Currents In Smooth Muscle Cells Research Articles

High-energy photons vs protons in their action on vascular function in rats

The goal of this work was to compare the effects of a photon (PTI) and proton/hadron (HTI) irradiation on rat’s cardiovascular system. Cardiovascular functions were studied in rats after PTI and HTI impact in the equivalent total absorbed dose of 6 Gy. Photons were delivered using 60Co gamma-rays (0.8 Gy min-1). The particle irradiation was done by using a 9,6×10-12 J proton beam accelerated in the U-240 isochronous cyclotron. Both PTI and HTI decreased the acetylcholine-induced relaxation in rat’s aorta smooth muscle (SM) and outward potassium currents in aortic SM cells on the 9th day post-irradiation but HTI appeared to produce a more profound effect. HTI had no significant effect on systolic blood pressure (SBP) in rats while PTI produced clearly defined systemic hypertension. HTI, unlike PTI, significantly increased the left ventricle pressure in Langendorff - perfused rat’s heart. Thus, the biological effects of PTI and HTI on rat’s aorta endothelium-dependent relaxation and net potassium currents in the SM cells appear to be similar, although the effects of HTI are more pronounced. However, PTI, unlike HTI, produced significant systemic hypertension.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Correction: Voltage-dependent inward currents in smooth muscle cells of skeletal muscle arterioles.

[This corrects the article DOI: 10.1371/journal.pone.0194980.].

Voltage-dependent inward currents in smooth muscle cells of skeletal muscle arterioles.

Voltage-dependent inward currents responsible for the depolarizing phase of action potentials were characterized in smooth muscle cells of 4th order arterioles in mouse skeletal muscle. Currents through L-type Ca2+ channels were expected to be dominant; however, action potentials were not eliminated in nominally Ca2+-free bathing solution or by addition of L-type Ca2+ channel blocker nifedipine (10 μM). Instead, Na+ channel blocker tetrodotoxin (TTX, 1 μM) reduced the maximal velocity of the upstroke at low, but not at normal (2 mM), Ca2+ in the bath. The magnitude of TTX-sensitive currents recorded with 140 mM Na+ was about 20 pA/pF. TTX-sensitive currents decreased five-fold when Ca2+ increased from 2 to 10 mM. The currents reduced three-fold in the presence of 10 mM caffeine, but remained unaltered by 1 mM of isobutylmethylxanthine (IBMX). In addition to L-type Ca2+ currents (15 pA/pF in 20 mM Ca2+), we also found Ca2+ currents that are resistant to 10 μM nifedipine (5 pA/pF in 20 mM Ca2+). Based on their biophysical properties, these Ca2+ currents are likely to be through voltage-gated T-type Ca2+ channels. Our results suggest that Na+ and at least two types (T- and L-) of Ca2+ voltage-gated channels contribute to depolarization of smooth muscle cells in skeletal muscle arterioles. Voltage-gated Na+ channels appear to be under a tight control by Ca2+ signaling.

Tributyltin Affects Rat Vascular Contractility Through L-Type Calcium Channels

The humans being exposed to tributyltin (TBT), through the ingestion of contaminated diet, particularly seafood, and through the ingestion of indoor dust. TBT is one of the most studied organotins and some reports demonstrated that this compound interferes with the physiology of the cardiovascular system; however, the exact mechanisms involved are still under discussion. Hence, this study aims to evaluate the effects of TBT on the vascular function. The evaluation of TBT effects on the contractility of rat aorta was performed using the organ bath technique using two different contractile agents: noradrenaline (NA) and potassium chloride (KCl). The whole-cell configuration of the patch clamp technique was performed to evaluate the TBT effects on the calcium currents of A7r5 cell line. The results demonstrate that TBT interferes with the vascular system as it elicits relaxation of the rat aorta contracted by NA or by KCl and inhibits L-type calcium currents in smooth muscle cells.

