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A den Hertog

Publications and source records attributed to A den Hertog.

At least 19 recordsLinked to original sources
PubMed1996

Inotropic effects of propofol, thiopental, midazolam, etomidate, and ketamine on isolated human atrial muscle.

BACKGROUND: Cardiovascular instability after intravenous induction of anesthesia may be explained partly by direct negative inotropic effects. The direct inotropic influence of etomidate, ketamine, midazolam, propofol, and thiopental on the contractility of isolated human atrial tissue was determined. Effective concentrations were compared with those reported clinically. METHODS: Atrial tissue was obtained from 16 patients undergoing coronary bypass surgery. Each fragment was divided into three strips, and one anesthetic was tested per strip in increasing concentrations (10(-6) to 10(-2) M). Strips were stimulated at 0.5 Hz, and maximum isometric force was measured. Induction agents were studied in two groups, group 1 (n = 7) containing thiopental, midazolam, and propofol, and group 2 (n = 9) consisting of etomidate, ketamine, and propofol. RESULTS: The tested anesthetics caused a concentration-dependent depression of contractility resulting in complete cessation of contractions at the highest concentrations. The IC50s (mean +/- SEM; microM) for inhibition of the contractility were: thiopental 43 +/- 7.6, propofol 235 +/- 48 (group 1), and 246 +/- 42 (group 2), midazolam 145 +/- 54, etomidate 133 +/- 13, and ketamine 303 +/- 54. CONCLUSIONS: This is the first study demonstrating a concentration-dependent negative inotropic effect of intravenous anesthetics in isolated human atrial muscle. No inhibition of myocardial contractility was found in the clinical concentration ranges of propofol, midazolam, and etomidate. In contrast, thiopental showed strong and ketamine showed slight negative inotropic properties. Thus, negative inotropic effects may explain in part the cardiovascular depression on induction of anesthesia with thiopental but not with propofol, midazolam, and etomidate. Improvement of hemodynamics after induction of anesthesia with ketamine cannot be explained by intrinsic cardiac stimulation.

Anesthetics, Intravenous↗
PubMed1996

Different mechanisms of Ca2(+)-handling following nicotinic acetylcholine receptor stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization in C2C12 myotubes.

1. The increase in intracellular CA2+ on nicotinic acetylcholine receptor (nAChR) stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization was investigated in mouse C2C12 myotubes by use of fura-2 fluorescence to characterize the intracellular organisation of Ca2+ releasing stores and Ca(2+)-entry process. 2. Stimulation of nAChRs with carbachol induced a rapid rise in internal Ca2+ (EC50 = 0.85 +/- 0.09 microM), followed by a sustained phase. The Ca2+ response evoked by carbachol (10 microM) was completely blocked by the nAChR antagonist, pancuronium (3 microM), but was not affected by the muscarinic antagonist, atropine (3 microM), or under conditions when Ca2+ entry was blocked by La3+ (50 microM) or diltiazem (10 microM). Addition of pancuronium (3 microM) during the sustained phase of the carbachol-evoked response did not affect this phase. 3. Stimulation of P2U purinoceptors with ATP (1 mM) induced a somewhat higher biphasic Ca2+ response (EC50 of the rapid phase: 8.72 +/- 0.08 microM) than with carbachol. Pretreatment with La3+ abolished the sustained phase of the ATP-induced Ca2+ response, while the response was unaffected by diltiazem or pancuronium. 4. Stimulation of the cells with high K+ (60 mM), producing the same depolarization as with carbachol (10 microM), induced a rapid monophasic Ca2+ response, insensitive to diltiazem, pancuronium or La3+. 5. Under Ca(2+)-free conditions, the sustained phase of the carbachol- and ATP-evoked responses were abolished. Pre-emptying of depolarization-sensitive stores by high K+ under Ca(2+)-free conditions did not affect the carbachol- or ATP-evoked Ca2+ mobilization and vice versa. Preincubation of the cells with ATP in the absence of extracellular Ca2+ decreased the amplitude of the subsequent carbachol-induced Ca2+ response to 11%, while in the reverse procedure the ATP-induced response was decreased to 65%. Ca2+ mobilization evoked by simultaneous addition of optimal concentrations of carbachol and ATP was increased compared to levels obtained with either agonist. 6. Preincubation with high K+ under normal conditions abolished the sustained phase of the ATP-evoked Ca2+ response. The carbachol response consisted only of the sustained phase in the presence of high K+. 7. The carbachol-induced Ca2+ response was completely abolished under low Na+/Ca(2+)-free conditions, while under low Na+ conditions only a sustained Ca2+ response was observed. The ATP- and K(+)-induced responses were changed compared to Ca(2+)-free conditions. 8. ATP (300 microM) induced the formation of Ins(1,4,5)P3 under Ca(2+)-free conditions with a comparable time course to that found for the rise in internal Ca2+. In contrast to ATP, carbachol (10 microM) did not affect Ins(1,4,5)P3 levels under Ca(2+)-free conditions. 9. It is concluded that the Ca2+ release from discrete stores of C2C12 myotubes is induced by stimulation of nAChRs, P2U-purinoceptors and by high K+. Only the P2U-purinoceptor and nAChR activated stores show considerable overlap in releasable Ca2+. Sustained Ca(2+)-entry is activated by stimulation of nAChRs and P2U-purinoceptors via separate ion-channels, which are different from the skeletal muscle nAChR-coupled cation-channel.

