Farnesol inhibits L-type Ca2+ channels in vascular smooth muscle cells

Earlier experiments with animal and human arteries have shown that farnesol, a natural 15-carbon (C15) isoprenoid, is an inhibitor of vasoconstriction (Roullet, J.-B., Xue, H., Chapman, J., McDougal, P., Roullet, C. M., and McCarron, D. A. (1996) J. Clin. Invest. 97, 2384-2390). We report here that farnesol reduced KCl- and norepinephrine-dependent cytosolic Ca2+ transients in fura-2-loaded intact arteries. An effect on Ca2+ signaling was also observed in cultured aortic smooth muscle cells (A10 cells). In these cells, farnesol reduced KCl-induced [Ca2+]i transients and mimicked the inhibitory effect of Ca2+-free medium on the [Ca2+]i response to both 12,13-phorbol myristate acetate, a protein kinase C activator, and thapsigargin, a specific endoplasmic reticulum ATPase inhibitor. Perforated patch-clamp experiments further showed in two vascular smooth muscle cell lines (A10 and A7r5), a reversible, dose-dependent inhibitory effect of farnesol on L-type Ca2+ currents (IC50 = 2.2 microM). Shorter (C10, geraniol) and longer (C20, geranylgeraniol) isoprenols were inactive. L-type Ca2+ channel blockade also occurred under tight (gigaohm) seal configuration using cell-attached, single-channel analysis, thus suggesting a possible action of farnesol from within the intracellular space. We finally demonstrated that farnesol did not affect Ca2+-sensitive pathways implicated in smooth muscle contraction, as tested with alpha-toxin permeabilized arteries. Altogether, our results indicate that farnesol is an inhibitor of vascular smooth muscle Ca2+ signaling with plasma membrane Ca2+ channel blocker properties. The data have implications for the endogenous and pharmacological regulation of vascular tone by farnesol or farnesol analogues.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

Voltage-dependent Ca2+ channels in arterial smooth muscle cells

The past years have seen some significant advances in our understanding of the functional and molecular properties of voltage-dependent Ca2+ channels in arterial smooth muscle. Molecular cloning and expression studies together with experiments on native voltage-dependent Ca2+ channels revealed that these channels are built upon a molecular structure with properties appropriate to function as the main source for Ca2+ entry into arterial smooth muscle cells. This Ca2+ entry regulates intracellular free Ca2+, and thereby arterial tone. We summarize several avenues of recent research that should provide significant insights into the functioning of voltage-dependent Ca2+ channels under conditions that occur in arterial smooth muscle. These experiments have identified important features of voltage-dependent Ca2+ channels, including the steep steady-state voltage-dependence of the channel open probability at steady physiological membrane potentials between -60 and -30 mV, and a relatively high permeation rate at physiological Ca2+ concentrations, being about one million Ca2+ ions/s at -50 mV. This calcium permeation rate seems to be a feature of the pore-forming Ca2+ channel alpha1 subunit, since it was identical for native channels and the expressed alpha1 subunit alone. The channel activity is regulated by dihydropyridines, vasoactive hormones and intracellular signaling pathways. While the membrane potential of smooth muscle cells primarily regulates arterial muscle tone through alterations in Ca2+ influx through dihydropyridine-sensitive voltage-dependent ('L-type') Ca2+ channels, the role of these channels in the differentiation and proliferation of vascular smooth muscle cells is less clear. We discuss recent findings suggesting that other Ca2+ permeable ion channels might be important for the control of Ca2+ influx in dedifferentiated vascular smooth muscle cells.     

