Hristo Gagov

Role of KCNQ Channels in Skeletal Muscle Arteries and Periadventitial Vascular Dysfunction

KCNQ channels have been identified in arterial smooth muscle. However, their role in vasoregulation and chronic vascular diseases remains elusive. We tested the hypothesis that KCNQ channels contribute to periadventitial vasoregulation in peripheral skeletal muscle arteries by perivascular adipose tissue and that they represent novel targets to rescue periadventitial vascular dysfunction. Two models, spontaneously hypertensive rats and New Zealand obese mice, were studied using quantitative polymerase chain reaction, the patch-clamp technique, membrane potential measurements, myography of isolated vessels, and blood pressure telemetry. In rat Gracilis muscle arteries, anticontractile effects of perivascular fat were inhibited by the KCNQ channel blockers XE991 and linopirdine but not by other selective K+ channel inhibitors. Accordingly, XE991 and linopirdine blocked noninactivating K+ currents in freshly isolated Gracilis artery smooth muscle cells. mRNAs of several KCNQ channel subtypes were detected in those arteries, with KCNQ4 channels being dominant. In spontaneously hypertensive rats, the anticontractile effect of perivascular fat in Gracilis muscle arteries was largely reduced compared with Wistar rats. However, the vasodilator effects of KCNQ channel openers and mRNA expression of KCNQ channels were normal. Furthermore, KCNQ channel openers restored the diminished anticontractile effects of perivascular fat in spontaneously hypertensive rats. Moreover, KCNQ channel openers reduced arterial blood pressure in both models of hypertension independent of ganglionic blockade. Thus, our data suggest that KCNQ channels play a pivotal role in periadventitial vasoregulation of peripheral skeletal muscle arteries, and KCNQ channel opening may be an effective mechanism to improve impaired periadventitial vasoregulation and associated hypertension.

Urocortin relaxes rat tail arteries by a PKA-mediated reduction of the sensitivity of the contractile apparatus for calcium

Urocortin is an endogenous vasodilator although the mechanism of vasorelaxation is not completely understood. The hypothesis that an alteration of smooth muscle calcium concentration is involved was tested using isometric tension recording and calcium fluorimetry. The relationship between contraction and intracellular calcium was also estimated. Urocortin produced a concentration dependent relaxation (pD2 8.59±0.06, n=6) of vessels pre‐contracted with a physiological salt solution containing 42 mM KCl (42 mM K‐PSS). Removal of the endothelium did not alter the effect of urocortin, pD2 was 8.49±0.11, n=5. Corticotropin‐releasing factor relaxed 42 mM K‐PSS pre‐contracted vessels with less potency compared to urocortin (pD2 6.99±0.28, n=5). Urocortin at 100 nM relaxed vessels pre‐contracted with 42 mM K‐PSS by 59.6±4.6% (n=8) and vessels pre‐contracted with 500 nM noradrenaline by 25.2±6.8% (n=6). Both effects were not accompanied by a change in the intracellular calcium concentration. Urocortin at 100 nM produced a significant rightward shift of 0.33±0.07 units of normalized intracellular calcium (n=5) of the relationship between tension and intracellular calcium. The urocortin‐induced relaxation was considerably reduced in the presence of 0.3 mM Rp‐8‐CPT‐cAMPS, a cyclic AMP‐dependent protein kinase (PKA) inhibitor. The PKA‐activator Sp‐5,6‐DCl‐cBIMPS relaxed 42 mM K‐PSS pre‐contracted vessels (pD2 4.98±0.07, n=6). Sp‐5,6‐DCl‐cBIMPS at 0.1 mM relaxed vessels by 85.3±2.5% (n=5), but did not change the intracellular calcium concentration. In conclusion, the data show that urocortin is a potent, endothelium‐independent dilator of rat tail arteries and suggest that this effect is mediated by PKA causing a reduction of the sensitivity of the contractile apparatus for calcium.