The angiotensin II receptor type 1b is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

The angiotensin II receptor type 1b (AT1 Rb ) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane-stretch induced stimulation of AT1 Rb activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT1 Ra or AT1 Rb with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT1 Rb mRNA is ∼30-fold higher than AT1 Ra in whole cerebral arteries and ∼45-fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT1 Rb for pressure-induced vasoconstriction. ABSTRACT: Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT1 R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT1 R - AT1 Ra and AT1 Rb - and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT1 Ra increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT1 Ra knockout animals compared with controls. In patch-clamp experiments using native cerebral arterial myocytes, membrane stretch-induced cation currents were blocked by the TRPM4 inhibitor 9-phenanthrol in both groups. Further, the AT1 R blocker losartan (1μm) diminished myogenic tone and blocked stretch-induced cation currents in cerebral arteries from both groups. Activation of AT1 R with angiotensin II (30nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT1 Rb mRNA was ∼30-fold greater than AT1 Ra in cerebral arteries, and knockdown of AT1 Rb selectively diminished myogenic constriction. We conclude that AT1 Rb , acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

ATP‐Sensitive K+ Channels in Smooth Muscle Cells from Rat Mesenteric Lymph Vessels VESSELS

The ATP‐sensitive potassium (KATP) channel is expressed in plasma membranes of many types of smooth muscle cells (SMCs). It is disinhibited when intracellular ATP concentrations are reduced during conditions of metabolic stress, and the resulting K+ efflux through KATP channels mediates membrane hyperpolarization and SMC relaxation. However, in the lymphatic smooth muscle cells (LSMCs) of rhythmically contracting lymphatic vessels, KATP channels reportedly play an important role in regulating the basal rhythmic contractions that establish lymph flow even in the absence of overt metabolic challenge. The KATP channels in LSMCs also may participate in the dilator responses of lymphatic vessels (LVs) to nitric oxide, inflammatory mediators, and compromise lymph flow in other pathologies associated with oxidative stress1,2,3. Although it is recognized that the properties and the subunit composition of KATP channels show tissue‐specificity, the identity and properties of KATP channels in LSMCs are unknown. The goal of this study was to begin to define the function and properties of KATP channels in rat mesenteric LVs. Isolated rat mesenteric LVs were cannulated on glass pipettes, pressurized at 4.5–6 mmHg, and edge detection was used to continuously monitor changes in vessel diameter. Addition of the KATP channel agonist cromakalim (0.01 μM‐100 μM; half‐log units) progressively increased LV diameter and abolished rhythmic contractions (n=3). In contrast, addition of the KATP channel antagonist, glibenclamide (0.01 μM‐100 μM; half‐log units) resulted in no change in LV diameter but suppressed rhythmic contractions. The latter finding confirmed the important role of KATP channels as regulators of LV contractile function under basal conditions. Subsequently, we have begun to characterize KATP channel currents in SMCs freshly isolated from rat mesenteric LVs. Patch‐clamp studies in the cell‐attached mode reveal unitary K+ currents in symmetrical 145 K+ recording solutions at negative patch potentials near the resting membrane potential of LSMCs. Unitary amplitudes calculated over a range of negative patch potentials from −80 mV to −30 mV indicate a single‐channel conductance of ~ 37 pS. Thus, we have tentatively identified the KATP channel in rat LSMCs as a first step toward defining it's regulation by endogenous substances and pharmacological interventions that may alter lymph flow.Support or Funding InformationSupported by T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR) from the National Institutes of Health.