Adenosine Triphosphate↗
PubMed1996

The effect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+ entry in DDT1 MF-2 cells.

1. The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+]i), and on histamine H1 receptor- and thapsigargin-mediated increases in [Ca2+]i in DDT1 MF-2 smooth muscle cells. 2. After pretreatment of cells with GF109203X (5 microM, 45 min), the histamine (100 microM)-induced initial rise in [Ca2+]i, representing Ca2+ mobilization from internal stores, was inhibited (by 59 +/- 7%). The slowly declining phase of the histamine induced Ca2+ response, reflecting Ca2+ entry, was enhanced (83 +/- 26%) in the presence of the PKC inhibitor. 3. The histamine induced release of Ca2+ from internal stores, measured after blocking Ca2+ entry with LaCl3 was inhibited by GF109203X in a concentration-dependent manner (IC50: 3.1 +/- 1.1 microM). 4. Histamine-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was not changed in the presence of GF109203X. 5. The PKC activating phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM), strongly reduced histamine-induced Ins(1,4,5)P3 formation (58 +/- 16%). This effect was reversed by GF109203X (5 microM). Furthermore, PMA diminished histamine evoked Ca2+ release (50 +/- 6%) and blocked Ca2+ entry completely. 6. The rise in [Ca2+]i caused by blocking endoplasmic reticulum Ca2(+)-ATPase with thapsigargin (1 microM), was strongly reduced (57 +/- 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long-term pretreatment of cells with PMA (1 microM, 48 h) did not abolish this effect of GF109203X (48 +/- 3% inhibition). 7. In permeabilized DDT, MF-2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 microM) was markedly reduced (42 +/- 9%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5)P3 receptor function. 8. Uptake of 45Ca2+ by intact cells, representing Ca2+ entry, was enhanced by GF109203X (65 +/- 11%), by histamine (24 +/- 6%) and also by thapsigargin (121 +/- 10%). The GF109203X- and the thapsigargin-induced uptake of 45Ca2+ were not additive. 9. These data suggest that GF109203X reduces the filling-state of intracellular Ins(1,4,5)P3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake into these stores, thereby promoting store-dependent (capacitive) Ca2+ entry.

Animals↗
PubMed1995

Arachidonic acid is functioning as a second messenger in activating the Ca2+ entry process on H1-histaminoceptor stimulation in DDT1 MF-2 cells.

