Internal Ca2+ ions inactivate and modify ATP-sensitive potassium channels in adult mouse skeletal muscle.

The effects of internal Ca2+ ions on single ATP-sensitive potassium channels (KATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel activity was high when patches were excised in a Ca(2+)-free, Na(+)-rich solution and declined irreversibly within seconds in the presence of internal Ca2+ (0.1 and 2 mM). After Ca(2+)-dependent inactivation of the channels, the ATP concentration-response curve was steeper and 50% channel blockage occurred at a lower ATP concentration than before inactivation. ATP (50 microM) in a K(+)-rich solution bathing the intracellular membrane surface reduced the open-probability of KATP channels to 18% before and to 10% after exposure of the patch to internal Ca2+ (0.1 mM). The block of KATP channels by ATP (50 microM) was also enhanced by internal Ca2+ at a concentration of 13 microM. It is concluded that internal Ca2+ ions can both inactivate KATP channels and modify the active channels. KATP channels modified by Ca2+ are blocked more strongly by ATP than unmodified channels.

[Treatment of incomplete meniscal lesions in athletes]. / Die Therapie des inkompletten Meniskusrisses beim Sportler.

PURPOSE: Arthroscopic treatment of complete meniscal lesions is well established. Nevertheless there is discussion, how to treat incomplete meniscal tears, especially in younger and active patients. This study was designed to evaluate our standard-therapy without refixation of the meniscus. METHOD: Between 7/89 and 3/01 in 47 patients (48 knees, Ø age 29 years) an incomplete meniscal lesion following sports injury was found. The lesions were revitalized by "needling" or shaving. We performed no refixation. All patients had a postoperative flexion limit in an orthosis for 6 weeks. The follow-up examination was performed 6.5 (2 - 14) years postoperative. RESULTS: The avarage Lysholm-Score increased significantly from 55 points preoperative to 92 points at the follow-up examination. The Tegner-Score increased from 3.3 points preoperative to 6.2 points at the follow-up examination. The overall result was rated "exellent" and "good" by 83 % of the patients, "fair" by 15 % of the patients and "poor" by 2 % of the patients. Return to sports activity was possible at an avarage of 7 (3 - 12) months postoperative. CONCLUSIONS: In summary we found, that shaving and "needling" of an incomplete meniscal lesion in combination with partial synovectomi and standardized postoperative treatment leads to a high healing rate. A limited flexion for 6 weeks postoperativ in an orthosis at full weight bearing is recommended. In case of complete healing of the lesion the chondroprotective and joint stabilizing function of the meniscus, especially in young and active patients is obtained.

Diverse effects of pinacidil on KATP channels in mouse skeletal muscle in the presence of different nucleotides.

OBJECTIVE: The potassium channel opener pinacidil relaxes smooth muscle and exerts cardioprotective effects. The aim of the study was to investigate the actions of pinacidil on ATP sensitive potassium channels (KATP channels) in mammalian skeletal muscle and to explore the interrelations of this drug with various nucleotides. METHODS: Single skeletal muscle fibres were prepared enzymatically from flexor digitorum brevis muscles of adult mice. Membrane patches of the inside-out configuration were excised in a Ca(2+)-free solution, and currents through single KATP channels were recorded at -40 mV. The cytoplasmic face of the patch was exposed to a K(+)-rich solution with MgCl2 (1 mM), and pinacidil (0.1 or 0.4 mM) and the nucleotides (0.1 mM) were added to this internal solution. RESULTS: KATP channels were not activated by pinacidil in the presence of the nonhydrolysable ATP analogue AMP-PNP, in contrast to the reported channel activation by pinacidil and ATP. KATP channels had a high activity in the control and were blocked by ADP; the subsequent addition of pinacidil did not enhance the open probability of KATP channels. Pinacidil in the presence of a mixture of AMP-PNP and ADP activated KATP channels. CONCLUSIONS: The diverse effects of pinacidil are interpreted with a model of the KATP channel containing a binding site for pinacidil and two sites for nucleotides, one activatory (A) site and one inhibitory (I) site. Occupation of the A site by ATP or ADP activates the channel, while occupation of the I site by ATP, AMP-PNP, ADP closes the channel. Pinacidil activates the channel and displaces blockers from the I site only if the A site is occupied.

Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes.

Intracellular Ca2+ ([Ca2+]i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca2+]i transients vary with Ca2+ load. In addition to a rapid initial decay, large (> 500 nM) [Ca2+]i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca2+]i transients, consistent with stopping uptake of Ca2+ by mitochondria. CCCP causes a small increase of [Ca2+]i at rest. After a large Ca2+ entry the amount is much larger, consistent with release from a mitochondrial Ca2+ pool that fills during cytoplasmic Ca2+ loading. The rate of Ca2+ uptake by mitochondria is dependent upon [Ca2+]i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca2+]i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN-) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca(2+)-uniporter) in the pipette, GnRH does not induce rhythmic [Ca2+]i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca2+ loads in gonadotropes and participate in GnRH-induced periodic [Ca2+]i oscillations.