Intracellular renin increases the inward calcium current in smooth muscle cells of mesenteric artery of SHR. Implications for hypertension and vascular remodeling

The influence of intracellular renin on the inward calcium current in isolated smooth muscle cells from SHR mesenteric arteries was investigated. Measurements of calcium current were performed using the whole cell configuration of pCLAMP. The results indicated that: 1) renin (100nM) dialyzed into smooth muscle cells, increased the inward calcium current; 2) verapamil (10–9M) administered to the bath inhibited the effect of renin on the inward calcium current; 3) concurrently with the increase of calcium current a depolarization of 6.8+/−2.1mV (n=16)(P<0.05) was found in cells dialyzed with renin; 4) intracellular dialysis of renin (100nM) into smooth muscle cells isolated from mesenteric arteries of normal Wystar Kyoto rats showed no significant change on calcium current; 5) aliskiren (10–9M) dialyzed into the cell together with renin (100nM) abolished the effect of the enzyme on the calcium current in SHR; 6) Ang II (100nM) dialyzed into the smooth muscle cell from mesenteric artery of SHR in absence of renin, decreased the calcium current-an effect greatly reduced by valsartan (10–9M) added to the cytosol; 7) administration of renin (100nM) plus angiotensinogen (100nM) into the cytosol of muscles cells from SHR rats reduced the inward calcium current; 8) extracellular administration of Ang II (100nM) increased the inward calcium current in mesenteric arteries of SHR. Conclusions: intracellular renin in vascular resistance vessels from SHR due to internalization or expression, contributes to the regulation of vascular tone and control of peripheral resistance-an effect independently of Ang II. Implications for hypertension and vascular remodeling are discussed.

Abstract 134: Increased Ca2+ Influx Through Cav1.2 in Vascular Smooth Muscle Cells Causes Hypertension Due to Impairment of Acetylcholine-dependent Vascular Relaxation

Calcium channel plays an important role in smooth muscle contraction and relaxation and L-type calcium channel (Cav1.2) antagonist is widely used for treating hypertension. However, whether increasing Ca2+ influx into smooth muscle cells results in hypertension (through increasing smooth muscle cell contractility) or hypotension (through activating BKCa channels) is unclear. Aiming to answer this question, we established a transgenic (TG) mouse model with smooth muscle cell specific (SM22-α promoter driven) overexpression of a splicing variant of Cavβ2 subunit, Cavβ2a, tagged with GFP. Methods and Results: Immunostaining showed that Cavβ2a-GFP expressed in smooth muscle cells specifically and L-type Ca2+ current in smooth muscle cells from the 4 th branch of mesenteric arteries of transgenic mice was significantly increased (5.1±0.7pA/pF in TG, n=12 vs. 2.2±0.5pA/pF in control, n=10). Telemetric blood pressure measurement showed that 24h-averaged systolic pressure was significantly increased (135.2±9.8mmHg, n=25) compared to that of that of control c57bl/6 mice (110.2±7.8mmHg, n=16). The diastolic pressure was also significantly elevated in TG mice than in control mice. Maximum contractility of the 4 th branch mesenteric arteries measured with 40mM KCl was not different between control and TG groups. Sodium nitroprusside relaxed the phenylephrine-precontracted mesenteric artery in the same dose-dependent manner However, acetylcholine induced mesenteric artery relaxation was signficantly impaired in TG mesenteric arteries. Conclusions: Increasing Ca2+ influx through the L-type Ca2+ channel in smooth muscle cells causing hypertension via impairment of acetylcholine-dependent relaxation.

Equol increases cerebral blood flow in rats via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells

The present study was designed to investigate the effect of equol on cerebral blood flow and the underlying molecular mechanisms. The regional cerebral blood flow in parietal lobe of rats was measured by using a laser Doppler flowmetry. Isolated cerebral basilar artery and mesenteric artery rings from rats were used for vascular reactivity measurement with a multi wire myography system. Outward K+ current in smooth muscle cells of cerebral basilar artery, large-conductance Ca2+-activated K+ (BK) channel current in BK-HEK 293 cells stably expressing both human α (hSlo)- and β1-subunits, and hSlo channel current in hSlo-HEK 293 cells expressing only the α-subunit of BK channels were recorded with whole cell patch-clamp technique. The results showed that equol significantly increased regional cerebral blood flow in rats, and produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Both paxilline and iberiotoxin, two selective BK channel blockers, significantly inhibited equol-induced vasodilation in cerebral arteries. Outward K+ currents in smooth muscle cells of cerebral basilar artery were increased by equol and fully reversed by washout or blockade of BK channels with iberiotoxin. Equol remarkably enhanced human BK current in BK-HEK 293 cells, but not hSlo current in hSlo-HEK 293 cells, and the increase was completely abolished by co-application of paxilline. Our findings provide the first information that equol selectively stimulates BK channel current by acting on its β1 subunit, which may in turn contribute to the equol-mediated vasodilation and cerebral blood flow increase.

