Biomedical subjects

J Vereecke

Publications and source records attributed to J Vereecke.

At least 19 recordsLinked to original sources

PubMed2003

Intercellular Ca(2+)-transient propagation in normal and high glucose solutions in rat retinal epithelial (RPE-J) cells during mechanical stimulation.

We investigated the effect of high glucose and modulation of protein kinase C (PKC) on the intercellular propagation of Ca(2+)-waves in a rat retinal pigment epithelial cell line (RPE-J cells) in order to compare its properties with the properties previously investigated in primary LE-RPE cells. The intercellular propagation of the Ca(2+)-waves in RPE-J cells was analyzed by fluorescence imaging confocal microscopy and fluorescence recovery after photobleaching (FRAP). In control conditions the maximal normalized fluorescence in the mechanically stimulated (MS) cell and the propagation towards the neighboring RPE-J cells were similar to LE-RPE cells. As in LE-RPE cells, the propagation was reduced by the gap junction (GJ) blocker halothane, and FRAP experiments demonstrated the presence of functional GJ coupling. Similar to the effect in LE-RPE cells, the propagation of the Ca(2+)-transient was reduced by 25 mM glucose. However, unlike LE-RPE cells, the neighboring RPE-J cells presented a Ca(2+)-rise of amplitude similar to that in normal glucose levels. PKC activation with 1 microM PMA for 30 min resulted in inhibition of the Ca(2+)-wave propagation, which could be overcome by PKC downregulation as in LE-RPE cells. Cells grown for 72 h in a high glucose solution in which PKC activity was downregulated, did not develop the inhibitory effect on Ca(2+)-wave propagation that was induced by elevated glucose levels. However, the effects were not as pronounced as in LE-RPE cells. We concluded that despite marked similarities, the transduction and the modulation of intercellular propagation of the Ca(2+)-transients in RPE-J cells are not identical to the mechanisms in primary LE-RPE cells.

Animals↗

PubMed2001

Effects of hyperglycemia and protein kinase C on connexin43 expression in cultured rat retinal pigment epithelial cells.

Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial (RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons (RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46 were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively. Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 microM phorbol 12-myristate 13-acetate (PMA). The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions.

Animals↗

PubMed2001

Mechanism of intracellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured endothelial CPAE cells.

Intra- and intercellular Ca(2+)-signaling during mechanical stimulation in calf pulmonary artery endothelial cells (CPAE) was investigated with digital fluorescence microscopy. Mechanical stimulation of a CPAE cell in a Ca(2+)-containing solution revealed a rise of the free intracellular Ca(2+)-concentration ([Ca(2+)](i)) in the mechanically stimulated cell (MS) proceeding to the neighboring (NB) cells as an intercellular Ca(2+)-wave. Experiments in Ca(2+)-free solution, containing 2mM EGTA, demonstrated that a detectable [Ca(2+)](i)-transient in the MS cell is not always a requisite for intercellular communication (IC). The Ca(2+)-wave propagation was not affected by changes in membrane potential and was not mediated by voltage-dependent Ca(2+)-channels. Ca(2+)-influx through the Ni(2+)-sensitive Ca(2+)-pathway occurred in the MS as could be assessed by Mn(2+)-quenching experiments. The intra- and intercellular Ca(2+)-wave was triggered by the release of thapsigargin-sensitive intracellular Ca(2+)-stores. Phospholipase C (PLC) inhibition by U73122 reduced the Ca(2+)-amplitude of the MS cell and almost completely inhibited the IC, indicating that the Ca(2+)-release in the MS and NB cells is PLC/inositol 1,4,5-trisphosphate (IP(3)) mediated.

Animals↗

PubMed2001

Intercellular communication upon mechanical stimulation of CPAE- endothelial cells is mediated by nucleotides.

