Thomas Noack

Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells

The hypothesis that protein kinase C (PKC) is able to regulate the whole cell Ca-activated K (KCa) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog 1,2-dioctanoyl- sn-glycerol (DOG) at 10 μM attenuated the current-voltage ( I- V) relationship of the KCa current significantly and reduced the KCacurrent at +70 mV by 70 ± 4% ( n = 14). In contrast, 10 μM DOG after pretreatment of the cells with 1 μM calphostin C or 1 μM PKC inhibitor peptide, selective PKC inhibitors, and 10 μM 1,3-dioctanoyl- sn-glycerol, an inactive diacylglycerol analog, did not significantly alter the KCa current. Furthermore, the catalytic subunit of PKC (PKCC) at 0.1 U/ml attenuated the I- Vrelationship of the KCa current significantly, reduced the KCacurrent at +70 mV by 44 ± 3% ( n = 17), and inhibited the activity of single KCa channels at 0 mV by 79 ± 9% ( n = 6). In contrast, 0.1 U/ml heat-inactivated PKCC did not significantly alter the KCacurrent or the activity of single KCa channels. Thus these results suggest that PKC is able to considerably attenuate the KCa current of freshly isolated rat tail artery smooth muscle cells independently of effects of PKC on local Ca release events, most likely by a direct effect on the KCa channel.The hypothesis that protein kinase C (PKC) is able to regulate the whole cell Ca-activated K (KCa) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) at 10 microM attenuated the current-voltage (I-V) relationship of the KCa current significantly and reduced the KCa current at +70 mV by 70 +/- 4% (n = 14). In contrast, 10 microM DOG after pretreatment of the cells with 1 microM calphostin C or 1 microM PKC inhibitor peptide, selective PKC inhibitors, and 10 microM 1,3-dioctanoyl-sn-glycerol, an inactive diacylglycerol analog, did not significantly alter the KCa current. Furthermore, the catalytic subunit of PKC (PKCC) at 0.1 U/ml attenuated the I-V relationship of the KCa current significantly, reduced the KCa current at +70 mV by 44 +/- 3% (n = 17), and inhibited the activity of single KCa channels at 0 mV by 79 +/- 9% (n = 6). In contrast, 0.1 U/ml heat-inactivated PKCC did not significantly alter the KCa current or the activity of single KCa channels. Thus these results suggest that PKC is able to considerably attenuate the KCa current of freshly isolated rat tail artery smooth muscle cells independently of effects of PKC on local Ca release events, most likely by a direct effect on the KCa channel.

Characterization of potassium currents modulated by BRL 38227 in rat portal vein.

1 Smooth muscle cells of the rat portal vein were dispersed by enzymatic treatment and recordings of whole‐cell membrane potassium currents were made by the voltage‐clamp technique. In isolated cells by use of combined voltage‐ and current‐clamp the effect of BRL 38227 on membrane potential and ionic currents was also studied. 2 BRL 38227 (0.1 to 10 μm) induced a non‐inactivating potassium current (IKCO) which developed slowly (900 s to 300 s, respectively) to its full size. These effects of BRL 38227 were reversible. 3 In addition to its K‐channel opening properties, BRL 38227 (1 to 10 μm) inhibited the amplitude and changed the activation and inactivation characteristics of a slowly‐inactivating, calcium influx‐independent, outward potassium current (ITO). 4 Application of stationary fluctuation analysis to IKCO, showed a mean single channel current of 0.65 pA at −10 mV under a quasi‐physiological potassium gradient. 5 In a combined voltage‐clamp/current‐clamp configuration, BRL 38227 (1 μm) induced a mean hyperpolarization of 22 mV. 6 The induction of IKCO by BRL 38227 and the associated hyperpolarization were suppressed by glibenclamide (1 to 10 μm) in a concentration‐dependent manner. Glibenclamide (1 μm) had no effect on the inhibition of ITO by BRL 38227 (1 μm).

