Doris Wilhelm

Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus.

AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.

The role of α1-adrenoceptor subtypes in the phasic and tonic responses to phenylephrine in the longitudinal smooth muscle of the rat portal vein

SummaryThe purpose of this investigation was to determine whether α1-adrenoceptor subtypes (co)exist in the rat portal vein and, if so, whether they could be functionally associated with the phasic and tonic types of contraction as a response to α1-adrenoceptor stimulation by phenylephrine.A low Ca2+ concentration (0.9 mmol/l) in the Tyrode solution enabled us to quantify changes both in the phasic myogenic activity and in the basal tone of the rat portal vein preparation very precisely. We used both competitive and non-competitive α-adrenoceptor antagonists which have been employed successfully by other investigators to discriminate between α1-adrenoceptor subtypes in vascular and other tissues. Schild analysis showed that the competitive α-adrenoceptor antagonists prazosin, phentolamine, yohimbine, corynanthine, idazoxan, rauwolscine and 5-methyl-urapidil could not distinguish between the phasic and tonic responses to phenylephrine and/or different α1-adrenoceptor subtypes in the rat portal vein. However, when we compared our pA2 values with those found to be representative indicators according to subclassifications based on the use of selective antagonists in different tissues, the α1-adrenoceptors in the rat portal vein appeared to belong to the α1L- or α1a-subtype. This subclassification was not in accordance with the data obtained with the irreversible α-cadrenoceptor antagonist chloroethylclonidine. However, the validity of this alkylating agent as a tool for receptor classification was restricted, at least in the rat portal vein, by its effects on receptor reserve. In contrast to the competitive α-adrenoceptor antagonists, the irreversible α-adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine could indeed discriminate between the phasic and tonic types of contraction in response to α1-adrenoceptor stimulation by phenylephrine, indicating two different receptor reserves for phenylephrine for the two types of responses.In conclusion, both the phasic and tonic types of contraction elicited by phenylephrine in the longitudinal smooth muscle of the rat portal vein appear to be mediated by one particular α1-adrenoceptor subtype as defined by Schild analysis with selective, competitive α-adrenoceptor antagonists. However, using the method of receptor alkylation with phenoxybenzamine, two different affinity constants for the two types of responses could be calculated for phenylephrine. This may reflect the involvement of two different subtypes of α1-adrenoceptors or more probably, the existence of only one α1-adrenoceptor subtype, which is coupled with two different intrinsic efficacies to the effector pathways mediating the phasic and tonic responses, respectively.

The effect of some α-adrenoceptor antagonists on spontaneous myogenic activity in the rat portal vein and the putative involvement of ATP-sensitive K+ channels

In the present study we showed that the α-adrenoceptor antagonists phentolamine, yohimbine, prazosin, corynanthine and idazoxan, when cumulatively applied in high concentrations (1-100 μmol/l), can increase spontaneous myogenic activity in the rat portal vein. 5-Methyl-urapidil and rauwolscine were ineffective in this respect. Pretreatment with phenoxybenzamine in a concentration of 1 μmol/l (20 min), which results in alkylation of all functional α-adrenoceptors in the rat portal vein, was unable to antagonize the increase in spontaneous myogenic activity elicited by phentolamine. Antazoline (1-100 μmol/l), a H1antagonist and 2-substituted imidazoline which is devoid of α-adrenoceptor blocking properties, exhibited similar effects on spontaneous myogenic activity as its structurally closely related analogue phentolamine. Since phentolamine is reported to interact with ATP-sensitive K+channels we investigated the role of K+channels in more detail. The K+channel openers cromakalim and diazoxide elicited a decrease in spontaneous myogenic activity. Glibenclamide (0.3-3 μmol/l), a selective blocker of ATP-sensitive K+channels in cardiac and pancreatic tissues, and phentolamine (1-10 μmol/l) shifted the concentration-response curves of cromakalim and diazoxide concentration dependently to the right. Yohimbine showed only a modest effect in the highest concentration (100 μmol/l) applied. E-4031 (0.01-0.3 μmol/l), a sotalol derivative and one of the most selective blockers of the delayed rectifier current (I(k)) in cardiac tissue, was a potent contractile agent when added to the rat portal vein in the same way as the α-adrenoceptor antagonists. All other α-adrenoceptor antagonists as well as E-4031, when tested in concentrations which maximally stimulated spontaneous myogenic activity, failed to influence the relaxations induced by cromakalim and diazoxide. The results of the present study cannot be explained on the basis of α-adrenoceptor blockade. Phentolamine, in contrast to the other α-adrenoceptor antagonists, can block glibenclamide-sensitive K+channels in the rat portal vein. However, it seems unlikely that this property can explain its potent effects on spontaneous myogenic activity, since glibenclamide itself was inactive.

