Calcium release from internal stores is required for the generation of spontaneous hyperpolarizations in dopaminergic neurons of neonatal rats.
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We recently have demonstrated the existence of spontaneous hyperpolarizations in midbrain dopaminergic neurons of neonatal but not adult rats. These events are mediated by the opening of apamin-sensitive K(+) channels after a rise in the intracellular concentration of Ca(2+). They are resistant to tetrodotoxin in most cases and are probably endogenous (i.e., not synaptically activated). Here their mechanism was investigated. Cyclopiazonic acid (10 microM), a specific inhibitor of endoplasmic reticulum Ca(2+) ATPases, reversibly abolished the events. Caffeine, which promotes Ca(2+) release from intracellular stores, had concentration-dependent effects. At 1 mM, it markedly and steadily increased the frequency and the amplitude of the hyperpolarizations. At 10 mM, it induced a transient increase in their frequency followed by their cessation. All these effects were quickly reversible. Ryanodine (10 microM), which decreases the conductance of Ca(2+) release channels, irreversibly blocked the spontaneous hyperpolarizations. Dantrolene (100 microM), a blocker of Ca(2+) release from sarcoplasmic reticulum of striated muscle, did not affect the events. On the other hand, Cd(2+) (100-300 microM), a broad antagonist of membrane voltage-gated Ca(2+) channels, significantly reduced the amplitude and the frequency of the hyperpolarizations. However, when the frequency of the events was increased by 1 mM caffeine, Cd(2+) affected them to a smaller extent, whereas cyclopiazonic acid still abolished them. We conclude that internal stores are the major source of Ca(2+) ions that induce the K(+) channel openings underlying the spontaneous hyperpolarizations of these neurons. (+info)
PACAP and nitric oxide inhibit contractions in the proximal intestine of the atlantic cod, Gadus morhua.
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The possible inhibitory roles of pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP) and nitric oxide in the control of intestinal motility were investigated in the Atlantic cod, Gadus morhua. Circular and longitudinal smooth muscle preparations developed spontaneous contractions that were inhibited by atropine (10(-)(5 )mol l(-)(1)). PACAP 27 and PACAP 38 (10(-)(7 )mol l(-)(1)) reduced the amplitude of the contractions but did not usually affect the resting tension. In the circular preparations, the mean active force developed (above resting level; +/- s.e.m.) was reduced from 0. 62+/-0.18 mN to 0.03+/-0.03 mN (N=10) by PACAP 27 and from 0.53+/-0. 20 mN to 0.31+/-0.13 mN (N=7) by PACAP 38, while neither cod nor mammalian VIP (10(-)(10)-10(-)(6 )mol l(-)(1)) had any effect. In the longitudinal preparations, PACAP 27 reduced the force developed from 1.58+/-0.22 mN to 0.44+/-0.25 mN (N=8) and PACAP 38 reduced it from 1.61+/-0.47 mN to 0.75+/-0.28 mN (N=5). The nitric oxide donor sodium nitroprusside (NaNP) almost abolished the contractions in the circular preparations, reducing the mean force developed from 0. 47+/-0.05 mN to 0.02+/-0.06 mN (10(-)(6 )mol l(-)(1); N=9) and 0+/-0. 07 mN (10(-)(5 )mol l(-)(1); N=8). In the longitudinal preparations, NaNP reduced the force developed from 2.03+/-0.36 mN to 0.33+/-0.22 mN (10(-)(6 )mol l(-)(1); N=8) and 0.19+/-0.30 mN (10(-)(5 )mol l(-)(1); N=8). The L-arginine analogue N(G)-nitro-L-arginine methyl ester (L-NAME; 3x10(-)(4 )mol l(-)(1)) enhanced the contractions in both circular and longitudinal preparations, increasing the mean force developed from 0.51+/-0.12 mN to 0.94+/-0.21 mN (N=8) and from 1.49+/-0.36 mN to 3.34+/-0.67 mN (N=7), respectively. However, preincubation with L-NAME before a second addition of PACAP 27 (10(-)(7 )mol l(-)(1)) did not affect the response to PACAP, neither did preincubation with the guanylate cyclase inhibitor 6-anilinoquinoline-5,8-quinone (LY83583; 10(-)(5 )mol l(-)(1)), while the inhibitory response to NaNP (3x10(-)(7 )mol l(-)(1)) was abolished by LY83583. The PACAP analogue PACAP 6-27 (3x10(-)(7 )mol l(-)(1)) had no effect on the response to either NaNP (3x10(-)(7 )mol l(-)(1)) or PACAP 27 (10(-)(8 )mol l(-)(1)) in the circular preparations. These findings indicate the presence of both a cholinergic and a nitrergic tonus in the smooth muscle preparations of the cod. Although PACAP and NaNP both inhibit contractions, there is no evidence of any interactions between the two substances. In addition, NaNP, but not PACAP, probably acts via stimulating the production of cyclic GMP. In conclusion, both PACAP and nitric oxide may act as inhibitory transmitters, using distinct signalling pathways, in the control of intestinal motility in the Atlantic cod. (+info)
Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes.