This study was carried out to identify the cellular component activating the histamine-stimulated Ca2+ entry in vas-deferens-derived DDT1 MF-2 cells. H1-histaminoceptor stimulation resulted in a rise in intracellular Ca2+ concentration, caused by Ca2+ release from inositol phosphate-sensitive Ca2+ stores and Ca2+ entry from the extracellular space, accompanied by a transient Ca(2+)-activated outward K+ current. The histamine-evoked K+ current was still observed after preventing inositol phosphate-induced Ca2+ mobilization by intracellularly applied heparin. This current was activated by Ca2+ entry from the extracellular space, because it was abolished in the presence of the Ca(2+)-channel blocker La3+ or under Ca(2+)-free conditions. H1-histaminoceptor-activated Ca2+ entry was also observed in the presence of intracellularly applied Ins(1,4,5)P3 and Ins(1,3,4,5)P4, depleting their respective Ca2+ stores and pre-activating the inositol phosphate-regulated Ca2+ entry. Thus the ability of histamine to activate Ca2+ entry independently of Ca2+ mobilization and the formation of inositol phosphates suggests that another component is involved to initiate the Ca(2+)-entry process. It was observed that H1-histaminoceptor stimulation resulted in a pronounced release of arachidonic acid (AA) in DDT1 MF-2 cells. Exogenously applied AA induced a concentration-dependent increase in internal Ca2+ due to activation of Ca2+ entry from the extracellular space. Slow inactivation of the AA-sensitive Ca2+ channels is suggested by the slow decline in Ca2+ entry. In accord, the histamine-induced Ca2+ entry was not observed with AA-pre-activated Ca2+ channels. Inhibition of the lipoxygenase and cyclo-oxygenase pathway did not affect the AA-induced Ca2+ and the concomitant K+ current were decreased in the presence of AA and caused by Ca2+ mobilization from internal stores. Blocking this internal Ca2+ release by heparin, in the presence of AA, resulted in abolition of the histamine-induced Ca(2+)-regulated K+ current. These observations show that AA, released on H1-histaminoceptor stimulation in DDT1 MF-2 cells, is functioning as a second messenger to activate plasma-membrane Ca2+ channels promoting Ca2+ entry from the extracellular space.

Arachidonic Acid↗
PubMed1995

Regulation of histamine- and UTP-induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca2+ by cyclic AMP in DDT1 MF-2 cells.

1. Stimulation of P2U-purinoceptors with UTP or histamine H1-receptors with histamine gave rise to the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in DDT1 MF-2 smooth muscle cells. 2. Stimulation of P2U-purinoceptors or histamine H1-receptors caused an increase in cytoplasmic Ca2+, consisting of an initial peak, representing the release of Ca2+ from internal stores and a sustained phase representing Ca2+ influx. 3. The P2U-purinoceptor-mediated Ca(2+)-entry mechanism was more sensitive to UTP than Ca(2+)-mobilization (EC50: 3.3 microM +/- 0.4 microM vs 55.1 microM +/- 9.2 microM), in contrast to these processes activated by histamine H1-receptors (EC50: 5.8 microM +/- 0.6 microM vs 3.1 microM +/- 0.5 microM). 4. Pre-stimulation of cells with several adenosine 3':5'-cyclic monophosphate (cyclic AMP) elevating agents, reduced the histamine H1-receptor-mediated formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin completely inhibited Ins(1,4,5)P3 formation (IC50: 158 +/- 24 nM) whereas Ins(1,3,4,5)P4 formation was inhibited by only 45% (IC50: 173 +/- 16 nM). The P2U-purinoceptor-mediated production of these inositol phosphates was not affected by cyclic AMP. 5. Forskolin and isoprenaline reduced the histamine-induced increase in cytoplasmic Ca2+, as measured in Ca2+ containing medium and in nominally Ca(2+)-free medium but did not change the UTP-induced increase in cytoplasmic Ca2+. 6. These results clearly demonstrate that cyclic AMP differentially regulates components of the histamine induced phospholipase C signal transduction pathway. Furthermore, cyclic AMP does not affect the phospholipase C pathway activated by stimulation of P2U-purinoceptors in DDT1 MF-2 cells.

Adenylyl Cyclases↗
PubMed1995

Ca(2+)-dependent and -independent mechanism of cyclic-AMP reduction: mediation by bradykinin B2 receptors.

1. Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 +/- 4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 +/- 1.1 nM). 2. The Ca2+ ionophore, ionomycin, mimicked both the bradykinin-induced transient reduction in the forskolin-stimulated cyclic AMP level and the sustained reduction in the isoprenaline-stimulated cyclic AMP level. 3. The Ca(2+)-dependent effect on cyclic AMP induced by bradykinin was mediated solely by Ca2+ release from internal stores, since inhibition of Ca2+ entry with LaCl3 did not reduce the response to bradykinin. 4. The involvement of calmodulin-dependent enzyme activities, protein kinase C or an inhibitory GTP binding protein in the bradykinin-induced responses was excluded since a calmodulin inhibitor, calmidazolium, a PKC inhibitor, staurosporine and pertussis toxin, respectively did not affect the decline in the cyclic AMP level. 5. Bradykinin enhanced the rate of cyclic AMP breakdown in intact cells, which effect was not mimicked by ionomycin. This suggested a Ca(2+)-independent activation of phosphodiesterase activity by bradykinin in DDT1 MF-2 cells. 6. The bradykinin B1 receptor agonist, desArg9-bradykinin, did not affect cyclic AMP formation in isoprenaline prestimulated cells, while the bradykinin B2 receptor antagonists, Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK) and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK completely abolished the bradykinin response in both forskolin and isoprenaline prestimulated cells. 7. Bradykinin caused an increase in intracellular Ca2+, which was antagonized by the bradykinin B2 receptor antagonists, Hoe 140 and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK. The bradykinin B2 receptor agonist,desArg9-bradykinin, did not evoke a rise in cytoplasmic Ca2 .8. It is concluded, that stimulation of bradykinin B2 receptors causes a reduction in cellular cyclic AMP in DDT1, MF-2 cells. This decline in cyclic AMP is partly mediated by a Ca2+/calmodulin independent activation of phosphodiesterase activity. The increase in [Ca2+], mediated by bradykinin B2 receptors inhibited forskolin- and isoprenaline-activated adenylyl cyclase differently, most likely by interfering with different components of the adenylyl cyclase signalling pathway.

Animals↗
PubMed1994

The phospholipase C activating P2U purinoceptor also inhibits cyclicAMP formation in DDT1 MF-2 smooth muscle cells.

The P2U purinoceptor mediated effect on cellular cAMP was investigated in DDT1 MF-2 smooth muscle cells. Stimulation of these receptors by ATP or UTP caused a pronounced decrease of about 50% in cellular cAMP levels in forskolin or isoprenaline pretreated cells. This action of the nucleotides was concentration dependent with an IC50 of 9.4 +/- 0.2 microM and 29.0 +/- 0.5 microM for UTP and ATP, respectively and was inhibited by the P2-purinoceptor antagonist suramin. The cAMP level appeared to be modified by intracellular Ca2+, represented by an initial decline in cAMP. Neither inactivation of protein kinase C by staurosporine nor elevated cytoplasmic Ca2+ concentrations interfered with the sustained decrease in cAMP levels induced by ATP or UTP, showing that this effect is not mediated via the phospholipase C pathway known to be activated after P2U purinoceptor stimulation in DDT1 MF-2 cells. Pertussis toxin inhibited the action of these nucleotides on the cellular cAMP level. It can be concluded that the P2U purinoceptor in DDT1 MF-2 cells is coupled to different G-proteins, activating phospholipase C and inhibiting adenylyl cyclase activity.

Adenosine Triphosphate↗
PubMed1994

Induction of Na+/K(+)-ATPase activity by long-term stimulation of nicotinic acetylcholine receptors in C2C12 myotubes.

1. To investigate the role of long-term stimulation of nicotinic acetylcholine receptors (AChRs) on the regulation of membrane potential, non-contracting C2C12 myotubes were stimulated for 1-4 days with carbachol (10 microM) and membrane potentials were measured by the intracellular microelectrode technique after washing out of the drug. 2. The membrane potential (-45.7 mV) gradually increased by 10.1 mV to -55.8 mV during 4 days treatment, which was caused by enhanced electrogenic Na+/K(+)-pumping. 3. The concentration-dependent enhancement of Na+/K(+)-ATPase activity in long-term carbachol-treated myotubes (4 days, EC50 = 5.3 microM) was prevented by co-treatment with the competitive nicotinic AChR antagonist, pancuronium but not by the muscarinic antagonist, atropine. 4. Enhanced Na+/K(+)-ATPase activity still developed in carbachol-stimulated myotubes during co-treatment (4 days) with the nicotinic AChR-channel blocker, chlorpromazine (1 microM). Membrane depolarization as such, obtained by incubation in high K+ medium (40 mM, 4 days) did not enhance Na+/K(+)-ATPase activity. 5. Non-treated myotubes possessed a high-affinity ouabain binding site (Kd = 119 nM) in association with the low Na+/K(+)-pumping activity. Long-term stimulation of myotubes (4 days) with carbachol or with a combination of carbachol and chlorpromazine was accompanied by the development of an additional low-affinity ouabain binding site (Kd = 13 microM). 6. Binding of monoclonal antibodies directed against either alpha 1- or alpha 2-subunit of Na+/K(+)-ATPase were both increased in myotubes treated with carbachol (4 days). 7. These results support the concept that nicotinic AChRs regulate Na+/K(+)-ATPase activity, independent of the functionality of the receptor-operated ion-channel.