Regulation of K+ and Ca++ channels by a family of neuropeptide Y receptors

Chronic treatment of C6 glioma cells stably expressing the rat delta opioid receptor (C6delta) with full agonists resulted in receptor down-regulation. Chronic [D-Ser2,L-Leu5]enkephalyl-Thr treatment caused a decrease in cell surface as well as a decrease in agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding. Treatment with full agonists for 12 hr resulted in a 90% decrease in receptor number that was paralleled by a decrease in the ability of agonist to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibit forskolin-stimulated adenylyl cyclase. Of the remaining receptors, a smaller fraction of receptors (41 +/- 4 vs. 56 +/- 4% in control) exhibited high affinity for agonist as compared to receptors in control membranes. Elimination of functional guanosine triphosphate binding protein (G protein) by Pertussis toxin pretreatment did not alter the ability of agonist to down regulate receptor. We hypothesized that agonist affinity (not efficacy) would be a predictor of an agonist's ability to down-regulate receptor. However, we found that only full agonists were able to down-regulate receptor number, G protein activation and adenylyl cyclase inhibition. Chronic exposure to partial agonist 7-spiroindinooxymorphone, which has a very high affinity for the receptor, as well as morphine, did not cause receptor down-regulation. Taken together, these results suggest that full agonists alter receptor conformation such that the altered conformation is recognized by G protein as well as proteins involved in receptor down-regulation. In addition, down-regulation is independent of agonist-mediated G protein activation and subsequent down-stream signaling.     

Inhibitory effects of diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells

BACKGROUND: Benzodiazepines have a direct bronchodilator action in airway smooth muscle, but the mechanisms by which these agents produce muscle relaxation are not fully understood. The current study was performed to identify the effects of the benzodiazepines diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells. METHODS: Whole-cell patch-clamp recording techniques were used to evaluate the effects of the benzodiazepines diazepam (10(-8) to 10(-3) M) and midazolam (10(-8) to 10(-3) M) on inward Ca2+ and outward K+ channel currents in dispersed canine tracheal smooth muscle cells. The effects of the antagonists flumazenil (10(-5) M) and PK11195 (10(-5) M) on these channels were also studied. RESULTS: Each benzodiazepine tested significantly inhibited Ca2+ currents in a dose-dependent manner, with 10(-6) M diazepam and 10(-5) M midazolam each causing approximately 50% depression of peak voltage-dependent Ca2+ currents. Both benzodiazepines promoted the inactivated state of the channel at more-negative potentials. The Ca2+-activated and voltage-dependent K+ currents were inhibited by diazepam and midazolam (> 10(-5) M and > 10(-4) M, respectively). Flumazenil and PK11195 had no effect on these channel currents or on the inhibitory effects of the benzodiazepines. CONCLUSIONS: Diazepam and midazolam had inhibitory effects on voltage-dependent Ca2+ channels, which lead to muscle relaxation. However, high concentrations of these agents were necessary to inhibit the K+ channels. The lack of antagonized effects of their antagonists is related to the non-gamma-aminobutyric acid-mediated electrophysiologic effects of benzodiazepines on airway smooth muscle contractility.     

Stimulation of large-conductance Ca2+-activated K+ channels by Evans blue in cultured endothelial cells of human umbilical veins

The effect of Evans blue (EB) on large-conductance Ca2+-activated K+ (BKCa) channels was investigated in cultured endothelial cells of human umbilical veins. In whole-cell configuration, EB (50 microM) reversibly increased the amplitude of K+ outward currents (IK). When the patch pipettes were filled with 10 mM EGTA, its stimulatory effect on IK was unaltered. Further application of EB in the presence of suramin, a blocker of P2-purinergic receptor, or AOPCP, an inhibitor of 5'-nucleotidase, still increased IK. However, charybdotoxin (100 nM) suppressed EB-induced increase in IK. In inside-out configuration, bath application of EB (50 microM) did not change single channel conductance but significantly increased the activity of BKCa channels. The EB-induced increase in the activity of BKCa channels was independent on internal Ca2+. EB (50 microM) shifted the activation curve of BKCa channels to less positive membrane potentials by approximately 20 mV. The change in the kinetic behavior of BKCa channels caused by EB in these cells is due to an increase in mean open time and a decrease in mean closed time. These results indicate that EB can stimulate the activity of BKCa channel in endothelial cells. This effect is unrelated to its blockade of P2-purinergic receptors or inhibition of 5'-nucleotidase. The direct stimulation of these ionic channels by EB may contribute to its effect on capillary permeability.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