UTP activates small-conductance Ca2+-activated K+ channels in murine detrusor PDGFRα+ cells.

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction is likely due to activation of inward currents in smooth muscle cells, and prolonged relaxation may be due to activation of small-conductance Ca(2+)-activated K(+) (SK) channels via P2Y1 receptors expressed by detrusor PDGF receptor (PDGFR)α(+) cells. We investigated whether other subtypes of P2Y receptors are involved in the activation of SK channels in PDGFRα(+) cells of detrusor muscles. Quantitative analysis of transcripts revealed that P2ry2, P2ry4, and P2ry14 are expressed in PDGFRα(+) cells of P2ry1-deficient/enhanced green fluorescent protein (P2ry1(-/-)/eGFP) mice at similar levels as in wild-type mice. UTP, a P2Y2/P2Y4 agonist, activated large outward currents in detrusor PDGFRα(+) cells. SK channel blockers and an inhibitor of phospholipase C completely abolished currents activated by UTP. In contrast, UTP activated nonselective cation currents in smooth muscle cells. Under current-clamp (current = 0), UTP induced significant hyperpolarization of PDGFRα(+) cells. MRS2500, a selective P2Y1 antagonist, did not affect UTP-activated outward currents in PDGFRα(+) cells from wild-type mice, and activation of outward currents by UTP was retained in P2ry1(-/-)/eGFP mice. As a negative control, we tested the effect of MRS2693, a selective P2Y6 agonist. This compound did not activate outward currents in PDGFRα(+) cells, and currents activated by UTP were unaffected by MRS2578, a selective P2Y6 antagonist. The nonselective P2Y receptor blocker suramin inhibited UTP-activated outward currents in PDGFRα(+) cells. Our data demonstrate that P2Y2 and/or P2Y4 receptors function, in addition to P2Y1 receptors, in activating SK currents in PDGFRα(+) cells and possibly in mediating purinergic relaxation responses in detrusor muscles.

Purinergic inhibitory regulation of murine detrusor muscles mediated by PDGFRα+ interstitial cells

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction could be due to activation of inward currents in smooth muscle cells, but the mechanism of purinergic relaxation has not been determined. We recently reported a new class of interstitial cells in detrusor muscles and showed that these cells could be identified with antibodies against platelet-derived growth factor receptor-α (PDGFRα(+) cells). The current density of small conductance Ca(2+)-activated K(+) (SK) channels in these cells is far higher (∼100 times) than in smooth muscle cells. Thus, we examined purinergic receptor (P2Y) mediated SK channel activation as a mechanism for purinergic relaxation. P2Y receptors (mainly P2ry1 gene) were highly expressed in PDGFRα(+) cells. Under voltage clamp conditions, ATP activated large outward currents in PDGFRα(+) cells that were inhibited by blockers of SK channels. ATP also induced significant hyperpolarization under current clamp conditions. A P2Y1 agonist, MRS2365, mimicked the effects of ATP, and a P2Y1 antagonist, MRS2500, inhibited ATP-activated SK currents. Responses to ATP were largely abolished in PDGFRα(+) cells of P2ry1(-/-) mice, and no response was elicited by MRS2365 in these cells. A P2X receptor agonist had no effect on PDGFRα(+) cells but, like ATP, activated transient inward currents in smooth muscle cells (SMCs). A P2Y1 antagonist decreased nerve-evoked relaxation. These data suggest that purines activate SK currents via mainly P2Y1 receptors in PDGFRα(+) cells. Our findings provide an explanation for purinergic relaxation in detrusor muscles and show that there are no discrete inhibitory nerve fibres. A dual receptive field for purines provides the basis for inhibitory neural regulation of excitability.

