presence of ketoconazole. The rate of absorption into plasma of (R)- and (S)-verapamil increased despite the low dose of ketoconazole added, indicating an inhibition of the gut wall metabolism of (R/S)-verapamil by ketoconazole. CONCLUSIONS: Ketoconazole did not affect the jejunal Peff of (R/S)-verapamil, but it did increase the overall transport into the systemic circulation (bioavailability), probably by inhibition of the gut wall metabolism of verapamil. This might be due to ketoconazole being less potent as an inhibitor of P-glycoprotein than of CYP3A4 in vivo in humans. (+info)
Drug concentration-dependent expression of multidrug resistance-associated protein and P-glycoprotein in the doxorubicin-resistant acute myelogenous leukemia sublines.
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The multidrug resistance of cancer cells can be mediated by an overexpression of the human MDR1 and MRP genes, which encode the transmembrane efflux pumps, the 170 kDa P-glycoprotein (Pgp) and the 190 kDa multidrug resistance-associated protein (MRP), respectively. In this study, we investigate which protein is preferentially overexpressed in the function of doxorubicin concentrations in the acute myelogenous leukemia cell line (OCI/AML-2). Multidrug-resistant AML-2 sublines were isolated in doxorubicin concentrations of 20, 100, 250, and 500 ng/ml. MRP was at first expressed at low concentrations of less than 5 x IC50 (100 ng/ml) of doxorubicin followed by the overexpression of Pgp with concentrations of more than 12.5 x IC50 (250 ng/ml) of doxorubicin. In addition, it appeared that increased amounts of MRP and its mRNA in AML-2/DX20 and /DX100 decreased gradually in both AML-2/DX250 and /DX500 overexpressing Pgp. In conclusion, it is thought that the overexpression of MRP or Pgp is dependent upon drug concentrations. It could be implicated that the overexpression of MRP might be negatively related to that of Pgp. (+info)
Repeated oral rifampicin decreases the jejunal permeability of R/S-verapamil in rats.
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The main purpose of this rat study was to investigate the effect of rifampicin on the effective permeability (P(eff)) of R/S-verapamil in the rat jejunum. In addition the effect on metabolism of R/S-verapamil to R/S-norverapamil was examined. In situ single-pass perfusions of the rat jejunum were performed in animals pretreated with oral rifampicin (250 mg/kg/day) or saline (control) over various time periods (1, 4, 7, and 14 days). The jejunal P(eff) of each of the enantiomers of verapamil and D-glucose was estimated. The appearance ratios of the CYP3A-formed metabolites R- and S-norverapamil were also estimated in the outlet jejunal perfusate. The jejunal P(eff) of both R- and S-verapamil decreased as an effect of the oral pretreatment with rifampicin. The appearance of R- and S-norverapamil in the jejunum was also affected by the oral pretreatment with rifampicin, with increasing concentrations of R/S-norverapamil being evident after 14 days of rifampicin pretreatment. There was no stereoselectivity in either the P(eff) of R- and S-verapamil or the metabolic appearance of R- and S-norverapamil. Treatment with oral rifampicin decreased the P(eff) of R/S-verapamil, which is in accordance with an induction of P-glycoprotein activity in the apical enterocyte membrane. The increase in appearance of R/S-norverapamil in jejunum is in accordance with an induction of CYP3A metabolism in the rat. (+info)
Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.
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Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein. (+info)