The contribution of coagulation factors and fibrinolytic variables to the development of ischaemic arterial disease is still not clearly established. The PRIME study is a prospective cohort study of myocardial infarction in men aged 50-59 years and recruited from three MONICA field centers in France (Lille, Strasbourg and Toulouse) and the center in Northern Ireland (Belfast). Baseline examination included measurement of plasma fibrinogen, factor VII, and PAI-1 activity in over 10,500 participants. We investigated the associations of these haemostatic variables with cardiovascular risk factors, prevalent atherosclerotic disease and geographical area. Fibrinogen level increased with age, smoking, waist-to-hip ratio, LDL-cholesterol, and it decreased with educational level, leisure physical activity, alcohol intake and HDL-cholesterol. Factor VII activity increased with body mass index, waist-to-hip ratio, triglycerides. HDL- and LDL-cholesterol. PAI-1 activity increased with body mass index, waist-to-hip ratio, triglycerides, alcohol intake, smoking, and decreased with leisure physical activity. PAI-1 level was higher in diabetic subjects than in subjects without diabetes. Cardiovascular risk factors explained 8%, 9%, and 26% of the total variance in fibrinogen, factor VII, and PAI-1, respectively. Compared with participants without prevalent cardiovascular disease, those with previous myocardial infarction (n = 280), angina pectoris (n = 230), or peripheral vascular disease (n = 19) had significantly higher levels of fibrinogen. but those with stroke (n = 67) had not. PAI-1 activity showed a similar pattern of association. The odds ratio for cardiovascular disease associated with a rise of a one standard deviation in fibrinogen and PAI-1 was 1.31 (95% confidence interval: 1.20 to 1.42, p <0.001) and 1.38 (95% confidence interval: 1.27 to 1.49, p<0.001), respectively. After adjustment for cardiovascular risk factors, these associations were attenuated but remained highly significant. There was no significant association between factor VII activity and prevalent cardiovascular disease. Fibrinogen level and, to a lesser extent, factor VII and PAI-1 activity were higher in Northern Ireland than France after adjustment for the main cardiovascular risk factors. These geographical variations are consistent with the 2 to 3-fold higher incidence of myocardial infarction in Northern Ireland than France. Our results provide further epidemiological evidence for a possible role of fibrinogen and PAI-1 in the pathogenesis of coronary heart disease.     

Protein kinase C inhibits the Ca(2+)-activated K+ channel of cultured porcine coronary artery smooth muscle cells

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.     

Effects of intracellular N+, M2+ and metabolites on C2+-activated K+ channels in pulmonary and ear arterial smooth muscle cells of the rabbit

We studied the distribution of the homeodomain proteins Pdx-1 and Nkx 6.1 in the developing rat pancreas. During early development, nuclear staining for both Pdx-1 and Nkx 6.1 occurred in most epithelial cells of the pancreatic anlage. Subsequently, Nkx 6.1 became more beta-cell-restricted, and Pdx-1 also occurred in other islet cell types and in the duodenal epithelium. During early pancreatic development, cells co-storing insulin and glucagon were regularly detected. The vast majority of these did not possess nuclear staining for either Pdx-1 or Nkx 6.1. Subsequently, cells storing insulin only appeared. Such cells displayed strongly Pdx-1- and Nkx 6.1-positive nuclei. Therefore, Nkx 6.1, like Pdx-1, may be an important factor in pancreatic development and in mature insulin cell function.     

Differential effects of Ca2+ channel blockers on Ca2+ transients and cell cycle progression in vascular smooth muscle cells

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV > Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.     