Animals↗
PubMed1993

Prolonged paralysis after long-term, high-dose infusion of pancuronium in anaesthetized cats.

We have studied the neuromuscular effects of a 48-h infusion of high-dose pancuronium (400 micrograms kg-1 h-1) in four cats anaesthetized with pentobarbitone, using contraction of tibialis anterior muscles after direct and indirect stimulation. After cessation of the pancuronium infusion, prolonged paralysis existed. The first twitch in the train-of-four stimuli (TOF) reappeared 8-12 h after termination of the pancuronium infusion. Twenty-four hours after termination of the infusion, TOF ratios were less than 0.08 and twitch contraction averaged 39 (SE 8)% of initial values. Twitch contraction after direct stimulation did not differ from initial values. Antagonism of paralysis was accomplished with neostigmine 60 micrograms kg-1 in two animals and neostigmine 90 micrograms kg-1 and 4-aminopyridine 500 micrograms kg-1 in the others. Steady-state plasma concentration of pancuronium (2000 ng ml-1) decreased rapidly after termination of the infusion, but then stabilized at about 130 ng ml-1. These results indicate that prolonged paralysis after long-term administration of high-dose pancuronium is caused primarily by failure of neuromuscular transmission, most likely caused by the persistent plasma concentrations of the drug in the pharmacologically active range.

4-Aminopyridine↗
PubMed1993

Characterization of P2-purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes.

1. The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2C12 myotubes. 2. ATP (100 microM) and ATP gamma S (100 microM) caused a sustained increase in cyclic AMP content of the cells, reaching a maximum after 10 min. The cyclic AMP content reached a maximum in the presence of 100 microM ATP, followed by a decline at higher ATP concentrations. ATP-induced cyclic AMP formation was inhibited by the P2-purinoceptor antagonist, suramin. 3. Myotubes hydrolysed ATP to ADP at a rate of 9.7 +/- 1.0 nmol mg-1 protein min-1. However, further hydrolysis of ADP to AMP and adenosine was negligible. 4. The cyclic AMP formation induced by ADP (10 microM-1 mM) showed similar characteristics to that induced by ATP, but a less pronounced decline was observed than with ATP. ADP-induced cyclic AMP formation was blocked by suramin, while cyclic AMP formation elicited by adenosine (10 microM-1 mM) was insensitive to suramin. 5. The ATP analogue, alpha,beta-methylene-ATP also induced a suramin-sensitive cyclic AMP formation, while 2-methylthio-ATP and the pyrimidine, UTP, did not affect cyclic AMP levels. 6. Stimulation of the myotubes with ATP or UTP (10 microM-1 mM) caused a concentration-dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 microM-1 mM) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels. 7. Incubation of the cells with UTP (30 microM- 1 mM) inhibited the ATP- and ADP-induced cyclic AMP formation, suggesting that stimulation of the 'nucleotide' type P2-receptor inhibits P2-purinoceptor mediated cyclic AMP formation in C2C12 myotubes. In contrast, UTP (30 microM-I mM) enhanced adenosine-induced cyclic AMP formation.8. Adenosine-sensitive P1-purinoceptors activating cyclic AMP formation were found in C2C12 myotubes.Further, a novel P2-purinoceptor is postulated, sensitive to ATP, ADP and ATPgammaS, which also activates the formation of cyclic AMP in C2C12 myotubes.

Adenine Nucleotides↗
PubMed1993

Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2-purinoceptors in C2C12 myotubes.

1. The presence of a nucleotide receptor and a discrete ATP-sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP-sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+. 2. The nucleotides ATP and UTP caused a concentration-dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 +/- 2 microM, UTP 80 +/- 4 microM). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1-, P2X- and P2Y-purinoceptors, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP, appeared to be ineffective. 3. Under Ca(2+)-free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP- and UTP-induced formation of Ins(1,4,5)P3 was diminished. 4. The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP- and UTP-induced Ins(1,4,5)P3 formation showed cross-desensitization, whereas cross-desensitization was absent in responses elicited by one of the nucleotides and bradykinin. 5. The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP- and UTP-stimulated myotubes showed slopes greater than unity (1.63 +/- 0.09 and 1.37 +/- 0.11, respectively). Apparent pA2 values were 4.50 +/- 0.48 and 4.41 +/- 0.63 for ATP and UTP, respectively. 6. Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+. The ATP and UTP-evoked increase in intracellular Ca2+ was not additive and showed cross-desensitization. Cross-desensitization was absent in myotubes stimulated with one of the nucleotides and bradykinin.7. These results show that ATP- and UTP-induced formation of Ins(1,4,5)P3, Ca2+ release from internal stores and Ca2+-influx from the extracellular space are mediated exclusively via the nucleotide type P2-purinoceptor in mouse C2C12 myotubes.

