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| 2. |
- Birnir, Bryndis, et al.
(författare)
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Bicuculline, pentobarbital and diazepam modulate spontaneous GABA(A) channels in rat hippocampal neurons
- 2000
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Ingår i: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 131:4, s. 695-704
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Tidskriftsartikel (refereegranskat)abstract
- Spontaneously opening, chloride-selective channels that showed outward rectification were recorded in ripped-off patches from rat cultured hippocampal neurons and in cell-attached patches from rat hippocampal CA1 pyramidal neurons in slices. In both preparations, channels had multiple conductance states and the most common single-channel conductance varied. In the outside-out patches it ranged from 12 to 70 pS (Vp=40 mV) whereas in the cell-attached patches it ranged from 56 to 85 pS (-Vp=80 mV). Application of GABA to a patch showing spontaneous channel activity evoked a rapid, synchronous activation of channels. During prolonged exposure to either 5 or 100 microM GABA, the open probability of channels decreased. Application of GABA appeared to have no immediate effect on single-channel conductance. Exposure of the patches to 100 microM bicuculline caused a gradual decrease on the single-channel conductance of the spontaneous channels. The time for complete inhibition to take place was slower in the outside-out than in the cell-attached patches. Application of 100 microM pentobarbital or 1 microM diazepam caused 2 - 4 fold increase in the maximum channel conductance of low conductance (<40 pS) spontaneously active channels. The observation of spontaneously opening GABA(A) channels in cell-attached patches on neurons in slices suggests that they may have a role in neurons in vivo and could be an important site of action for some drugs such as benzodiazepines, barbiturates and general anaesthetics.
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| 3. |
- Bouw, M. René, et al.
(författare)
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Blood-brain barrier transport and brain distribution of morphine-6-glucuronide in relation to the antinociceptive effect in rats : pharmacokinetic/pharmacodynamic modelling
- 2001
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Ingår i: British Journal of Pharmacology. - : The British Pharmacological Society. - 0007-1188 .- 1476-5381. ; 134:8, s. 1796-1804
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Tidskriftsartikel (refereegranskat)abstract
- 1. The objective of this study was to investigate the contribution of the blood-brain barrier (BBB) transport to the delay in antinociceptive effect of morphine-6-glucuronide (M6G), and to study the equilibration of M6G in vivo across the BBB with microdialysis measuring unbound concentrations. 2. On two consecutive days, rats received an exponential infusion of M6G for 4 h aiming at a target concentration of 3000 ng ml(-1) (6.5 microM) in blood. Concentrations of unbound M6G were determined in brain extracellular fluid (ECF) and venous blood using microdialysis and in arterial blood by regular sampling. MD probes were calibrated in vivo using retrodialysis by drug prior to drug administration. 3. The half-life of M6G was 23+/-5 min in arterial blood, 26+/-10 min in venous blood and 58+/-17 min in brain ECF (P<0.05; brain vs blood). The BBB equilibration, expressed as the unbound steady-state concentration ratio, was 0.22+/-0.09, indicating active efflux in the BBB transport of M6G. A two-compartment model best described the brain distribution of M6G. The unbound volume of distribution was 0.20+/-0.02 ml g brain(-1). The concentration-antinociceptive effect relationships exhibited a clear hysteresis, resulting in an effect delay half-life of 103 min in relation to blood concentrations and a remaining effect delay half-life of 53 min in relation to brain ECF concentrations. 4. Half the effect delay of M6G can be explained by transport across the BBB, suggesting that the remaining effect delay of 53 min is a result of drug distribution within the brain tissue or rate-limiting mechanisms at the receptor level.
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| 4. |
- Dreja, Karl, et al.