Intercellular Ca(2+)-signaling, after mechanical stimulation of calf pulmonary artery endothelial cells (CPAE), was investigated with fluorescence video imaging. Mechanical stimulation evoked an intracellular Ca(2+)-response in the mechanically stimulated (MS) cell, proceeding to the neighboring (NB) cells as a Ca(2+)-wave. The intercellular propagation of the Ca(2+)-wave was unaffected by the gap junction blockers halothane or heptanol. Therefore the intercellular communication (IC) pathway of the Ca(2+)-wave in CPAE cells does not depend on gap junctional communication but is most likely mediated by release of an extracellular mediator. Continuous unilateral flow experiments confirmed the presence of a diffusible mediator: the Ca(2+)-rise in upstream NB cells is significantly lower than in control experiments. After desensitization of purinergic receptors by pretreatment of CPAE cells with ATP (100mM), UTP (100 microM), 2MeSATP (100microM) or ADPbS (100 microM), the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation was significantly inhibited. Also suramin (200 and 400 microM), a non-specific purinergic receptor blocker, reduced the IC. Application of the nucleotidase apyrase VI (10U/ml), which has a high ATPase/ADPase ratio, enhanced Ca(2+)-signaling and IC. In contrast, apyrase VII (10U/ml), which has a high ADPase/ATPase ratio, significantly depressed the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation. Our experiments therefore demonstrate that the IC, evoked by a mechanical stimulus of CPAE cells, is mediated via release of nucleotides in the extracellular space. The data indicate that the diffusible messenger, responsible for the propagation of a Ca(2+)-wave, is mainly ADP or a combination of ADP/ATP.

Adenosine Triphosphate↗

PubMed2001

Intra- and intercellular Ca(2+)-transient propagation in normal and high glucose solutions in ROS cells during mechanical stimulation.

Experiments using confocal laser microscopy on the rat osteosarcoma cell line (ROS 17/2.8) indicate that mechanical stimulation elicits pronounced [Ca2+](i)transients in the MS (mechanically stimulated) cell, which then propagate to the NB (neighbouring) cells. Experiments with Ca(2+)-free solutions or gadolinium suggest that Ca(2+)-influx through stretch-sensitive channels is required. When intracellular stores are depleted with thapsigargin, mechanical stimulation was able to evoke a Ca(2+)transient of reduced amplitude that disappeared entirely after subsequent blocking of Ca(2+)-influx. Heptanol inhibited intercellular propagation of the Ca(2+)transient, demonstrating the involvement of gap junctions in the propagation of the Ca(2+)transient in ROS cells. PKC activation has only a small inhibitory effect, while inhibition of PKC or tyrosine kinase was ineffective. PKA activation reduced the amplitude of the [Ca2+](i)-rise in NB cells, and decreased the percentage of responsive cells. Cells grown in 50mM glucose for 72h presented only a very limited decrease of the Ca(2+)-rise during mechanical stimulation in the MS and NB cells compared to control conditions. PKC downregulation in high glucose did not modulate this effect. The results of our experiments indicate that PKC or sustained high glucose concentrations do not affect gap junctional communication in ROS cells, while activation of PKA has an inhibitory effect. This might indicate that osteoblastic dysfunction in diabetes could be directly related to the high glucose concentrations and not to inhibition of the intercellular communication.

Adenosine Triphosphate↗

PubMed2000

The effect of external pH on the delayed rectifying K+ current in cardiac ventricular myocytes.

The effects of extracellular pH (pHe) on the delayed rectifying K+ current iKr in rabbit ventricular myocytes were studied using the whole-cell-clamp technique. Since a variety of results have been reported on the effect of pH on expressed hERG channels, our aim was to investigate the effects of pH on iKr channels in their native environment. iKr is reduced by extracellular acidification and its deactivation is faster. Extracellular acidification results in a marked shift of the steady-state activation curve to more positive potentials, while alkalinization does not produce a significant shift. E1/2= - 11.3 mV, -20.2 mV, -21.4 mV at pHe 6.5, 7.4, 8.5 respectively; the slope factor is 6.2 mV, and is not affected by pHe. Deactivation of iKr is biexponential, with time constants of the order of 0.5 s and 10 s at -50 mV. Both time constants decrease with external acidification. Also the contribution of the fast component to the total amplitude becomes larger with acidification. Acidification also decreases the fully activated iKr current. Our experiments demonstrate that extracellular acidification reduces iKr by increasing the rate of deactivation, causing a shift of the voltage dependence of activation and producing a voltage-dependent block of the fully activated iKr current.

Acids↗

PubMed2000

[Intra- and intercellular Ca(2+)-signal transduction].