Urocortin-Induced Decrease in Ca2+ Sensitivity of Contraction in Mouse Tail Arteries Is Attributable to cAMP-Dependent Dephosphorylation of MYPT1 and Activation of Myosin Light Chain Phosphatase

Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 &mgr;mol/L noradrenaline was associated with a ≈2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with &agr;-toxin were relaxed with urocortin by 39±3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 &mgr;mol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 ≈2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca2+-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.

Protons Inhibit the BKCa Channel of Rat Small Artery Smooth Muscle Cells

The regulation of the activity of calcium-activated potassium (BK_Ca) channels by intracellular proton ions (pH_i) was investigated using the patch-clamp technique in smooth muscle cells freshly isolated from rat tail small arteries. Single-channel conductance and voltage dependence of activation were not different at pH_i 7.0, 7.4 and 7.8. However, the membrane potential at which channel open probability reached 0.5 was 74 ± 5 mV (n = 6) (mean ± SE) at pH_i 7.4 and 54 ± 2 mV (n = 4) at pH_i 7.8 under conditions of pCa 5.9, and 30 ± 5 mV (n = 5) at pH_i 7.4 and 62 ± 4 mV (n = 5) at pH_i 7.0 under conditions of pCa 5.4. Furthermore, at a membrane potential of 0 mV, the pD₂ for intracellular calcium ions was 5.19 ± 0.04 (n = 26) (mean ± SD) at pH_i 7.8, 5.02 ± 0.05 (n = 28) at pH_i 7.4, and 4.82 ± 0.05 (n = 30) at pH_i 7.0. In addition, an alteration of pH_i resulted in a profound change in the amplitude of BK_Ca currents in intact cells; it reversibly attenuated the current-voltage relationship decreasing the current by 55 ± 3% (n = 7) (p < 0.001) at 70 mV after lowering the extracellular NH₄Cl concentration to decrease the calculated pH_i from 7.2 to 6.8. Thus, alterations of pH_i in the range from 7.0 to 7.8 did not affect single-channel conductance and voltage dependence of activation but markedly altered single BK_Ca channel activity as well as intact cell BK_Ca current amplitude, where an increase of the intracellular proton concentration inhibited this channel.

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.

Urocortin hyperpolarizes stomach smooth muscle via activation of Ca2+-sensitive K+ currents

The effect of urocortin (Uro), a recently discovered neuropeptide with selectivity towards corticotropin-releasing hormone type 2 receptor, was tested on whole cell currents expressed by guinea-pig gastric antrum smooth muscle cells. Uro (1 pmol/l − 1 nmol/l) caused a concentration-dependent increase of Ca2+-sensitive K currents (IK) up to 500% as compared to control currents and did not affect the kinetics and voltage-dependence of inward Ca2+ currents. The IK-increasing effect of Uro was fully antagonized by preliminary emptying of intracellular Ca2+ stores with ryanodine and cyclopiazonic acid, as well as by bath application of selective blockers of adenylyl cyclase and cAMP-dependent protein kinase (PKA), but not by inhibitors of guanylyl cyclase, cGMP-dependent protein kinase, and protein kinase C. Comparable IK increase was obtained by forskolin (activator of adenylyl cyclase), Sp-cAMPS (activator of PKA), or by intracellular application of the catalytic subunit of PKA. It was concluded that Uro binds to a selective receptor in antral smooth muscle cells where it stimulates IK via PKA-dependent increase of Ca2+ concentration near the plasma membrane due to enhanced release from intracellular calcium stores.

Ovarian cancer as a genetic disease.

Ovarian cancer is characterized by the highest mortality rate among gynecologic malignancies. Therefore, there is a growing need for innovative therapies and techniques for monitoring and prevention of this disease. The exact cause of most ovarian tumors usually remains unknown. Ovarian cancer is believed to be caused by a range of different variables. This review is an attempt to summarize some genetic factors involved in the disruption of certain signaling pathways responsible for ovarian tumor transformation and development. Those factors considerably contribute to accurate diagnostics, treatment and prognosis in ovarian cancer.

