Hai-Lei Zhu

Reduced KCNQ4-Encoded Voltage-Dependent Potassium Channel Activity Underlies Impaired β-Adrenoceptor–Mediated Relaxation of Renal Arteries in Hypertension

KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to &bgr;-adrenoceptor–mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to &bgr;-adrenoceptor–mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired &bgr;-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

Cytoskeletal reorganization evoked by Rho-associated kinase- and protein kinase C-catalyzed phosphorylation of cofilin and heat shock protein 27, respectively, contributes to myogenic constriction of rat cerebral arteries.

Background: The myogenic response of cerebral arteries to intravascular pressure regulates blood flow to the brain. Results: Pressurization reduced smooth muscle G-actin and increased phospho-cofilin and -HSP27 content by a mechanism blocked by ROK or PKC inhibitors. Conclusion: ROK- and PKC-mediated control of cofilin and HSP27 contributes to actin polymerization in myogenic constriction. Significance: Knowledge of cytoskeletal dynamics is crucial for understanding myogenic control of cerebral arterial diameter. Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20-kDa regulatory light chain subunits (LC20) of myosin II, which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC20, calponin, caldesmon, cofilin, and HSP27, as well as G-actin content, were determined. A decline in G-actin content was observed following pressurization from 10 mm Hg to between 40 and 120 mm Hg and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase and PKC prevented the decline in G-actin; reduced cofilin and HSP27 phosphoprotein content, respectively; and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.

Actions of two main metabolites of propiverine (M-1 and m-2) on voltage-dependent L-type Ca2+ currents and Ca2+ transients in murine urinary bladder myocytes

The anticholinergic propiverine (1-methyl-4-piperidyl diphenylpropoxyacetate), which is used for the treatment of overactive bladder syndrome, has functionally active metabolites [M-1 (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide) and M-2 (1-methyl-4-piperidyl benzilate N-oxide)], but the site of actions of these metabolites is uncertain. Propiverine is rapidly absorbed after oral administration and is extensively biotransformed in the liver, giving rise to several active metabolites (M-1 and M-2). This study determines the effect of M-1 and M-2 on voltage-dependent nifedipine-sensitive inward Ca2+ currents (ICa) using patch-clamp techniques and fluorescent Ca2+ imaging [after electrical field stimulation (EFS) and acetylcholine (ACh)] in the murine urinary bladder. In conventional whole-cell recording, propiverine and M-1 but not M-2 inhibited the peak amplitude of ICa in a concentration-dependent manner at a holding potential of –60 mV (propiverine, Ki = 10 μM; M-1, Ki = 118 μM). M-1 shifted the steady-state inactivation curve of ICa to the left at –90 mV by 7 mV. Carbachol (CCh) reversibly inhibited ICa. This inhibition probably occurred through muscarinic type 3 receptors, coupling with G-proteins, because nanomolar concentrations of 4-diphenylacetoxy-N-methyl-piperidine greatly reduced this inhibition, whereas pirenzepine or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) at concentrations up to 1 μM was almost ineffective. In the presence of M-2, the CCh-induced inhibition of ICa was blocked. In fluorescent Ca2+ imaging, M-2 inhibited EFS-induced and ACh-induced Ca2+ transients. These results suggest that M-1 acts, at least in part, as a Ca2+ channel antagonist (as it inhibited ICa), whereas M-2 has more direct antimuscarinic actions.

Actions of veratridine on tetrodotoxin-sensitive voltage-gated Na+ currents, NaV1.6, in murine vas deferens myocytes

Background and purpose:  The effects of veratridine, an alkaloid found in Liliaceae plants, on tetrodotoxin (TTX)‐sensitive voltage‐gated Na+ channels were investigated in mouse vas deferens.