(författare)
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Rat arterial smooth muscle devoid of ryanodine receptor function: effects on cellular Ca2+ handling
- 2001
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Ingår i: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 132:8, s. 1957-1966
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Tidskriftsartikel (refereegranskat)abstract
- The roles of intracellular Ca2+ stores and ryanodine (Ry) receptors for vascular Ca2+ homeostasis and viability were investigated in rat tail arterial segments kept in organ culture with Ry (10 100 M) for up to 4 days. Acute exposure to Ry or the non-deactivating ryanodine analogue C10-Oeq glycyl ryanodine (10 M) eliminated Ca2+ release responses to caffeine (20 mM) and noradrenaline (NA, 10 M), whereas responses to NA, but not caffeine, gradually returned to normal within 4 days of exposure to Ry. Ry receptor protein was detected on Western blots in arteries cultured either with or without Ry. Brief Ca2+ release events (sparks) were absent after culture with Ry, whereas Ca2+ waves still occurred. The propagation velocity of waves was equal (19 m s-1) in tissue cultured either with or without Ry. Inhibition of Ca2+ accumulation into the sarcoplasmic reticulum (SR) by culture with caffeine (5 mM), cyclopiazonic acid or thapsigargin (both 10 M) decreased contractility due to Ca2+-induced cell damage. In contrast, culture with Ry did not affect contractility. Removal of Ca2+ from the cytosol following a Ca2+ load was retarded after Ry culture. Thapsigargin reduced the rate of Ca2+ removal in control cultured rings, but had no effect after Ry culture. It is concluded that intracellular Ca2+ stores recover during chronic Ry treatment, while Ry receptors remain non-functional. Ry receptor activity is required for Ca2+ sparks and for SR-dependent recovery from a Ca2+ load, but not for Ca2+ waves or basal Ca2+ homeostasis.
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| 6. |
- Hansen-Schwartz, Jacob, et al.
(författare)
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Protein kinase mediated upregulation of endothelin A, endothelin B and 5-hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery.
- 2002
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Ingår i: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 137:1, s. 118-126
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Tidskriftsartikel (refereegranskat)abstract
- 1 Organ culture has been shown to upregulate both endothelin (ET) and 5-hydroxytryptamine 1B/1D (5-HT(1B/1D)) receptors in rat cerebral arteries. The purpose of the present study was to investigate the involvement of protein kinases, especially protein kinases C (PKC) and A (PKA) in this process. 2 The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhitor), RO 31-7549 (specific inhibitor of classical PKC's) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments with ET-1 (unspecific ET(A) and ET(B) agonist), S6c (specific ET(B) agonist) and 5-CT (5-HT(1) agonist). Levels of mRNA coding for the ET(A), ET(B), 5-HT(1B) and 5-HT(1D) receptors were analysed using real-time RT-PCR. 3 Classical PKC's are critically involved in the appearance of the ET(B) receptor; co-culture with RO 31-7549 abolished the contractile response (6.9+/-1.8%) and reduced the ET(B) receptor mRNA by 44+/-4% as compared to the cultured control. Correlation between decreased ET(B) receptor mRNA and abolished contractile function indicates upstream involvement of PKC. 4 Inhibition of PKA generally had an enhancing effect on the induced changes giving rise to a 7-25% increase in E(max) in response to ET-1, S6c and 5-CT as compared to the cultured control. 5 Staurosporine inhibited the culture induced upregulation of the response of both the ET(A) and the 5-HT(1B/1D) receptors, but had no significant effect on the mRNA levels of these receptors. This lack of correlation indicates an additional downstream involvement of protein kinases. British Journal of Pharmacology (2002) 137, 118-126. doi:10.1038/sj.bjp.0704838
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| 7. |
- Henriksson, Marie, et al.
(författare)
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Importance of ERK1/2 in upregulation of endothelin type B receptors in cerebral arteries
- 2004
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Ingår i: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 142:7, s. 1155-1161
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Tidskriftsartikel (refereegranskat)abstract
- 1 This study examines the importance of mitogen-activated protein kinases (MAPKs) in upregulation of endothelin type B (ETB) receptors. 2 Rat middle cerebral arteries (MCAs) were incubated for 24 h with or without kinase inhibitors. Vessel segments were mounted in myographs and the contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; ETB receptor agonist) were studied. We used real-time PCR to measure the receptor mRNA levels. An ELISA assay showed the activation of ERK1/2 kinases after 3 h. Immunohistochemistry revealed the presence of ETB receptors on the vessels. 3 After organ culture, S6c induced vasoconstriction. Incubation with the MEK/ERK inhibitors U0126 and SB386023 diminished the contractile response to S6c. The p38 MAPK inhibitor SB239063 did not affect the S6c-induced contraction. 4 The ET-1-induced vasoconstriction was increased after incubation with SB386023 or SB239063, while unaffected by U0126. 5 The ETB receptor mRNA levels were diminished by SB386023 and U0126. The ETA receptor mRNA levels were unaffected. 6 The levels of activated ERK1/2 kinases were significantly higher after 3 h of organ culture as compared to fresh vessels. 7 The level of ETB receptor protein on the smooth muscle cells of the MCA, visualised by immunohistochemistry, was somewhat diminished by SB386023. 8 Our results show that the ERK1/2 MAPK is important in the upregulation of contractile ETB receptors in MCA after organ culture. Since there is a similar upregulation in models of focal ischaemia and subarachnoid haemorrhage, this may be an important pathophysiological event.