Calcium is one of the most universal signal-transduction elements in a large variety of cells ranging from bacteria to specialized neurons. Ca2+ acts as a second messenger controlling such processes as secretion, cell differentiation or signal transmission. In order to be able to execute their specific functions and to react in a coordinated way to stimuli, multicellular organs need a precise orchestration of cellular functions. For this purpose cells have developed different forms of intercellular communication (IC). In this study we investigated a number of mechanisms of intracellular propagation and IC using experiments with fluorescent Ca(2+)-indicators, confocal microscopy and digital imaging techniques. In ROS 17/2.8 osteoblasts, retinal pigment epithelial cells (RPE) and CPAE endothelial cells, a small mechanical deformation of the plasma membrane results in a transient increase of free cytoplasmic Ca2+ concentration ([Ca2+]i). This Ca(2+)-rise starts at the site of stimulation and propagates concentrically to neighboring cell layers. The intracellular Ca(2+)-wave in RPE and ROS cells is caused by Ca(2+)-influx followed by Ca(2+)-release from the intracellular stores and by intercellular propagation of the Ca(2+)-wave. The [Ca2+]i-transient upon mechanical stimulation of LLC-PK1 epithelial cells, C6 glioma cells and MLO-Y4 osteocytes was limited and/or variable. In CPAE cells only the intracellular release is important for evoking the Ca(2+)-transient, and is followed by IC. IC can occur via gap junctions (GJ) consisting of membrane-spanning proteins, connexins (Cx). It was demonstrated that IC and GJ in RPE and ROS cells can be reversibly blocked by gap-junction inhibitors such as heptanol or halothane. We demonstrated important differences in modulation of gap junctional communication between these cell types. While in RPE cells stimulation of PKC activity was able to inhibit IC, this was not the case in ROS cells. We screened LE-RPE cDNA via PCR using specific primers for different connexins and found no effect of high glucose solutions, which cause decreased intercellular communication, on the Cx-isoforms expressed. Cx43 is the only Cx-isoform present at the protein level for which Western blot analysis revealed the presence of different forms corresponding to different phosphorylated states. Increased phosphorylation of Cx43 was only seen after direct PKC activation by PMA, but not by indirect PKC activation by high glucose levels. The decreased communication by high glucose concentrations was however associated by a decreased expression of cellular Cx43 to about 3/4 of the level in control conditions. High glucose concentrations therefore decrease Cx43 at the protein level via a PKC effect that appears to be independent of the direct activation of PKC by phorbolesters. Mechanical stimulation did not evoke intercellular Ca(2+)-waves in LLC-PK1 epithelial cells, C6 glioma cells and MLO-Y4 osteocytes. In CPAE-endothelial cells, the contribution of gap junctions to IC following mechanical stimulation is negligible, and modulation of gap junctions via phosphorylation or high glucose solutions is absent. Perfusion experiments and pharmacological studies demonstrated that IC following mechanical stimulation of these cells occurs via release of an extracellular mediator. Our experiments provide strong evidence in favor of purinergic agonists as mediators, such as ATP but mainly ADP. In conclusion we can say that cells contain a wide spectrum of mechanisms for intra- and intercellular communication, and that widely different mechanisms can evoke the same phenomenon of intra- and intercellular Ca(2+)-waves.

Animals↗

PubMed1999

Intra- and intercellular Ca2+ signaling in retinal pigment epithelial cells during mechanical stimulation.

The intercellular communication (IC) was investigated between cultured rat retinal pigment epithelial (RPE) cells isolated from Long-Evans (LE) or dystrophic Royal College of Surgeons (RCS) rats and grown in solutions containing normal and high glucose concentrations, or after modulation of protein kinase C (PKC). This was performed by studying the conduction of the free Ca2+-concentration ([Ca2+]i) wave elicited by mechanical stimulation and by analyzing the fluorescence recovery after photobleaching (FRAP). Mechanical stimulation of LE-RPE cells triggers Ca2+ influx, mediated by stretch-sensitive cation channels followed by intracellular Ca2+ release. A regenerative [Ca2+]i wave was found with a lower propagation rate in RCS-RPE cells. This rate could be increased by PKC down-regulation. Mechanical stimulation caused a [Ca2+]i increase in the mechanically stimulated (MS) cell followed after a delay by a [Ca2+]i rise in the adjacent cell layers. The intercellular [Ca2+]i wave propagation could be blocked by gap junction blockers such as halothane or PKC activation. An inhibition of the [Ca2+]i-wave propagation similar to that induced by halothane could be observed in cells grown in solutions containing 14 mM or higher concentrations of glucose. PKC down-regulated cells grown in glucose-rich medium did not develop this inhibitory effect on gap junction communication (GJC). FRAP experiments confirmed that the observed changes were consistent with a PKC-mediated inhibitory effect of high glucose concentrations on GJC.