Time-irreversible subconductance gating associated with Ba2+ block of large conductance Ca2+-activated K+ channels

Ba2+ block of large conductance Ca2+-activated K+ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba2+ ion would dissociate to the external solution (150 mM N-methyl-D-glucamine+o, 500 mM K+i, 10 microM Ba2+i, +30 mV, and 100 microM Ca2+i to fully activate the channel), Ba2+ blocks with a mean duration of approximately 2 s occurred, on average, once every approximately 100 ms of channel open time. Of these Ba2+ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at approximately 0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in approximately 10,000 Ba2+ blocks. Thus, the preopenings represent Ba2+-induced time-irreversible subconductance gating. The fraction of Ba2+ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba2+]i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba2+ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba2+o and no added Ba2+i. Adding K+, Rb+, or Na+ to the external solution decreased the fraction of Ba2+ blocks with preopenings, with K+ and Rb+ being more effective than Na+. These results are consistent with models in which the blocking Ba2+ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba2+ blocking site and acts directly as the preopening gate.     

Ca(2+)-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins

Despite recent progress in the molecular characterization of high-conductance Ca(2+)-activated K+ (maxi-K) channels, the molecular identities of intermediate conductance Ca(2+)-activated K+ channels, including that of mature erythrocytes, remains unknown. We have used various peptide toxins to characterize the intermediate conductance Ca(2+)-activated K+ channels (Gardos pathway) of human and rabbit red cells. With studies on K+ transport and on binding of 125I-charybdotoxin (ChTX) and 125I-kaliotoxin (KTX) binding in red cells, we provide evidence for the distinct nature of the red cell Gardos channel among described Ca(2+)-activated K+ channels based on (i) the characteristic inhibition and binding patterns produced by ChTX analogues, iberiotoxin (IbTX) and IbTX-like ChTX mutants, and KTX (1-37 and 1-38 variants); (ii) the presence of some properties heretofore attributed only to voltage-gated channels, including inhibition of K transport by margatoxin (MgTX) and by stichodactyla toxin (StK); (iii) and the ability of scyllatoxin (ScyTX) and apamin to displace bound 125I-charybdotoxin, a novel property for K+ channels. These unusual pharmacological characteristics suggest a unique structure for the red cell Gardos channel.     

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Neuregulins stimulate the functional expression of Ca2+-activated K+ channels in developing chicken parasympathetic neurons

The developmental expression of macroscopic Ca2+-activated K+ currents (IK[Ca]) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM beta1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in IK[Ca] in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type alpha transforming growth factor but not by 10 nM alpha2-neuregulin peptide or by neurotrophins at 40 ng.ml-1. beta1-neuregulin treatment for 12 hr also confers Ca2+ sensitivity onto large-conductance (285 pS) K+ channels observed in inside-out patches. beta-Neuregulins have no effect on voltage-activated Ca2+ currents of CG neurons. These data support the hypothesis that beta-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons.     