An approach to differentiate between noradrenaline-elicited contractile processes in the rat isolated aorta

SummaryThe aim of the present study was to assess the different processes contributing to the contraction induced by noradrenaline (NA, 1 gmol/l) in the rat isolated aorta. Pretreatment with maximally effective concentrations of nifedipine or cromakalim reduced the NA-induced contraction to 80 ± 3.5% or 63 ± 2.0%, respectively, without alteration of the shape of the response. After pretreatment with Mn2+, NA caused a transient phasic contraction followed by a sustained tonic component, comparable to the response obtained in “Ca2+-free” medium. Ryanodine — in the presence of extracellular Ca2+ — caused a slight increase of resting tension, but did not modify the NA-induced contraction. In “Ca2+-free” medium the contraction elicited by NA consisted of a transient phasic and a sustained tonic component. The amplitude of the phasic contraction decreased exponentially with the time of exposure to “Ca2+-free” medium. The phasic component was identified as elicited by Ca2+ released from the sarcoplasmic reticulum (SR) by means of ryanodine. If Ca2+ depleted tissues (80 min in “Ca2+-free” solution) were exposed to Ca2+ in the presence of Mn2+ or cromakalim, the NA-induced phasic response was inhibited, suggesting that Mn2+ and cromakalim blocked the refilling of the store. It can be concluded that activation of α1-adrenoceptors in the rat aorta by NA elicits Ca2+-entry processes which have a different sensitivity to nifedipine, cromakalim and Mn2+. The Ca2+ released from SR contributes about 20% to the overall contractile response. Our data suggest that the depleted SR can be refilled from the extracellular space via a direct cromakalim- and Mn2+-sensitive pathway.

R 56865 Differentiates between Contractile Agents with Respect to the Nifedipine-Sensitive Component in the Isolated Rat Aorta

The interaction of the benzothiazolamine R 56865 with the nifedipine-sensitive component of the serotonin (5-HT)-, angiotensin II (AII)- and arginine-vasopressin (AVP)-induced contractions was studied in the isolated rat aorta. Nifedipine caused concentration-dependently (10(-9)-10(-6) mol/l) a slight rightward shift accompanied by a limited depression of the maximum of the concentration-response curves for 5-HT-, AII- and AVP-induced contractions. R 56865 (10(-5) mol/l) antagonized the contraction elicited by 5-HT and AII in a similar manner as nifedipine. The effect of R 56865 on 5-HT- and AII-induced contractions was no longer observed after pretreatment with nifedipine. The AVP-induced contraction was not affected by R 56865 (10(-5) mol/l). As shown previously, R 56865 is a weak inhibitor of potential-operated channels but inactive on Ca2+ channels activated by NA. In conclusion, R 56865 does not only differentiate between depolarization and receptor-stimulation, but also between the activation of Ca2+ channels by different types of receptors. We propose that R 56865 may interact with Ca2+ channels at a site which plays a role in their activation.