Adenosine Triphosphate↗
PubMed1992

The nucleotide receptors on mouse C2C12 myotubes.

1. The response of C2C12 mouse myotubes to stimulation with adenosine triphosphate (ATP) and other nucleotides was studied by measuring changes in membrane potential. 2. A transient hyperpolarization followed by a slowly declining depolarization of the cells was observed in the presence of ATP (10 microM-1 mM). 3. The hyperpolarization was not observed in the absence of external calcium, and was abolished in the presence of tetraethylammonium (20 mM) or the bee toxin, apamin (0.1 microM). The depolarization was reduced under low sodium conditions. 4. A biphasic change in membrane potential was also recorded in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) and the pyrimidine uridine triphosphate (UTP), while the ATP derivatives and analogues, adenosine diphosphate, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP and the nucleotides, guanosine triphosphate and cytidine triphosphate, did not affect the membrane potential of the myotubes. 5. The hyperpolarization elicited by ATP gamma S or UTP was also blocked by apamin and abolished under Ca(2+)-free conditions. 6. In contrast to ATP and ATP gamma S, the depolarization evoked by UTP was unaffected under low Na+ and less sensitive to the antagonistic action of suramin. 7. The ATP and UTP responses at maximal concentration were not additive after simultaneous application. ATP elicited a depolarization if applied after UTP, while UTP did not change membrane potential following the application of ATP. 8. The concentration-response curves of the effective nucleotides were shifted to the right in the presence of suramin, suggesting competitive antagonism.9. These results can be explained by the presence of 'nucleotide receptors' mediating the ATP/UTPinduced hyperpolarization and depolarization in C2C12 myotubes. Furthermore, an increase in Na+-conductivity can be exclusively activated by ATP.

Adenosine Triphosphate↗
PubMed1991

Voltage-dependent sodium and potassium, but no calcium conductances in DDT1 MF-2 smooth muscle cells.

Voltage-dependent inward and outward membrane currents were investigated in the DDT1 MF-2 smooth muscle cell line using the whole-cell patch-clamp technique. Application of a pulse protocol with subsequent depolarizing voltage steps elicited an inactivating inward current and a non-inactivating outward current. The outward current was activated at membrane potentials more positive than -35 mV, with tau act = 30 -40 ms. The outward current was blocked by tetraethylammonium (NEt4Cl) and 3,4-aminopyridine in a dose-dependent manner (EC50 of 5 mM and 0.5 mM, respectively). The amplitude of the outward current was linked to the potassium equilibrium potential (Vek), and tail currents reversed near Vek. The outward current was completely abolished when intracellular potassium was substituted by 106 mM caesium and 20 mM NEt4Cl. The inward current was activated at potentials more positive than -30 mV with tau act of 1.6-2.5 ms, and with tau inact of 1.7-3.0 ms. Steady-state inactivation was 50% at a holding potential of -40 mV. The inward current was blocked by tetrodotoxin (EC50 of 0.15 microM) and dependent on the reversal potential for sodium. Voltage-dependent calcium currents could not be detected. Further, the cytoplasmic free calcium concentration, as measured using Indo-1 fluorescence, was not changed during high-potassium (40 mM)-induced depolarization. In contrast, contraction of freshly obtained hamster vas deferens tissue elicited by high-potassium(40 mM)-induced depolarization was largely inhibited by diltiazem (20 microM). These findings showed that voltage-dependent calcium channels are not functional in DDT1 MF-2 smooth muscle cells in contrast to freshly obtained Syrian hamster vas deferens smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopyridines↗
PubMed1990

P2 purinoceptor-mediated inositol phosphate formation in relation to cytoplasmic calcium in DDT1 MF-2 smooth muscle cells.