TREK-1 currents in smooth muscle cells from pregnant human myometrium.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Protein Kinase C-Dependent Modulation of Heterologously Expressed Homomeric Kv7.4, Kv7.5 and Heteromeric Kv7.4/7.5; Differential Sensitivities to Low Concentrations of Vasopressin and PMA in A7R5 Vascular Smooth Muscle Cells

Kv7.4 and Kv7.5 voltage-activated potassium channels are proposed to contribute to the maintenance of resting membrane voltage in smooth muscle cells. We had previously provided evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels when natively or exogenously expressed in vascular smooth muscle cells. Endogenous Kv7 currents in smooth muscle cells are suppressed upon activation of Gq coupled receptors. It remained to be elucidated if both Kv7.4 and Kv7.5 respond similarly to low concentrations of vasopressin (AVP), an agonist of the V1a Gq-coupled receptor. using patch-clamp techniques, we measured currents through human Kv7.4 and Kv7.5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells and compared their sensitivity to AVP (100 pM and 500 pM) and to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1nM). AVP (100 pM) and PMA suppressed currents through Kv7.4, Kv7.5 and Kv7.5/7.5. Currents were reduced with different voltage dependencies and potencies in the rank order: Kv7.5 > Kv7.4/7.5 > Kv7.4. Both AVP and PMA increased the steepness of Kv7.5 voltage-dependent activation and dramatically decreased Gmax. In contrast, AVP and PMA induced a rightward shift of the Kv7.4 activation curve with only a slight reduction in maximal conductance (Gmax). The modulation of Kv7.4/7.5 activation by AVP and PMA had intermediate biophysical characteristics that were distinct from the modulation of either of the homomeric configurations. These findings reinforce the significance of PKC-dependent regulation of the Kv7 channels and suggest a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation.

Functional expression of SK channels in murine detrusor PDGFRα+ cells

We sought to characterize molecular expression and ionic conductances in a novel population of interstitial cells (PDGFRα(+) cells) in murine bladder to determine how these cells might participate in regulation of detrusor excitability. PDGFRα(+) cells and smooth muscle cells (SMCs) were isolated from detrusor muscles of PDGFRα(+)/eGFP and smMHC/Cre/eGFP mice and sorted by FACS. PDGFRα(+) cells were highly enriched in Pdgfra (12 fold vs. unsorted cell) and minimally positive for Mhc (SMC marker), Kit (ICC marker) and Pgp9.5 (neuronal marker). SK3 was dominantly expressed in PDGFRα(+) cells in comparison to SMCs. αSlo (BK marker) was more highly expressed in SMCs. SK3 protein was observed in PDGFRα(+) cells by immunohistochemistry but could not be resolved in SMCs. Depolarization evoked voltage-dependent Ca(2+) currents in SMCs, but inward current conductances were not activated in PDGFRα(+) cells under the same conditions. PDGFRα(+) cells displayed spontaneous transient outward currents (STOCs) at potentials positive to -60 mV that were inhibited by apamin. SK channel modulators, CyPPA and SKA-31, induced significant hyperpolarization of PDGFRα(+) cells and activated SK currents under voltage clamp. Similar responses were not resolved in SMCs at physiological potentials. Single channel measurements confirmed the presence of functional SK3 channels (i.e. single channel conductance of 10 pS and sensitivity to intracellular Ca(2+)) in PDGFRα(+) cells. The apamin-sensitive stabilizing factor regulating detrusor excitability is likely to be due to the expression of SK3 channels in PDGFRα(+) cells because SK agonists failed to elicit resolvable currents and hyperpolarization in SMCs at physiological potentials.