Characterization of the interaction of R 56865 with cardiac Na- and L-type Ca channels

1 In isolated cardiac muscle, submicromolar concentrations of R 56865 (N‐[1‐[4‐(4‐fluorophenoxy)‐butyl]‐4‐piperidinyl]‐N‐methyl‐2‐benzothiazolamine) have been shown to attenuate the toxicity of cardiac glycosides. 2 We studied the influence of R 56865 on calcium and sodium currents in single isolated ventricular cardiomyocytes. The effect of R 56865 on action potential and contractile force in the presence of increased sodium load was also tested by exposing papillary muscles to veratridine or Anemonia sulcata toxin ATX II. 3 The calcium current was not affected by R 56865 as assessed in slow action potentials of papillary muscles and current measurements in ventricular cardiomyocytes. 4 In papillary muscles, R 56865 (1 μmol l−1) abolished veratridine‐induced aftercontractions and afterdepolarizations without affecting the profound prolongation of the action potential. When pretreated with R 56865, the occurrence of afterdepolarizations was prevented and the decline of the resting membrane potential was attenuated. 5 Pretreatment with R 56865 (1 μmol l−1) did not counteract the ATX II‐induced prolongation of the action potential. 6 The sodium current (Nao 30 mmol l−1) was concentration‐dependently decreased by R 56865 (0.1–10 μmol l−1). The blocking effect was more pronounced at less negative holding potentials. 7 Our results demonstrate that the protective effect of R 56865 against veratridine‐induced electrical and mechanical oscillations is not due to a direct effect on the calcium current. A potential‐dependent inhibition of the sodium current may contribute. Additional sites of action, like interference with intracellular calcium release and inhibition of potassium currents, remain to be investigated.

Measurement of slowly exchanging 45Ca in rat aorta without using EGTA or lanthanum and its application to quantify the effects of the calcium entry blockers nifedipine and verapamil

In rat aortic strips a method was developed to measure a fraction of slowly exchanging 45Ca, which correlates with contraction and the cytosolic Ca pool that is enhanced by K+-induced depolarization. In this method no EGTA or lanthanum are used, but the strips are washed for 45 min with a Tyrode solution at 4 degrees C. The K+ depolarization induced increase in 45Ca and contraction was concentration-dependently inhibited by verapamil and nifedipine. Since lanthanum and EGTA affect cellular membranes, this method may allow a more physiological approach to the measurements of slowly exchanging 45Ca.

Different effects of R 56865 and calcium entry blockers on K+- and noradrenaline-induced contractions and 45Ca uptake in rat aorta.

The effects of R 56865, nifedipine, verapamil, diltiazem and flunarizine on K+- and NA-induced contractions and K+-induced 45Ca uptake were compared in the isolated rat aorta. The calcium entry blockers concentration dependently inhibited the K+-induced contraction and 45Ca uptake over the same dose-range. R 56865 inhibited the K+-induced 45Ca uptake, but only partly inhibited the K+-induced contraction. The calcium entry blockers caused a slight rightward shift and a depression of the maximum of the concentration-response curve for the NA-induced contraction. In contrast, R 56865 caused a strong, dose-dependent rightward shift and a depression of the maximum, 10(-6) and 10(-5) M being equieffective. The effects of R 56865 and nifedipine were independent of each other. Nevertheless, the NA-induced increase in 45 Ca uptake, a putative model for Ca influx, was attenuated by R 56865. In conclusion, R 56865 is a weak inhibitor of the K+-induced Ca influx but is without effect on the NA-induced Ca influx. The discrepancy between its effects on K+-induced contractions and 45Ca uptake may be explained by an inhibition of the uptake of 45Ca from the cytosol into the 45Ca pool. The interaction between R 56865 and the alpha 1-adrenoceptor-mediated contractions may be explained by an action at a site that is distinct from the NA-binding-site on the alpha 1-adrenoceptor.