Intracellular divalent cations block smooth muscle K+ channels

The patch-clamp technique was used to examine the sensitivity of delayed rectifier K+ channels to changes in intracellular divalent cations (Mg2+ and Ca2+). During voltage-step and ramp depolarizations, a delayed rectifier K+ current (IK(dr)) was identified in renal, pulmonary, coronary, and colonic smooth muscle cells as a low-noise outward current that activated near -40 mV, was sensitive to 4-aminopyridine (4-AP), and was insensitive to charybdotoxin. During whole-cell voltage-clamp experiments in each of the cell types, the 4-AP-sensitive IK(dr) was significantly less in cells dialyzed with 10 mM Mg2+ as compared with cells in which no Mg2+ was added to the internal dialysis solution (P < or = .05, n > or = 4). In coronary artery cells, 100 microM 2-(2-aminoethyl)pyridine (an H1 receptor agonist) or 10 microM ryanodine, agents that cause an increase in [Ca2+]i, also caused a significant reduction of the 4-AP-sensitive IK(dr) similar to that produced by Mg2+. 4-AP (5 mM) significantly depolarized single renal arterial cells that were dialyzed with Mg(2+)-free solution but not those dialyzed with 10 mM Mg2+ (P < .01, n = 4). In inside-out patches of renal arterial smooth muscle cells, with 200 nM charybdotoxin in the patch pipette to block large conductance Ca(2+)-activated K+ channels, a 59 +/- 10-picosiemen K+ channel that was sensitive to cytoplasmic Mg2+ was identified. In Mg(2+)-free solution, channel open probability was 0.028 +/- 0.012 (n = 8) and 0.095 +/- 0.011 (n = 8) at +40 and +80 mV, respectively. When the bath solution was changed to one containing 5 or 15 mM Mg2+, channel open probability was significantly reduced by 66% and 68% (+40 mV) or 93% and 96% (+80 mV), respectively. This decrease in the open probability of the delayed rectifier K+ channel resulted from a concentration- and voltage-dependent decrease in mean open time. At +40 mV, time constants for the open time distribution were significantly decreased from 5.5 +/- 0.52 to 1.2 +/- 0.14 milliseconds, whereas the closed time constant was significantly increased from 634 +/- 11.1 to 820 +/- 14.4 milliseconds (P < .01, n = 4). It is concluded that a 4-AP-sensitive delayed rectifier K+ channel in both vascular and visceral smooth muscle cells is modulated by changes in intracellular Ca2+ and Mg2+ that may alter membrane potential and the contractile state of smooth muscle.     

Increase in functional Ca2+ channels in cerebral smooth muscle with renal hypertension

The hypothesis that availability of functional Ca2+ channels in vascular smooth muscle is augmented in hypertension was tested in basilar artery cells from Wistar rats exhibiting stable systolic blood pressure (BPsys) for 2 to 11 weeks after partial renal artery ligation (Goldblatt 2-kidney 1-clip [2K1C] model). Cells were freshly isolated and patch-clamped using a nystatin-perforated patch method. BPsys ranged from 110 to 280 mm Hg and correlated with normalized kidney mass. Macroscopic current-voltage curves were fit to a Boltzmann function to obtain maximum conductance (gmax), steepness and midpoint potential for the voltage dependence of activation (k and E1/2, respectively), and extrapolated reversal potential for the chord conductance (Erev). Linear regression of normalized conductance (ng(max)=g(max)/cell capacitance) versus BPsys for 103 cells indicated a strong relationship, with a slope of 0.0019 nS x pF(-1) x mm Hg(-1) (P<0.0001). Similar analysis of data from 35 other cells exposed to 500 nmol/L Bay K 8644 gave a slope of 0.0041 nS x pF(-1) x mm Hg(-1) (P=0.001). Voltage-dependent parameters, k, E1/2, and Erev, were not significantly related to BPsys. Single-channel measurements in cell-attached patches revealed that the number of channels in 32 patches was significantly related to BPsys (P=0.0024) but that slope conductance, open dwell times at 0 mV, and distribution between 2 open states were not. Finally, in a subgroup of 61 cells from animals made hypertensive (180 mm Hg相似文献   

Inhibition of a K+ current by beta-dendrotoxin in primary and subcultured vascular smooth muscle cells

beta-Dendrotoxin (beta-DTX), a polypeptide component of Eastern Green Mamba snake venom, inhibits a slow voltage-activated 86Rb efflux from synaptosomes, suggesting that beta-DTX inhibits K+ channels. The effects of beta-DTX on the K+ currents in primary cultured and subcultured (passages 8-12) rat tail artery vascular smooth muscle cells (VSMCs) were studied using the whole-cell patch-clamp technique. A delayed rectifier K+ current was observed in both types of cells. The current, which was relatively insensitive to tetraethylammonium, was activated at -40 to -30 mV and showed almost no inactivation. beta-DTX (1-1000 nM) decreased the outward K+ current. The effect was concentration dependent and reversible by washout but did not depend on the frequency of stimulation (use dependence) or the membrane potential. beta-DTX was more effective in primary cultured cells than in subcultured cells. K+ channels in primary cultured cells were maximally (45%) inhibited by 1 microM beta-DTX compared with 35% inhibition in subcultured cells. The concentration producing half-maximal inhibition was 5.1 x 10(-8) M for primary cells and 7.1 x 10(-8) M for subcultured cells. The delayed rectifier current was not affected by alpha-DTX, a blocker of the fast-inactivating outward K+ current (IA). These results clearly demonstrate that beta-DTX is a novel antagonist of the delayed rectifier K+ current in primary and subcultured rat tail artery VSMCs.     