The effect of P2 purinoceptor stimulation on inositol phosphate (InsP) formation in relation to the intracellular Ca2+ concentration was measured in vas deferens DDT1 MF-2 smooth muscle cells. The different [3H]myo-inositol-labelled InsP fractions were analyzed by high performance liquid chromatography and intracellular Ca2+ was determined by measuring fluorescence using Indo-1 as indicator. Stimulation with ATP (10(-4) M) resulted in an enhanced formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP1, InsP2, InsP3 and InsP4), but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). The putative second messenger Ins(1,3,4,5)P4 rapidly increased after addition of the agonist, reaching a maximum after about 2 min. The isomer Ins(1,4,5)P3 showed a delayed rise starting after about 2 min. The formation of Ins(1,3,4,5)P4 in the presence of ATP (2 min) was concentration-dependent, reaching a half maximal value at about 50 microM of the agonist. The intracellular Ca2+ concentration showed an initial increase after P2 purinoceptor stimulation, reaching a plateau after 2 min. Both the top of the initial phase and the plateau value of the response reached a half maximal value at an ATP concentration of about 7 microM. This Ca2+ response could be evoked repeatedly by ATP and was not affected by diltiazem (10(-5) M). In the absence of external Ca2+, the internal Ca2+ concentration increased transiently in the presence of ATP without showing the plateau phase. This response could be evoked only once under Ca2(+)-free conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Alpha-adrenoceptor regulation of inositol phosphates, internal calcium and membrane current in DDT1 MF-2 smooth muscle cells.

The effect of alpha 1-adrenoceptor stimulation on inositol phosphates (InsPs), intracellular Ca2+ and membrane current was measured in vas deferens DDT1 MF-2 cells. The InsPs were analyzed after labelling the cells with [3H]myo-inositol using high performance liquid chromatography and the internal Ca2+ concentration was determined by measuring fluorescence using Indo-1 as probe. Noradrenaline stimulated the formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP, InsP2, InsP3 and InsP1) concentration-dependently in the presence of LiCl, but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). Various isomers of InsP3 and InsP4 were detected after noradrenaline (10(-5) M) stimulation (without LiCl). Only the formation of the putative second messenger Ins(1,4,5)P3 formation was increased in the presence of noradrenaline. The internal Ca2+ concentration was enhanced both in the presence and absence of external Ca2+ upon addition of noradrenaline. The response in the presence of extracellular Ca2+ was not affected by diltiazem (10(-5) M). The increase in cytoplasmic Ca2+ could be repeatedly evoked, but could be elicited only once under Ca2(+)-free conditions. The relation between the noradrenaline concentration and the rise in internal Ca2+ was similar to that obtained for the InsP formation. Half-maximal effects were obtained at about 1 microM. The membrane current measured in these cells by using the whole-cell patch-clamp method was not changed by the agonist (10(-5) M). These results suggest that noradrenaline acts on DDT1 MF-2 smooth muscle cells, represented by the formation of InsP3 and enhancement of internal Ca2+ originating from internal structures, via the alpha 1B-adrenoceptor subtype.

Animals↗
PubMed1989

Effect of cromakalim on smooth muscle cells of guinea-pig taenia caeci.

Cromakalim caused hyperpolarization and reduction of the electrotonic potential in a concentration-dependent manner in smooth muscle cells of guinea-pig taenia caeci. There was a relatively constant change in the electrotonic potential under calcium-free, low-sodium and low-chloride conditions in the presence of cromakalim as compared to control conditions with Krebs solutiuon. The effect of cromakalin (10(-5) M) was inhibited by glibenclamide (5 X 10(-5) M). These results indicate that cromakalim specifically promotes potassium efflux in smooth muscle cells of the guinea-pig taenia caeci via glibenclamide-sensitive potassium channels, to cause hyperpolarization, suppression of spike activity and relaxation.

Animals↗
PubMed1989

The inhibitory action of suramin on the P2-purinoceptor response in smooth muscle cells of guinea-pig taenia caeci.

The effect of suramin on the smooth muscle cell response of guinea-pig taenia caeci to P2-purinoceptor and alpha 1-adrenoceptor stimulation was measured. The ATP-induced relaxation in potassium (20 mM) pre-contracted taenia caeci was inhibited by suramin (3 x 10(-4) M). The P2-purinoceptor-induced hyperpolarization elicited by ATP both in the presence and absence of calcium was also reduced by suramin. The alpha 1-adrenoceptor-mediated relaxation evoked by phenylephrine was only affected by suramin at low concentrations of the agonist. The results indicate that suramin inhibits the ATP response by interacting with P2y-purinoceptors.

Adenosine Triphosphate↗