Animals↗

PubMed1996

Mechanism of L- and T-type Ca2+ channel blockade by flunarizine in ventricular myocytes of the guinea-pig.

Flunarizine is a substance known to block voltage-dependent Ca2+ channels in smooth muscle and neuronal cells. Reports on the effect on voltage-dependent cardiac Ca2+ channels are however sparse. Therefore, the mechanism of action of flunarizine on two types of voltage-dependent cardiac Ca2+ channels, the L- and T-type, in single ventricular myocytes of the guinea-pig was investigated using the whole-cell voltage clamp technique. Both channel types can be blocked by flunarizine in a time-, frequency-, voltage-, Ca(2+)-, and proton-dependent way. While the overall mechanism of action on cardiac myocytes is similar to the one reported for other cell types, we found that cardiomyocytes are less susceptible to block (Kd 3.3-11 mM). We also describe a complete analysis of the different components of block, together with evidence for open channel state block and drug-induced changes in channel gating. These findings provide new insights into the mechanism of action of flunarizine on voltage-dependent Ca2+ channels.

Animals↗

PubMed1995

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange.

Transient inward currents (Iti) during oscillations of intracellular [Ca2+] ([Ca2+]i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca2+-dependent membrane currents contribute to Iti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca2+]i oscillations and during caffeine-induced Ca2+ release from the sarcoplasmic reticulum in the absence of Na+. Membrane currents were recorded during whole-cell voltage clamp and [Ca2+]i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li+, Cs+ or N-methyl-D-glucamine (NMDG+) substituted for Na+, the cell could be loaded with Ca2+ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca2+]i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca2+ release was accompanied by a shift of the membrane current in the outward direction at potentials between -40 mV and +60 mV. This [Ca2+]i-dependent outward current shift was not abolished when NMDG+ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl-. It was however, sensitive to the blockade of ICa by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca2+ release represents [Ca2+]i-dependent transient inhibition of ICa. Similarly, during the [Ca2+]i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca2+-activated nonselective cation channel, or to a Ca2+-activated Cl- channel; however, transient Ca2+-dependent inhibition of ICa was again observed. We conclude that neither the Ca2+-activated nonselective cation channel nor the Ca2+-activated Cl- channel contribute significantly to the membrane currents during spontaneous [Ca2+]i oscillations in guinea-pig ventricular myocytes. However, in the voltage range between -40 mV and +60 mV Ca2+-dependent transient inhibition of ICa will contribute to the oscillations of the membrane current.

Animals↗

PubMed1994

Reversal of rectification and alteration of selectivity and pharmacology in a mammalian Kv1.1 potassium channel by deletion of domains S1 to S4.

1. A possible relation between the family of inwardly rectifying K+ channels and the Shaker superfamily of K+ channels was investigated using a deletion mutant (DelS1-S4) of a delayed rectifier Kv1.1 (RCK1) K+ channel. 2. The mutant DelS1-S4 was made by eliminating the sequence coding for transmembrane domains S1 to S4 of the Kv1.1 K+ channel, and re-ligating the sequence coding for the cytoplasmic amino terminus to transmembrane domain S5. Microelectrode voltage-clamp and patch-clamp experiments were performed on Xenopus laevis oocytes after injection of in vitro transcribed mRNA coding for mutant and wild-type channels. 3. The lack of transmembrane domains S1 to S4 converts a depolarization-activated wild-type Kv1.1 K+ channel with outward rectification into a hyperpolarization-activated channel with inward rectification. Although the pore region of the deletion mutant is identical to the wild-type channel, the mutant channel is a non-selective cation channel and is characterized by an altered pharmacology profile.

Animals↗

PubMed1994

Mode of regulation by G protein of the ATP-sensitive K+ channel in guinea-pig ventricular cell membrane.