Apamin-sensitive Ca(2+)-activated K+ channels regulate pacemaker activity in nigral dopamine neurons

Mesencephalic dopamine-containing neurons exhibit a Ca(2+)-dependent oscillation in membrane potential believed to underlie the ability of these cells to maintain spontaneous activity in the absence of afferent synaptic drive. In the present series of experiments, sharp electrode intracellular recording techniques were used in conjunction with an in vitro brain slice preparation to explore the ionic mechanisms underlying rhythmogenesis in nigral dopamine neurons in the rat. Our results indicate that the K+ channel producing the prolonged post-spike afterhyperpolarization exhibited by these neurons is also principally responsible for generating the falling phase of the autogenous pacemaker oscillation. Alterations in the expression of this conductance are associated with marked changes in neuronal firing pattern, indicating that modulation of ligand-gated Ca(2+)-activated K+ channels may constitute a functional means of altering temporal coding among the major mesotelencephalic dopamine systems.     

Effects of isoflurane on receptor-operated Ca2+ channels in rat aortic smooth muscle

OBJECTIVE: Map-guided procedures have been the accepted standard for ventricular tachycardia surgery. However, promising results of visually guided resections without mapping have been reported. The goal of this study was to evaluate the efficacy of large encircling cryoablation without mapping for ventricular tachycardia after anterior myocardial infarction. METHODS: Between 1985 and 1996, this procedure, along with aneurysmectomy, was performed on 38 patients for malignant ventricular tachycardia. The mean interval between the operation and myocardial infarction was 59.2 months; 7 patients (18.4%) were operated on within 1 month of myocardial infarction. The mean patient age was 62.1 +/-7.3 years and the mean left ventricular ejection fraction was 29.0% +/-7.2%. RESULTS: Hospital mortality was 2.6% (1 patient). The electrical success rate based on postoperative electrophysiologic studies was 94.5%. Overall electrical success rate was 89.1%. Freedom from ventricular tachycardia was 77% (95% CI 61%-94%) at both 5 and 7 years. Freedom from sudden cardiac death was 91% (95% CI 80%-100%) at both 5 and 7 years, with overall actuarial survivals at 5 and 7 years of 63% (95% CI 47%-80%) and 42% (95% CI 22%-63%), respectively. The main cause of late death was congestive heart failure in 62.6% of these patients. CONCLUSIONS: One can achieve good results without intraoperative mapping in the treatment of patients with ventricular tachycardia after anterior myocardial infarction by using large encircling cryoablation.     

A comparison of the effects of SCA40, NS 004 and NS 1619 on large conductance Ca(2+)-activated K+ channels in bovine tracheal smooth muscle cells in culture

1. The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca(2+)-activated K+ (BKCa) channels in inside-out and outside-out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BKCa channel openers, NS 004 and NS 1619. 2. The presence of BKCa channels on inside-out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K+, dependence of channel activity on [Ca2+]i, and sensitivity to the selective BKCa channel blocker, iberiotoxin. 3. NS 004 and ND 1619 (3-30 microM) induced concentration-related increases in open state probability of BKCa channels when applied to either inside-out or outside-out BTSM patches, thus confirming that these compounds are activators of the BKCa channel in this preparation. 4. SCA40 (0.1-10 microM) had no effect on the activity of BKCa channels when applied to either inside-out or outside-out patches which subsequently responded to the application of NS 004 (10-20 microM). 5. It is concluded that SCA40 does not have a direct effect on BKCa channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.