ATP-sensitive K+ channels in pig urethral smooth muscle cells are heteromultimers of Kir6.1 and Kir6.2

The inwardly rectifying properties and molecular basis of ATP-sensitive K(+) channels (K(ATP) channels) have now been established for several cell types. However, these aspects of nonvascular smooth muscle K(ATP) channels still remain to be defined. In this study, we investigated the molecular basis of the pore of K(ATP) channels of pig urethral smooth muscle cells through a comparative study of the inwardly rectifying properties, conductance, and regulation by PKC of native and homo- and heteroconcatemeric recombinant Kir6.x channels coexpressed with sulfonylurea receptor subunit SUR2B in human embryonic kidney (HEK) 293 cells by the patch-clamp technique (conventional whole-cell and cell-attached modes). In conventional whole-cell clamp recordings, levcromakalim (> or = 1 microM) caused a concentration-dependent increase in current that demonstrated strong inward rectification at positive membrane potentials. In cell-attached mode, the unitary amplitude of levcromakalim-induced native and recombinant heteroconcatemeric Kir6.1-Kir6.2 K(ATP) channels also showed strong inward rectification at positive membrane potentials. Phorbol 12,13-dibutyrate, but not the inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate, enhanced the activity of native and heteroconcatemeric K(ATP) channels at -50 mV. The conductance of the native channels at approximately 43 pS was consistent with that of heteroconcatemeric channels with a pore-forming subunit composition of (Kir6.1)(3)-(Kir6.2). RT-PCR analysis revealed the expression of Kir6.1 and Kir6.2 transcripts in pig urethral myocytes. Our findings provide the first evidence that the predominant K(ATP) channel expressed in pig urethral smooth muscle possesses a unique, heteromeric pore structure that differs from the homomeric Kir6.1 channels of vascular myocytes and is responsible for the differences in inward rectification, conductance, and PKC regulation exhibited by the channels in these smooth muscle cell types.

SMAD2 disruption in mouse pancreatic beta cells leads to islet hyperplasia and impaired insulin secretion due to the attenuation of ATP-sensitive K + channel activity

Aims/hypothesisThe TGF-β superfamily of ligands provides important signals for the development of pancreas islets. However, it is not yet known whether the TGF-β family signalling pathway is required for essential islet functions in the adult pancreas.MethodsTo identify distinct roles for the downstream components of the canonical TGF-β signalling pathway, a Cre-loxP system was used to disrupt SMAD2, an intracellular transducer of TGF-β signals, in pancreatic beta cells (i.e. Smad2β knockout [KO] mice). The activity of ATP-sensitive K+ channels (KATP channels) was recorded in mutant beta cells using patch-clamp techniques.ResultsThe Smad2βKO mice exhibited defective insulin secretion in response to glucose and overt diabetes. Interestingly, disruption of SMAD2 in beta cells was associated with a striking islet hyperplasia and increased pancreatic insulin content, together with defective glucose-responsive insulin secretion. The activity of KATP channels was decreased in mutant beta cells.Conclusions/interpretationThese results suggest that in the adult pancreas, TGF-β signalling through SMAD2 is crucial for not only the determination of beta cell mass but also the maintenance of defining features of mature pancreatic beta cells, and that this involves modulation of KATP channel activity.

Molecular and biophysical properties of voltage-gated Na+ channels in murine vas deferens

The biological and molecular properties of tetrodotoxin (TTX)-sensitive voltage-gated Na(+) currents (I(Na)) in murine vas deferens myocytes were investigated using patch-clamp techniques and molecular biological analyses. In whole-cell configuration, a fast, transient inward current was evoked in the presence of Cd(2+), and was abolished by TTX (K(d) = 11.2 nM), mibefradil (K(d) = 3.3 microM), and external replacement of Na(+) with monovalent cations (TEA(+), Tris(+), and NMDG(+)). The fast transient inward current was enhanced by veratridine, an activator of voltage-gated Na(+) channels, suggesting that the fast transient inward current was a TTX-sensitive I(Na). The values for half-maximal (V(half)) inactivation and activation of I(Na) were -46.3 mV and -26.0 mV, respectively. RT-PCR analysis revealed the expression of Scn1a, 2a, and 8a transcripts. The Scn8a transcript and the alpha-subunit protein of Na(V)1.6 were detected in smooth muscle layers. Using Na(V)1.6-null mice (Na(V)1.6(-/-)) lacking the expression of the Na(+) channel gene, Scn8a, I(Na) were not detected in dispersed smooth muscle cells from the vas deferens, while TTX-sensitive I(Na) were recorded in their wild-type (Na(V)1.6(+/+)) littermates. This study demonstrates that the molecular identity of the voltage-gated Na(+) channels responsible for the TTX-sensitive I(Na) in murine vas deferens myocytes is primarily Na(V)1.6.