1. The effect of G protein activation on the ATP-sensitive K+ (K+ATP) channel was examined in inside-out patches from guinea-pig ventricular myocytes. At low (0.3 mM) intracellular ATP concentration ([ATP]i) in the bathing solution, in the absence of agonists in the pipette, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or AlF4- applied to the intracellular side of the patch membrane gradually activated the K+ATP channel. The activation by GTP gamma S was irreversible, although high [ATP]i could completely close the channel. 2. In ATP-free media GTP gamma S did not increase further the activity of the fully active channel, and was unable to reactivate the channel in the non-operative state after rundown. [ATP]i-channel activity curves constructed before and after GTP gamma S application demonstrated that GTP gamma S shifts the half-inhibitory [ATP]i from 19.5 to 110 microM without changing the Hill coefficient. 3. When acetylcholine or adenosine was included in the pipette, intracellular GTP reversibly activated the K+ATP channel which was partially inhibited by [ATP]i. 4. These results indicate that G protein may stimulate myocardial K+ATP channels in the operative state by reducing the potency of ATP inhibition. The possible coupling of the G protein with muscarinic as well as A1 adenosine receptors is suggested.

Acetylcholine↗

PubMed1994

Effects of intracellular sodium and hydrogen ion on the sodium activated potassium channel in isolated patches from guinea pig ventricular myocytes.

OBJECTIVE: The Na+ activated K+ channel (IK(Na)) may be activated during ischaemia when the intracellular Na+ concentration is raised. As ischaemia is also associated with intracellular acidification, the influence of intracellular pH on this K+ channel was investigated. METHODS: The effects of intracellular Na+ and H+ on the IK(Na) channel were investigated in isolated patches from guinea pig ventricular myocytes by the patch clamp technique. RESULTS: Increase of intracellular Na+ increased the open probability of the channel. Intracellular acidification had no effect on the single channel conductance, but significantly decreased the open probability of the channel in the pH range 7.5-6.5. The effect of intracellular pH on open probability was about the same in a wide range of intracellular Na+ concentrations. The lower open probability induced by acidification seemed to be caused by prolonged closed times. CONCLUSIONS: Because the effects of increased intracellular Na+ and decreased pH on open probability are opposite, it is suggested that the IK(Na) channel might be important under conditions of raised intracellular Na+ with relatively unchanged intracellular pH. Consequently, it is hypothesised that this channel may be involved in adaptation of action potential duration at high heart rate and in action potential shortening in the border zone of regional ischaemic areas.

Action Potentials↗

PubMed1993

Acetylcholine-mediated K+ channel activity in guinea-pig atrial cells is supported by nucleoside diphosphate kinase.

We studied the role of nucleoside diphosphate kinase (NDPK) in acetylcholine-mediated muscarinic K+ channel activation in inside-out patches of guinea-pig atrial cells. NDPK-catalysed activation of the muscarinic K+ channels by adenosine triphosphate-Mg2+ (ATP-Mg2+) is not prevented by occupation of the muscarinic receptor [by acetylcholine (ACh) or atropine], nor by uncoupling of the receptor from the G protein by pertussis-toxin-catalysed adenosine diphosphate (ADP)-ribosylation of GK. In the presence of ACh, addition of 0.1 mM guanosine triphosphate (GTP) after activation of the channels by 4 mM ATP alone resulted in a moderate increase of channel activity (in contrast to block in the absence of ACh): NDPK-mediated direct transphosphorylation is uncoupled by the G nucleotide but agonist-induced guanosine diphosphate (GDP)-to-GTP exchange takes over activation of the channels. Moreover, ACh-dependent channel stimulation was possible in inside-out patches while ATP and GDP were present in the bathing solution (in contrast to the complete absence of channel activation in the absence of ACh). This indicates that NDPK synthesizes sufficient GTP to support channel activation by exchange. Hence, it is postulated that the main functional role of NDPK under physiological conditions is to provide a local supply of GTP (using GDP and ATP) in the immediate vicinity of the G protein, thereby maintaining a high local GTP/GDP ratio and ensuring adequate receptor-mediated regulation of muscarinic K+ channel activity.

Acetylcholine↗

PubMed1992

Intracellular protons inhibit inward rectifier K+ channel of guinea-pig ventricular cell membrane.