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Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to -50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca(2+) with equimolar Mn(2+) caused STOCs to "run down. " Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs" remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca(2+)-activated K(+) channels (SK channels). Application of ATP or 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, a P(2) receptor blocker. Similarly, pretreatment of cells with U-73122 (1 microM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P(2Y) receptors results in localized Ca(2+) release via PLC- and IP(3)-dependent mechanisms. Release of Ca(2+) is coupled to STOCs, which are composed of currents mediated by large-conductance Ca(2+)-activated K(+) channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation. (+info)
The pharmacology of hSK1 Ca2+-activated K+ channels expressed in mammalian cell lines.
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The pharmacology of hSK1, a small conductance calcium-activated potassium channel, was studied in mammalian cell lines (HEK293 and COS-7). In these cell types, hSK1 forms an apamin-sensitive channel with an IC(50) for apamin of 8 nM in HEK293 cells and 12 nM in COS-7 cells. The currents in HEK293 cells were also sensitive to tubocurarine (IC(50)=23 microM), dequalinium (IC(50)=0.4 microM), and the novel dequalinium analogue, UCL1848 (IC(50)=1 nM). These results are very different from the pharmacology of hSK1 channels expressed in Xenopus oocytes and suggest the properties of the channel may depend on the expression system. Our findings also raise questions about the role of SK1 channels in generating the apamin-insensitive slow afterhyperpolarization observed in central neurones. (+info)
Apamin-sensitive, non-nitric oxide (NO) endothelium-dependent relaxations to bradykinin in the bovine isolated coronary artery: no role for cytochrome P450 and K+.
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Since cytochrome P(450)-derived metabolites of arachidonic acid and K(+) have been implicated in endothelium-derived hyperpolarizing factor (EDHF)-dependent responses, the aim of this study was to determine whether such factors contribute to non-nitric oxide (NO), endothelium-dependent relaxation to bradykinin (BK) in bovine isolated coronary artery. In rings of artery contracted with U46619 and treated with indomethacin (3 microM) and N(G)-nitro-L-arginine (L-NOARG; 100 microM), relaxation to BK (0.01 nM-0.3 microM) was blocked by approximately 60% after inhibition of K(+) channels with either high extracellular K(+) (high [K(+)](o); 15 - 67 mM) or apamin (0.3 microM). Ouabain (1 microM), an inhibitor of Na(+)/K(+)-ATPase, decreased the sensitivity to BK without affecting the maximum response. In L-NOARG-treated rings, ouabain had no further effect on the relaxation to BK. An inhibitor of inward-rectifying K(+) channels, Ba(2+) (30 microM), had no effect on relaxations to BK in the absence or presence of either L-NOARG or ouabain. KCl (2.5 - 10 mM) elicited small relaxations ( approximately 20%) that were abolished by nifedipine (0.3 microM) and ouabain. Both the high [K(+)](o)/apamin-sensitive relaxation to BK, and the relaxation to the K(ATP) channel-opener, levcromakalim (0.6 microM), were unaffected by the cytochrome P(450) inhibitor, 7-ethoxyresorufin (10 microM), or by co-treatment with a phospholipase A(2) inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3); 3 microM) and a diacylglycerol (DAG)-lipase inhibitor, 1, 6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267; 30 microM). The non-NO/high [K(+)](o)-insensitive, approximately 40% relaxation to BK was, however, abolished by these treatments. Therefore, neither cytochrome P(450)-derived metabolites of arachidonic acid nor K(+) appear to mediate the EDHF-like relaxation to BK (i.e the non-NO, high [K(+)](o)/apamin-sensitive component) in bovine coronary arteries. Cytochrome P(450)-derived metabolites may be released at higher BK concentrations to act in parallel with NO and the high [K(+)](o)/apamin-sensitive mechanism. (+info)
The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K(+) channels.