Effect of calmodulin antagonists on contraction and 45Ca movements in rat aorta

To study the selectivity of calmodulin antagonists it was assumed that they should inhibit noradrenaline (NA)- and K(+)-induced contractions similarly without an accompanying inhibition of 45Ca uptake. Therefore, in isolated rat aorta the effects of W-7, calmidazolium and trifluoperazine on contraction and 45Ca uptake elicited by K+ and NA were investigated. Calmidazolium (10(-5)-10(-4) mol/l) elicited an incomplete inhibiton of K(+)- and NA-induced contraction and 45Ca uptake. Trifluoperazine inhibited the NA-induced contractions at lower concentrations (10(-8)-10(-6) mol/l) than the K(+)-induced contraction (10(-6)-10(-4) mol/l). The K(+)- and NA-induced 45Ca uptake was blocked by trifluoperazine (10(-5) mol/l). W-7 (10(-5)-10(-4) mol/l) inhibited the K(+)- and NA-induced contraction, however, in the same concentration range W-7 diminished the K(+)- and NA-induced 45Ca uptake. In conclusion, the results indicate that calmidazolium and trifluoperazine are hardly useful as calmodulin antagonists because of their additional properties, whereas W-7 seems to be the least unspecific of the calmodulin antagonists studies.

Functional study on the effects of nifedipine, cromakalim, and the absence of extracellular Ca2+on α1-adrenoceptor-mediated excitation-contraction coupling in isolated rat portal vein: Comparison with depolarization-mediated excitation-contraction coupling

The effects of Ca2+-entry blockade by nifedipine, K+ channel opening by cromakalim, and of omitting extracellular Ca2+ on the contractile response elicited by a supramaximum concentration of the “full” and selective α1-adrenoceptor agonist phenylephrine (10-4 M) were compared with those elicited by a supramaximal concentration of KC1 (50 mM) in isolated rat portal vein. The contractile response to phenylephrine appeared to be biphasically composed of an early “transient” phase and a slowly developing “sustained” phase that reached maximum values after 30 s and 5 min after initiation of contraction, respectively. The contractile response to KG (50 mM) exhibited a triphasic pattern consisting of “spike,” “transient”, and “sustained” components that peaked after 8 s, 25 s, and 10 min, respectively. Nifedipine was able to eliminate all components of the contractions in response to both phenylephrine and KC1 almost completely. Nifedipine was −10 times more potent at suppressing the slowly developing sustained components of the contractions in response to both stimuli than the early transient components. The spontaneous myogenic contractions were inhibited by nifedipine with intermediate potency. Cromakalim, in contrast to nifedipine, selectively eliminated the early transient components of the contractions in response to both phenylephrine and KCl. The sustained components of the contractions in response to both stimuli were relatively resistant to K+ channel opening, although higher concentrations (>1 μM) of cromakalim were capable of antagonizing the sustained response to phenylephrine accompanied by oscillations in tone. Cromakalim was most potent in counteracting spontaneous myogenic contractions. When phenylephrine and KCl were added with or without external Ca2+ after different periods of equilibration in nominally Ca2+-free medium, different washout kinetics for the different components of the contractions in response to both stimuli were observed. The early transient phases of tension development in response to both stimuli were completely lost after ~6 min of equilibration in nominally Ca2+ -free medium, whereas the slowly developing sustained components of the contractions were immediately lost after the change to nominally Ca2+ -free medium. Externally added Ca2+, when administered together with phenylephrine or KCl after the preparations had been exposed for different times to nominally Ca2+ -free medium, could not restore the early transient components. In isolated rat portal vein, apart from absolute differences in height and time course of tension development which are probably caused by a difference in release of intracellular second messengers during the two responses, the contraction mediated by strong α1-adrenoceptor stimulation could, as tested, hardly be distinguished from the one produced by a maximum effective concentration of KCl. As for KCl stimulation, the Ca2+ influx essential for contraction during the entire response to supramaximal α1,-adrenoceptor stimulation can be almost completely, though differentially, inhibited by nifedipine or short periods of absence of extracellular Ca2+. For nifedipine, this differential inhibition probably reflects the voltage and time dependency of the interaction of this 1,4-dihydropyridine with the L-type Ca2+ channel. As reflected by its slower washout kinetics, the transient component of the contraction in response to phenylephrine is, apart from Ca2+ influx, also caused by release of intracellularly stored Ca2+ This concept is strengthened first because the transient component was selectively.