The effect of intracellular protons (Hi+) on the inward rectifier K+ channel of the guinea-pig ventricular cell membrane was examined, using the patch-clamp technique. The inward single-channel current was recorded in "inside-out" and "outside-out" patch configurations, while the pH of the solution perfusing the intra- and extracellular side, respectively, was varied. Low intracellular pH (pHi), but not low extracellular pH, inhibited the channel. Low pHi reduced the unit amplitude, which was about 20% smaller at pHi 6.0 than that at pHi 7.4 at every voltage tested. The slope conductance decreased from 41.7 pS at pHi 7.4 to 35.1 pS at pHi 6.0. Low pHi also reduced the channel activity without apparent voltage dependence. The concentration/response curve indicated the half-maximum inhibition at pHi 6.11 and a Hill coefficient of 2.52. Lowering the pHi from 7.4 to 6.0 did not affect the distributions of the open times and the closed times below 50 ms, while the time constant of the histogram constructed from closings longer than 50 ms was approximately doubled. These results indicate that the inward rectifier K+ channel in ventricular myocytes is inhibited by H+ from the intracellular side. This might contribute to the depolarization of the resting membrane potential induced by intracellular acidosis during myocardial ischaemia.

Animals↗

PubMed1992

Membrane-bound nucleoside diphosphate kinase activity in atrial cells of frog, guinea pig, and human.

Muscarinic K+ channels in inside-out patches of atrial cells from guinea pig or rabbit can be activated by Mg(2+)-ATP in the absence of acetylcholine and GTP or GDP. The ATP-dependent activation involves a phosphorylation and is postulated to be due to the association of a membrane-bound nucleoside diphosphate kinase (NDPK) with the G protein GK: direct phosphorylation of the GK-bound GDP into GTP, catalyzed by NDPK, would result in activation of the G protein and, hence, activation of the channels. The aim of this study was to identify the presence of NDPK activity in atrial membranes by investigating the phosphate transfer between tritium-labeled nucleotides. We show that frog, guinea pig, and human atrial membranes contain a substantial NDPK activity since they catalyze the conversion from [3H]GDP+nucleoside triphosphate (NTP or NTP gamma S) to [3H]GTP (or [3H]GTP gamma S), from [3H]ADP+NTP to [3H]ATP, and from [3H]GTP+nucleoside diphosphate (NDP) to [3H]GDP. The phosphate transfer rates for the [3H]GDP+ATP to [3H]GTP conversion are 1.8, 0.5, and 2.4 mumol inorganic phosphate formation/mg per 10 minutes at 37 degrees C in frog, guinea pig, and human, respectively. The order of substrate efficiency for different NTPs was ATP greater than ITP approximately equal to GTP greater than UTP greater than CTP, which parallels the efficiency of these nucleotides in their activation of the muscarinic K+ channels. Addition of other nucleotides blocked the transphosphorylation reaction, indicating that the NTP-NDP conversion mechanism is aspecific, as is expected for an NDPK-catalyzed reaction. In conclusion, the data support the concept of NDPK involvement in the atrial muscarinic signal transduction cascade.

Adenosine Diphosphate↗

PubMed1991

Flunarizine inhibits a high-threshold inactivating calcium channel (N-type) in isolated hippocampal neurons.

The action of flunarizine on the high-threshold inactivating calcium channel (N-type) in hippocampal neurons of the rat was investigated using the whole-cell voltage clamp technique. Flunarizine reduced the currents at all test potentials, without shifting the peak of the current-voltage relationship along the voltage-axis. The drug did not affect the activation curve, but drastically decreased the slope conductance in the linear region of the current-voltage relationship. Block of the current by flunarizine occurred in a use-dependent way. Flunarizine was without effect when applied intracellulary, and the onset of action, when applied extracellularly, was slow (range of minutes). The Kd for the block by flunarizine obtained after 6 repetitive depolarizations at 0.2 Hz (pulse duration 150 ms) from -90 mV to 0 mV was 0.8 microM. In conclusion, we present electrophysiological evidence that flunarizine blocks the high-threshold inactivating Ca channel of hippocampal neurons of the rat. We discuss the possibility that flunarizine might inhibit neuronal transmitter release by means of this effect.

Animals↗

PubMed1991

Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats.

Stimulation of alpha 1-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca2+ current (ICa) and K+ currents were comparatively studied in either tissue. In atrial preparations, phenylephrine 10 mumol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 mumol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 mumol/l. In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 mumol/l did not affect the magnitude of ICa in either cell type. Transient and steady state K+ outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes. It is concluded that the positive inotropic effect of alpha 1-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K+ currents.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