Potassium channel modulation in rat portal vein by ATP depletion: a comparison with the effects of levcromakalim (BRL 38227)

1 The effects of levcromakalim and of adenosine 5′‐triphosphate (ATP) depletion on membrane potential and ionic currents were studied in freshly‐dispersed smooth muscle cells of rat portal vein by use of combined voltage‐ and current‐clamp techniques. 2 Levcromakalim (1 μm) induced a glibenclamide‐sensitive, non‐inactivating K‐current (IKCO) and simultaneously inhibited the slow, transient outward, delayed rectifier K‐current (ITO). Levcromakalim also hyperpolarized the portal vein cells by approximately 20 mV. 3 Reduction of intracellular ATP by removal of glucose and carboxylic acids from the recording pipette and of glucose from the bath fluid, induced a slowly‐developing, non‐inactivating and glibenclamide‐sensitive K‐current (Imet) within 60–300 s after breaking the membrane patch. Imet reached peak amplitude after 300–900 s, remained at a plateau for 200–800 s and then slowly ran down. At the peak of Imet, the cells were hyperpolarized by approximately 20 mV and their input conductance was increased by 42%. 4 At the time of maximum development of Imet, the delayed rectifier current, ITO, was reduced by 48%. 5 In the absence of glucose and carboxylic acids, addition of 1 μm free ATP to the recording pipette almost doubled the magnitude of Imet. At a holding potential of −10 mV, Imet was increased from 124 ± 11 pA to 228 ± 54 pA whereas the time‐course of development and run‐down of Imet was unaffected. 6 During the development and after the run‐down of Imet, levcromakalim (1–10 μm) failed to induce IKCO. 7 Stationary fluctuation analysis of the current noise associated with Imet revealed a unitary conductance of between 10–20 pS in a physiological potassium gradient. A second contaminating current with an underlying unitary conductance of approximately 150 pS remained after Imet had run down. 8 It is concluded that IKCO induced by levcromakalim and Imet are carried by the same population of relatively small conductance, glibenclamide‐sensitive K‐channels. The open state of these is increased by procedures designed to lower intracellular ATP concentrations. 9 The simultaneous inhibition of the delayed rectifier current (ITO) by both levcromakalim and during the development of Imet is highly significant. It suggests that levcromakalim could modify the interaction of ATP with sites linked to more than one type of K‐channel. This results in the opening of those channels which underlie IKCO (and which are normally inhibited by ATP binding) together with the modulation of phosphorylation‐dependent channels such as those which underlie ITO.

Multiple sclerosis and fatigue: A review on the contribution of inflammation and immune-mediated neurodegeneration.

Multiple sclerosis (MS) is an immune mediated disease of the central nervous system (CNS) and the leading cause of non-traumatic disability among young and middle-aged adults in the western world. One of its most prevalent and debilitating symptoms is fatigue. Despite the general acceptance of the idea of an immune pathogenesis of MS itself, the role of autoimmunity in the course of MS-fatigue is a matter of debate. Both immune-related processes (acute inflammation, chronic inflammation, immune-mediated neurodegeneration, immune-mediated alterations of endocrine functions related to fatigue) and presumably non-immune-mediated disturbances and factors (sleep disturbances, depression, cognitive alterations, chronic infections, adverse effects of medications) contribute to the clinical picture. Data from in vitro and animal experiments has provided evidence for a role of cytokines as IL-1 and TNF-alpha. This association could not be verified directly in blood samples from humans whereas whole blood stimulation protocols gave some indirect evidence for a role of cytokines in MS-fatigue. MRI being able to detect acute and chronic immune mediated damage to the CNS could depict that global atrophy of gray or white matter does not correlate with fatigue. Rather, distinctive clusters of lesions and atrophy at different locations, mostly bifrontal or in subcortical structures, correlate specifically with fatigue. Regardless of the difficulties in pinpointing the immunogenesis of MS-fatigue, an important role of autoimmunity is strongly supported by an indirect route: A growing amount of data shows that the highly effective immunotherapeutics which have been introduced to MS-treatment over the last years effectively and sustainably stabilize and ameliorate fatigue in parallel to their dampening effects on the neuroinflammatory process. This review summarizes the existing data on the relation between inflammation, patterns of CNS-lesions and the effects of immunotherapeutics on MS-fatigue.