Inhibition of ATP-sensitive K+ channels of mouse skeletal muscle by disopyramide.

Single ATP-sensitive K+ channels (KATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. The class Ia antiarrhythmic, disopyramide (5-100 microM), applied to the cytoplasmic membrane surface inhibited KATP channels at -40 and +40 mV. Channel inhibition by disopyramide started slowly and reached an almost stationary level within 1 min. Recovery from channel inhibition by disopyramide was incomplete. At pH 7.4, the disopyramide concentrations producing 50% channel inhibition were 8.1 microM at -40 mV and 7.1 microM at +40 mV. The Hill coefficients of the concentration-response curves were close to unity at both potentials. Raising the internal pH from 7.4 to 8.0 had no significant effect on the actions of disopyramide, but lowering the pH to 6.5 greatly potentiated the inhibition of KATP channels by the antiarrhythmic. Thus the open probabilities of KATP channels at -40 mV and in the presence of disopyramide (20 microM) were smaller by a factor of 18 at pH 6.5 than at pH 7.4. The results suggest that disopyramide interacts with KATP channels through the lipid phase of the membrane and that lowering the intracellular pH increases the affinity of KATP channels to disopyramide. Thus disopyramide at therapeutic concentrations (6-15 microM) affects muscular KATP channels, in particular at reduced intracellular pH values that occur under ischaemic conditions and during fatiguing exercise.

The cardiotonic bipyridine AWD 122-60 inhibits adenosine triphosphate-sensitive potassium channels of mouse skeletal muscle.

The inhibition of ATP-sensitive potassium channels in mouse skeletal muscle by the cardiotonic bipyridine AWD 122-60 was investigated with the patch-clamp technique. In excised patches of the inside-out configuration, internally applied AWD 122-60 (10(-6)-10(-3) mol/l) reversibly reduced the open-probability of single ATP-sensitive potassium channels. The agent shortened the periods of channel activity but did not affect the channel conductance. At positive membrane potentials channel inhibition by AWD 122-60 was more pronounced than at negative potentials, the drug concentrations producing 50% channel inhibition were 11 mumol/l at +40 mV and 29 mumol/l at -40 mV. The Hill coefficients of the concentration-response curves were in the range between 0.5 and 0.6 for both potentials. Internal application of another cardiotonic bipyridine, milrinone (10(-4) mol/l), had no effects on ATP-sensitive potassium channels in skeletal muscle. Possible effects of the inhibition of ATP-sensitive potassium channels in heart muscle by AWD 122-60 are discussed.

[Influence of the postoperative activity level on tibial bone tunnel enlargement and functional treatment results following anterior cruciate ligament reconstruction using a patellar tendon autograft]. / Der Einfluss des postoperativen Aktivitätsgrades auf die tibiale Bohrkanalerweiterung und das funktionelle Behandlungsergebnis nach vorderer Kreuzbandplastik mit Patellarsehnendrittel.

AIM: This study was performed to evaluate the influence of the postoperative activity level on tibial bone tunnel enlargement following anterior cruciate ligament reconstruction using a mid-third patellar tendon autograft. METHODS: A clinical and radiological assessment was performed on 50 patients (21 male, 29 female, mean age 32 years, range 18 to 57 years) following ACL reconstruction using a patellar tendon autograft. The average follow-up examination was performed 18 (12 to 30) months after the operation. RESULTS: 33 patients (66 %) developed a tibial bone tunnel enlargement > 1 mm. We found a positive correlation (+ 0.59) of the grade of activity and the muscle status (+ 0.56) to the tibial bone tunnel enlargement. Patients with a major tibial bone tunnel enlargement performed at a higher (p < 0.05) postoperative activity grade (5.2 versus 4.1 in the Tegner grading), rated higher in the Lysholm (88 versus 77 points) and IKDC scores (p < 0.05) and reported a better subjective functional outcome (p < 0.05). There was no significant correlation of the results of the knee stability tests and the age of the patients to the grade of tibial bone tunnel enlargement. CONCLUSIONS: In ACL reconstruction using a patellar tendon autograft we recommend early rehabilitation as the concomitant tibial bone tunnel enlargement does not significantly influence the clinical outcome or knee stability.

KATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP.

Single ATP-sensitive potassium channels (KATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated in the absence or presence of 1 mM MgCl2 with K(+)-rich solutions bathing the internal membrane surface. Currents through single. KATP channels were recorded at -40 and +40 mV. AMP-PNP (5 to 500 microM; Li salt) reduced the open-probability po of KATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of po at -40 mV was observed at nucleotide concentrations of 29 microM in the absence and of 39 microM in the presence of Mg2+. The steepness of the AMP-PNP concentration-response curves was strongly affected by Mg2+, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl2. The efficacies of channel blockage by AMP-PNP at -40 and +40 mV were not significantly different. The results indicate that a KATP channel can bind more divalent Mg(2+)-complexes of AMP-PNP than trivalent protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium ions to the observed results, we studied the effects of LiCl (0.8 to 10 mM) in the Mg(2+)-free solution on the single channel current i. At a Li+ concentration of 10 mM, i was hardly affected at -40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of KATP channels by internal Li+ ions.