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The effect of several K(+) channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium (TEA), 4-aminopyridine (4-AP) and cesium on the peripheral antinociceptive effect of morphine was evaluated by the paw pressure test in Wistar rats. The intraplantar administration of a carrageenan suspension (250 microg) resulted in an acute inflammatory response and a decreased threshold to noxious pressure. Morphine administered locally into the paw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be mediated by a peripheral site up to the 100 microg dose. The selective blockers of ATP-sensitive K(+) channels glibenclamide (20, 40 and 80 microg paw(-1)) and tolbutamide (40, 80 and 160 microg paw(-1)) antagonized the peripheral antinociception induced by morphine (100 microg paw(-1)). This effect was unaffected by ChTX (0. 5, 1.0 and 2.0 microg paw(-1)), a large conductance Ca(2+)-activated K(+) channel blocker, or by apamin (2.5, 5.0 and 10.0 microg paw(-1)), a selective blocker of a small conductance Ca(2+)-activated K(+) channel. Intraplantar administration of the non-specific K(+) channel blockers TEA (160, 320 and 640 microg), 4-AP (10, 50 and 100 microg) and cesium (125, 250 and 500 microg) also did not modify the peripheral antinociceptive effect of morphine. These results suggest that the peripheral antinociceptive effect of morphine may result from activation of ATP-sensitive K(+) channels, which may cause a hyperpolarization of peripheral terminals of primary afferents, leading to a decrease in action potential generation. In contrast, large conductance Ca(2+)-activated K(+) channels, small conductance Ca(2+)-activated K(+) channels as well as voltage-dependent K(+) channels appear not to be involved in this transduction pathway. British Journal of Pharmacology (2000) 129, 110 - 114 (+info)
Pharmacological characterization of small-conductance Ca(2+)-activated K(+) channels stably expressed in HEK 293 cells.
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Three genes encode the small-conductance Ca(2+)-activated K(+) channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole-cell patch clamp recordings. The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC(50) values of 3.3 nM and 83 pM, respectively. The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC(50) value of 80 nM and rSK2 at 287 pM. The potent small molecule blockers showed little differentiation between the channel subtypes. The bis-quinolinium cyclophane UCL 1684 blocked hSK1 with an IC(50) value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC(50) values of 444 nM and 162 nM, respectively. The nicotinic acetylcholine receptor antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC(50) values of 27 microM and 17 microM when measured at +80 mV. The inhibition by d-tubocurarine was voltage-dependent with increasing affinities at more hyperpolarized potentials. The GABA(A) receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage-dependent manner with IC(50) values of 15 and 25 microM when measured at +80 mV. In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin-insensitive afterhyperpolarization of neurones is mediated by hSK1. (+info)
SK2 encodes the apamin-sensitive Ca(2+)-activated K(+) channels in the human leukemic T cell line, Jurkat.
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T cells express two different types of voltage-independent Ca(2+)-activated K(+) channels with small (SK) and intermediate (IK) conductance that serve important roles in the activation of T lymphocytes. In contrast to the IK channels from T lymphocytes which are upregulated upon mitogen stimulation, SK channels of Jurkat T cells, a human leukemic T cell line, are constitutively expressed even in the absence of mitogenic stimulation. We have used patch-clamp recordings from transfected or injected mammalian cells to show that the cloned SK2 channel demonstrates the biophysical and pharmacological properties of the majority of K(Ca) channels in Jurkat T cells. The cloned and native channels are voltage-independent, Ca(2+)-activated, apamin-sensitive, show an equivalent voltage-dependent Ba(2+) block and possess a similar ion selectivity. In addition, we used the polymerase chain reaction to demonstrate the presence of SK2 mRNA in Jurkat T cells, whereas SK3 transcripts encoding the other cloned apamin-sensitive SK channel were not detected. These data suggest that the voltage-independent apamin-sensitive K(Ca) channel in Jurkat T cells represents the recently cloned SK2 channel. (+info)