4-Aminopyridine affects rat arterial smooth muscle BKCa currents by changing intracellular pH

The hypothesis whether or not 4‐AP can affect vascular smooth muscle BKCa currents was tested using the patch‐clamp technique, pH‐ and calcium‐fluorimetry, and freshly isolated rat arterial smooth muscle cells. Application of 4‐AP reversibly inhibited BKCa currents at an intracellular calcium ([Ca]i) of 250 nM with a half‐block of 2.5 mM at +50 mV. The presence of 2 μM thapsigargin, 10 μM heparin, and 10 μM ryanodine did not alter the effect of 4‐AP on BKCa currents at [Ca]i 250 nM. At [Ca]i<100 nM 4‐AP did not inhibit BKCa currents. Application of 4‐AP to the intracellular or extracellular side of excised BKCa channels did not alter channel activity or channel amplitude. Replacement of the pH‐sensitive calcium buffer EGTA by the pH‐insensitive calcium buffer BAPTA in the intracellular solution turned the 4‐AP‐induced inhibition of BKCa currents into a stimulation at [Ca]i 250 nM. Application of 4‐AP to single cells increased intracellular pH, which was accompanied by a reduction of [Ca]i in EGTA‐loaded cells and a stable [Ca]i in BAPTA‐loaded cells. Thus, these results suggest that in isolated vascular smooth muscle cells at [Ca]i>100 nM 4‐AP affects BKCa currents via an alteration of intracellular pH.

Proliferation of human lens epithelial cells (HLE-B3) is inhibited by blocking of voltage-gated calcium channels

Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage-gated calcium channels. T-type and L-type calcium channels are substantially present in human lens epithelial cell (hLEC), and total calcium currents are inhibited by mibefradil. Here, the hypothesis was tested if calcium influx via Cav channels regulates proliferation in epithelial cells. Cell proliferation was determined by cell culture assays using the L- and T-type Cav channel blockers mibefradil and verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench technique. Western blot experiments were accomplished under standard conditions using antibodies against MAPK 3. Mibefradil as well as verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of MAPK 3 was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. We conclude that the transmembrane influx of Ca2+ through Cav channels contributes to the regulation of hLEC proliferation, identifying Cav channel blockers as potential therapeutic substances in ocular diseases.

Effects of cicletanine on whole-cell currents of single smooth muscle cells from the guinea-pig portal vein

1 Smooth muscle cells of the guinea‐pig portal vein were dispersed by enzymatic treatment and recordings of membrane currents were made in the whole‐cell mode by the patch‐clamp technique. The effects of extracellular application of cicletanine‐hydrochloride on the whole‐cell currents of isolated smooth muscle cells from the guinea‐pig portal vein were studied in solutions containing a normal concentration of calcium (2.5 mm). 2 Cicletanine, 10 to 100 μm, reduced the voltage‐dependent inward calcium current with an IC50 of 250 μm. These effects of cicletanine were reversible. 3 The action of cicletanine on calcium currents can be interpreted as a decrease of the availability of calcium channels but not by an alteration of the time course or voltage‐dependency of inactivation. 3 The control calcium current was enhanced by application of Bay K 8644. On this enhanced inward current, cicletanine also exerted inhibitory effects which were not use‐dependent. 5 Cicletanine, 1 to 100 μm, did not enhance outward potassium currents. 6 It is concluded that at least one component of the vasorelaxant effects of cicletanine is produced by inhibition of calcium currents.

Comparison of the relaxing effects of cicletanine and cromakalim on vascular smooth muscle.

Summary: Cicletanine inhibited the spontaneous activity of portal vein preparations (rat, guinea pig, and rabbit) and that produced by norepinephrine or an increase in external K+ concentration to 20 mmol/L in seven types of vascular smooth muscle preparation (portal vein of rat, guinea pig, and rabbit; aorta of rat and guinea pig; and iliac artery and ear artery of rabbit). The cicletanine ECJ0 was approximately 10-4 mol/L. Contractions produced by K+ =80 mmol/L were also inhibited by cicletanine in most preparations. Only in aortic strips of rat and guinea pig and in rabbit iliac artery were the concentration-response relationships for cicletanine shifted to the right, yielding ECS0 values of approximately 3 x 10-4 mol/L. In contrast, the inhibitory effects of the potassium channel opener cromakalim (BRL 34915) were completely abolished during K+ = 80 mmol/L treatment in all preparations. The inhibitory effect of cromakalim under the other test conditions (above) was completely antagonized by application of glibenclamide, 10-5 mol/L, whereas this treatment had only negligible effects on cicletanine inhibition in most preparations. The results indicate that a potassium channel opening effect does not contribute significantly to the inhibitory effect of cicletanine on vascular smooth muscle.