Adriaan den Hertog

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 sup -6 to 10 sup -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 IC50 S (mean +/-SEM; micro Meter) 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.

Calcium and the action of adrenaline, adenosine triphosphate and carbachol on guinea-pig taenia caeci.

1. The action of adrenaline (in the presence of propranolol; 3 × 10−6 M), adenosine triphosphate (ATP) and carbachol on guinea‐pig taenia caeci, and the interaction between these agonists, was studied by measuring changes in membrane potential using the sucrose‐gap method in quiescent preparations at 22 °C.

Calcium and the α‐action of catecholamines on guinea‐pig taenia caeci

1. The involvement of calcium in the α‐action of adrenaline on guinea‐pig taenia caeci was studied by measuring the changes in membrane potential and muscle contraction, using the sucrose‐gap method, and by determining the 42K efflux, in the presence of a β‐blocker (propranolol, 1·8 × 10−6 m).

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 μm, UTP 80 ± 4 μm). 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, α,β‐methylene ATP and 2‐methylthio ATP, appeared to be ineffective. 3 Under Ca2+‐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.

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 μm, 45 min), the histamine (100 μm)‐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 μm). 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 μm), strongly reduced histamine‐induced Ins(1,4,5)P3 formation (58 ± 16%). This effect was reversed by GF109203X (5 μm). 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 μm), was strongly reduced (57 ± 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long‐term pretreatment of cells with PMA (1 μm, 48 h) did not abolish this effect of GF109203X (48 ± 3% inhibition). 7 In permeabilized DDT1 MF‐2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 μm) 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.

Suramin and the inhibitory junction potential in taenia caeci of the guinea-pig

The effect of surinam on the inhibitory junction potential evoked in smooth muscle cells of guinea-pig taenia caeci by stimulation of intramural nerves at 22 degrees C was investigated. The amplitude of the inhibitory junction potential was reduced concentration dependently by suramin. The suppression of this response by suramin became less pronounced when the number of stimuli increased (pulse rate: 20/s). These results indicate that suramin reduces the inhibitory junction response by interacting with P2-purinoceptors.

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 μm) and ATP7S (100 μm) 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 μm 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 μm–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 μm–1 mm) was insensitive to suramin. 5 The ATP analogue, α,β‐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 μm–1 mm) caused a concentration‐dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 μm–1 mm) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels. 7 Incubation of the cells with UTP (30 μm–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 μm–1 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 ATPγS, which also activates the formation of cyclic AMP in C2C12 myotubes.

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 μm−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 μm). 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) (ATPγS) and the pyrimidine uridine triphosphate (UTP), while the ATP derivatives and analogues, adenosine diphosphate, adenosine, α,β‐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 ATPγS or UTP was also blocked by apamin and abolished under Ca2+‐free conditions. 6 In contrast to ATP and ATPγS, 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/UTP‐induced hyperpolarization and depolarization in C2C12 myotubes. Furthermore, an increase in Na+‐conductivity can be exclusively activated by ATP.

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.

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 Ca2+‐entry mechanism was more sensitive to UTP than Ca2+‐mobilization (EC50: 3.3 μm ± 0.4 μm vs 55.1 μm ± 9.2 μm), in contrast to these processes activated by histamine H1‐receptors (EC50: 5.8 μm ± 0.6 μm VS 3.1 μm ± 0.5 μm). 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 Ca2+‐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.