Modulation of voltage-dependent Ba2+ currents in the guinea-pig gastric antrum by cyclic nucleotide-dependent pathways

We have investigated whether the activation of cAMP‐ and cGMP‐dependent pathways modifies the properties of voltage‐dependent Ba2+ currents (IBa) recorded from guinea‐pig gastric myocytes using patch‐clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea‐pig gastric antrum. Both dibutyryl cAMP (db‐cAMP, 0.1–1 mM), a membrane‐permeable ester of cAMP, and isoproterenol, a selective β‐stimulant, inhibited IBa in a concentration‐dependent manner. Forskolin, but not dideoxy‐forskolin, an inactive isomer of forskolin, inhibited the peak amplitude of IBa. In the presence of either Rp‐cAMP or the PKA (cAMP‐dependent protein kinase) inhibitor peptide 5‐24 (PKA‐IP), neither forskolin nor db‐cAMP inhibited IBa. After establishing a conventional whole‐cell recording, the peak amplitude of IBa gradually decreased when the catalytic subunit of PKA was included in the pipette. The further application of Rp‐cAMP reversibly enhanced IBa. Sodium nitroprusside (0.1–1 mM) and 8‐Br‐cGMP (0.1–1 mM) also inhibited IBa in a concentration‐dependent manner. The inhibitory effects of forskolin or db‐cAMP on IBa were not significantly changed by pretreatment with a cGMP‐dependent protein kinase (PKG) inhibitor. Similarly, the inhibitory actions of 8‐Br‐cGMP on IBa were not modified by PKA‐IP. The membrane‐permeable cyclic nucleotides db‐cAMP and 8‐Br‐cGMP caused little shift of the voltage dependence of the steady‐state inactivation and reactivation curves. Neither of the membrane‐permeable cyclic nucleotides db‐cAMP or 8‐Br‐cGMP had additive inhibitory effects on IBa. These results indicate that two distinct cyclic nucleotide‐dependent pathways are present in the guinea‐pig gastric antrum, and that both inhibited IBa in an independent manner.

Characterization of NaV1.6-mediated Na+ currents in smooth muscle cells isolated from mouse vas deferens.

Patch‐clamp experiments were performed to investigate the behavior of voltage‐activated inward currents in vas deferens myocytes from NaV1.6‐null mice (NaV1.6−/−) lacking the expression of the Na+ channel gene, Scn8a, and their wild‐type littermates (NaV1.6+/+). Immunohistochemistry confirmed expression of NaV1.6 in the muscle of NaV1.6+/+, but not NaV1.6−/−, vas deferens. PCR analysis revealed that the only β1‐subunit gene expressed in NaV1.6+/+ vas deferens was Scn1b. In NaV1.6+/+ myocytes, the threshold for membrane currents evoked by 20 msec voltage ramps (−100 mV to 60 mV) was −38.5 ± 4.6 mV and this was shifted to a more positive potential (−31.2 ± 4.9 mV) by tetrodotoxin (TTX). In NaV1.6−/− myocytes, the threshold was −30.4 ± 3.4 mV and there was no TTX‐sensitive current. The Na+ current (INa) in NaV1.6+/+ myocytes had a bell‐shaped current–voltage relationship that peaked at approximately −10 mV. Increasing the duration of the voltage ramps beyond 20 msec reduced the peak amplitude of INa. INa displayed both fast (τ ∼10 msec) and slow (τ ∼1 sec) recovery from inactivation, the magnitude of the slow component increasing with the duration of the conditioning pulse (5–40 msec). During repetitive activation (5–40 msec pulses), INa declined at stimulation frequencies > 0.5 Hz and at 10 Hz ≤ 50% of the current remained. These findings indicate that INa is due solely to NaV1.6 in NaV1.6+/+ myocytes. The gating properties of these channels suggest they play a major role in regulating smooth muscle excitability, particularly in response to rapid depolarizing stimuli. J. Cell. Physiol. 223: 234–243, 2010.

The actions of azelnidipine, a dihydropyridine-derivative Ca antagonist, on voltage-dependent Ba2+ currents in guinea-pig vascular smooth muscle

Although azelnidipine is used clinically to treat hypertension its effects on its target cells, Ca2+ channels, in smooth muscle have not been elucidated. Therefore, its effects on spontaneous contractions and voltage‐dependent L‐type Ca2+ channels were investigated in guinea‐pig portal vein.