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| 8. |
- Holt, Sandra, et al.
(författare)
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Effects of pH on the inhibition of fatty acid amidohydrolase by ibuprofen.
- 2001
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Ingår i: British Journal of Pharmacology. - : Wiley. - 0007-1188 .- 1476-5381. ; 133:4, s. 513-520
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Tidskriftsartikel (refereegranskat)abstract
- The pharmacological properties of fatty acid amidohydrolase (FAAH) at different assay pH values were investigated using [(3)H]-anandamide ([(3)H]-AEA) as substrate in rat brain homogenates and in COS-1 [corrected] cells transfected with wild type and mutant FAAH. Rat brain hydrolysis of [(3)H]-AEA showed pH dependency with an optimum around pH 8-9. Between pH 6.3 and 8.2, the difference in activity was due to differences in the V(max), rather than the K(M) values. For inhibition of rat brain [(3)H]-AEA metabolism by a series of known FAAH inhibitors, the potencies of the enantiomers of ibuprofen and phenylmethylsulphonyl fluoride (PMSF) were higher at pH 5.28 than at pH 8.37, whereas the reverse was true for oleyl trifluoromethylketone (OTMK) and arachidonoylserotonin. At both pH values, (-)ibuprofen was a mixed-type inhibitor of FAAH. The K(i)((slope)) and K(i)((intercept)) values for (-)ibuprofen at pH 5.28 were 11 and 143 microM, respectively. At pH 8.37, the corresponding values were 185 and 3950 microM, respectively. The pH dependency for the inhibition by OTMK and (-)ibuprofen was also seen in COS-1 [corrected] cells transiently transfected with either wild type, S152A or C249A FAAH. No differences in potencies between the wild type and mutant enzymes were seen. It is concluded that the pharmacological properties of FAAH are highly pH-dependent. The higher potency of ibuprofen at lower pH values raises the possibility that in certain types of inflamed tissue, the concentration of this compound following oral administration may be sufficient to inhibit FAAH.
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| 9. |
- Jonsson, Kent-Olov, et al.
(författare)
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Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide.
- 2001
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Ingår i: British Journal of Pharmacology. - : Wiley. - 0007-1188 .- 1476-5381. ; 133:8, s. 1263-1275
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Tidskriftsartikel (refereegranskat)abstract
- The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [3H]-anandamide ([3H]-AEA) has been investigated.Palmitoylethanolamide and homologues with chain lengths from 12–18 carbon atoms inhibited rat brain [3H]-AEA metabolism with pI50 values of ∼5. Homologues with chain lengths eight carbon atoms gave <20% inhibition at 100 μM.R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [3H]-AEA metabolism with pI50 values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively.With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [3H]-WIN 55,212-2 binding to human CB2 receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [3H]-CP 55,940 binding to human CB1 receptors expressed on CHO cells.Most of the compounds had little effect upon the uptake of [3H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 μM) and palmitoylisopropylamide (30 and 100 μM) produced more inhibition of [3H]-AEA uptake than expected to result from inhibition of [3H]-AEA metabolism alone.In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [3H]-ethanolamine from [3H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective.These data provide useful information upon the ability of palmitoylethanolamide analogues to act as 'entourage' compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB1 or CB2 receptors.
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| 10. |
- Krjukova, Jelena, et al.
(författare)
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Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation
- 2004
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Ingår i: British Journal of Pharmacology. - : Wiley. - 0007-1188 .- 1476-5381. ; 143:1, s. 3-7
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we have investigated responses to the phospholipase C (PLC) activator m-3M3FBS in SH-SY5Y human neuroblastoma cells. As measured using fura-2, m-3M3FBS caused a slowly developing - full response was obtained within 4-6 min - Ca(2+) elevation both in the presence and absence of extracellular Ca(2+), indicating Ca(2+) release from intracellular stores, putatively from endoplasmic reticulum and mitochondria. PLC activity was also measured using two methods, the classical ion-exchange separation and the more novel fluorescent real-time method. In the time frame in which m-3M3FBS caused Ca(2+) elevation (up to 7 min), no PLC activation was detected. Instead, more than 20 min were required to see any inositol phosphate generation in response to m-3M3FBS. m-3M3FBS also interfered with store-operated Ca(2+) influx and Ca(2+) extrusion. In conclusion, m-3M3FBS cannot be considered either potent or specific PLC activator.
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