Ca2+-induced Ca2+ release activates K+ currents by a cyclic GMP-dependent mechanism in single gastric smooth muscle cells

Abstract The participation of sarcoplasmic reticulum Ca 2+ release channels in the activation of Ca 2+ -sensitive K + currents ( I K(Ca) ) by cyclic dibutyryl GMP was investigated in smooth muscle cells from the circular layer of guinea-pig gastric fundus. All experiments were performed in the presence of 3 μM nicardipine into the bath and low Ca 2+ buffering capacity of the pipette-filling solution (pCa 7.4). Ruthenium red (10 μM) as well as its combination with 10 μM heparin abolished the cyclic GMP-induced activation of I K(Ca) , while 10 μM heparin remained ineffective. Ryanodine (10 μM) and the subsequently added 1 μM thapsigargin induced a relatively small increase in I K(Ca) amplitudes. The addition of 10 μM ryanodine to 1 μM thapsigargin-containing bath solution caused a vast increase in I K(Ca) . It is hypothesyzed that protein kinase G-induced vectorial Ca 2+ flux from the cell bulk and sarcoplasmic reticulum Ca 2+ stores toward the plasma membrane is realized by a spontaneous Ca 2+ -induced Ca 2+ release from a superficially situated Ca 2+ store.

Corticotropin-releasing hormone acts on guinea pig ileal smooth muscle via protein kinase A

Abstract In contraction studies corticotropin-releasing hormone (CRH) was found to relax ileal but not gastric and jejunal smooth muscles of the guinea-pig, precontracted with BaCl2. Under whole-cell patch-clamp conditions, CRH concentration-dependently activated Ca2+-sensitive K+ currents (IK) with ED50=20 pM at 100 nM and ED50=0.13 pM at 500 nM intracellular Ca2+ respectively. This increase was accompanied by significant hyperpolarization of the cell membranes. CRH 9–41 peptide fragment did not affect IK amplitude, membrane potential or contraction. The CRH-induced increase of IK densities was accelerated in the presence of high intracellular Ca2+ concentrations (500 nM) and was abolished by pretreatment of cells with either ryanodine or thapsigargin, which cause depletion of intracellular Ca2+ stores, as well as in cells treated under conditions prohibiting intracellular Ca2+ store refilling. The effect of CRH on IK was not affected by bath application of various selective inhibitors of membrane-bound phospholipases, protein kinase C, cGMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase II, but was effectively antagonized by blockers of protein kinase A (PKA) or adenylyl cyclase. Neither forskolin nor the catalytic subunit of PKA could mimic the effect of CRH on IK. Thus, it was suggested that CRH exerts its relaxing activity on ileal smooth muscle cells via PKA-dependent phosphorylation of some intracellular target coupled to sarcoplasmic reticulum Ca2+ storage machinery.

Inhibition of Ca2+ current in ileal cells by cyclopiazonic acid and ryanodine

The effects of cyclopiazonic acid and its combination with ryanodine on the inward Ca2+ current (ICa) were investigated in smooth muscle cells isolated from the circular layer of guinea-pig ileum. The ICa of these cells exhibited two components: a low-threshold, nicardipine (5 microM)-resistant, fast-inactivating component and a high-threshold, nicardipine-blockable and slowly inactivating component. Neither cyclopiazonic acid (up to 10 microM) nor ryanodine (10 microM) was able to affect both these components of ICa, when applied separately. Cyclopiazonic acid and ryanodine combination led to total abolishment of the high-threshold component, leaving the low-threshold component unaffected. The data presented suggest a process of Ca(2+)-dependent inactivation of the high-threshold component, elicited by an increase in the subplasmalemmal Ca2+ concentration due to Ca2+ released from the sarcoplasmic reticulum. It is considered that the combination of cyclopiazonic acid and ryanodine can be used as a valuable method to study the calcium sensitivity of both components of the ICa.