Negative cooperativity may explain flat concentration-response curves of ATP-sensitive potassium channels.

Blockage of ATP-sensitive K+ channels by various drugs has been reported to exhibit a weak concentration dependence with Hill coefficients below unity. This phenomenon is interpreted by a negative cooperativity between K+ channels whereby drug binding to one channel lowers the drug affinities of neighbouring channels. Results are presented for a dimeric and a tetrameric channel model and compared with published experimental data.

Expression studies and promoter analysis of the nuclear gene for mitochondrial transcription factor 1 (MTF1) in yeast.

The basal mitochondrial transcription apparatus of Saccharomyces cerevisiae consists of the core enzyme for mitochondrial RNA polymerase and the specificity factor. The core enzyme is homologous to those of bacteriophages T3, T7 and SP6 whereas the specificity factor shows similarities with bacterial sigma factors. Recently it was shown that the bacteriophage-type core enzyme is widespread among the eukaryotic lineage and a common picture for the mitochondrial transcription apparatus in eukaryotic cells is now emerging. In contrast to the situation for the core enzyme, the gene for the specificity factor has only been identified from S. cerevisiae and more recently from two other yeast species. As the specificity factor is the key component for initiation of transcription at the mitochondrial promoter we wanted to study in more detail gene expression, regulation, and the function of the promoter of the nuclear MTF1 gene. For this purpose the messenger RNA level for scMTF1 was investigated under a large number of different growth conditions and thereby exhibited a very low, but regulated and carbon source-dependent, expression. Deletion experiments identify the minimal promoter for functional complementation in yeast. To evaluate the functional conservation of the promoter elements the homologous MTF1 gene from the closely related yeast Saccharomyces douglasii was isolated and tested in heterologous complementation experiments. In spite of a highly conserved protein sequence these studies demonstrate that at low-copy number sdMTF1 is not able to substitute for scMTF1 in S. cerevisiae. Promoter exchange experiments with MTF1 from S. cerevisiae and S. douglasii demonstrate that differences in gene expression are responsible for the failure in heterologous complementation. This finding prompted us to compare the promoter regions of MTF1 from four different yeast species. For this purpose the sequences of the 5' regions from S. douglasii, S. kluyveri and Kluyveromyces lactis were determined. A comparison of these sequences identifies significant differences and rapid changes in the intergenic regions, even between closely related yeast species.

Individual subunits contribute independently to slow gating of bovine EAG potassium channels.

The bovine ether à go-go gene encodes a delayed rectifier potassium channel. In contrast to other delayed rectifiers, its activation kinetics is largely determined by the holding potential and the concentration of extracellular Mg2+, giving rise to slowly activating currents with a characteristic sigmoidal rising phase. Replacement of a single amino acid in the extracellular linker between transmembrane segments S3 and S4 (L322H) strongly reduced the prepulse dependence and accelerated activation by 1 order of magnitude. In addition, compared with the wild type, the half-activation voltage of this mutant was shifted by more than 30 mV to more negative potentials. We used dimeric and tetrameric constructs of the bovine eag1 gene to analyze channels with defined stoichiometry of mutated and wild-type subunits within the tetrameric channel complexes. With increasing numbers of mutated subunits, the channel activation was progressively accelerated, and the sigmoidicity of the current traces was reduced. Based on a quantitative analysis, we show that the slow gating, typical for EAG channels, is mediated by independent conformational transitions of individual subunits, which gain their voltage dependence from the S4 segment. At a given voltage, external Mg2+ increases the probability of a channel subunit to be in the slowly activating conformation, whereas mutation L322H strongly reduces this probability.

Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.

Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect.

Functional role of the slow activation property of ERG K+ channels.

ERG (ether-à-go-go-related gene) K+ channels are crucial in human heart physiology (h-ERG), but are also found in neuronal cells and are impaired in Drosophila 'seizure' mutants. Their biophysical properties include the relatively fast kinetics of the inactivation gate and much slower kinetics of the activation gate. In order to elucidate how the complex time- and voltage-dependent activation properties of ERG channels underlies distinct roles in excitability, we investigated different types of ERG channels intrinsically present in cells or heterologously expressed in mammalian cells or Xenopus oocytes. Voltage-dependent activation curves were highly dependent on the features of the eliciting protocols. Only very long preconditioning times produced true steady-state relationships, a fact that has been largely neglected in the past, hampering the comparison of published data on ERG channels. Beyond this technical aspect, the slow activation property of ERG can be responsible for unsuspected physiological roles. We found that around the midpoint of the activation curve, the time constant of ERG open-close kinetics is of the order of 10-15 s. During sustained trains of depolarizations, e.g. those produced in neuronal firing, this leads to the use-dependent accumulation of open-state ERG channels. Accumulation is not observed in a mutant with a fast activation gate. In conclusion, it is well established that other K+ channels (i.e. Ca2+-activated and M) control the spike-frequency adaptation, but our results support the notion that the purely voltage-dependent activation property of ERG channels would allow a slow inhibitory physiological role in rapid neuronal signalling.