Ranolazine-Induced Severe Bladder Hypotonia

To describe a case of acute urinary retention due to bladder hypotonia during ranolazine treatment. An 81-year-old male with multiple cardiovascular diseases was hospitalized for worsening angina and heart failure symptoms. Ranolazine 375 mg twice daily was started, in addition to ongoing therapy (clopidogrel 75 mg once daily, diltiazem 60 mg 3 times daily, isosorbide mononitrate 40 mg 3 times daily, carvedilol 6.25 mg twice daily, rosuvastatin 20 mg once daily, enoxaparin 5000 IU once daily, pentoxifylline 600 mg twice daily, pantoprazole 40 mg twice daily, enalapril 20 mg twice daily, furosemide 150 mg once daily, and spironolactone 37 mg once daily). Two months later, the ranolazine dose was increased to 500 mg twice daily; shortly thereafter, acute urinary retention occurred and persisted despite institution of α-lytic (alfuzosin) and antiandrogenic (dutasteride) therapy. A urodynamic study revealed that urinary retention was caused by severe hypocontractility of the detrusor muscle. Ranolazine was withdrawn and, within 2 days, the patient recovered his ability to void spontaneously; a second urodynamic study confirmed that detrusor contractility was substantially improved. Drug rechallenge was not performed due to the patient's clinical condition. Nevertheless, a phenotyping test to assess the activity of the cytochrome isoenzymes CYP3A4 and CYP2D6 (responsible for ranolazine metabolism) was performed, with dextromethorphan used as the probe drug. The urinary metabolic ratios indicated relatively low activity for CYP3A4 and intermediate activity for CYP2D6. The causal role of ranolazine in our case of bladder hypotonia is probable according to the Naranjo criteria. The mechanism of bladder dysfunction is tentatively ascribed to blockage of late sodium current in smooth muscle cells. Although drug plasma concentrations were not measured, they were probably elevated, since the metabolic activity of CYP3A4 was at the lower end of the reference range. Enzyme inhibition produced by diltiazem may have contributed to decreasing CYP3A4 activity. Acute urinary retention in elderly men taking ranolazine may be due to drug-induced bladder hypotonia.

Influence of methanandamide and CGRP on potassium currents in smooth muscle cells of small mesenteric arteries

Cannabinoids have potent vasodilatory actions in a variety of vascular preparations. Their mechanism of action, however, is complex. Apart from acting on vascular smooth muscle or endothelial cannabinoid receptors, several studies point to the activation of type 1 vanilloid (TRPV1) receptors on primary afferent perivascular nerves, stimulating the release of calcitonin gene-related peptide (CGRP). In the present study, the direct influence of the cannabinoid methanandamide and the neuropeptide CGRP on the membrane potassium ion (K(+)) currents of rat mesenteric myocytes was explored. Methanandamide (10μM) decreased outward K(+) currents, an effect similar to that observed in smooth muscle cells from the rat aorta. Conversely, CGRP (10 nM) significantly increased whole-cell K(+) currents and this effect was abolished by preexposure to tetraethylammonium chloride (1mM) or iberiotoxin (100 nM), inhibitors of large-conductance calcium-dependent K (BK(Ca)) channels but not by glibenclamide (10μM), an inhibitor of ATP-dependent K channels. In the presence of the CGRP receptor antagonist CGRP(8-37) (100 nM), the adenylyl cyclase inhibitor SQ22536 (100μM), or the protein kinase A inhibitor Rp-cAMPS (10μM), CGRP had no effect. These findings show that methanandamide does not increase membrane K(+) currents in smooth muscle cells of small mesenteric arteries, supporting an indirect mechanism for the reported hyperpolarizing influence in this vessel. Moreover, CGRP acts directly on these smooth muscle cells by increasing BK(Ca) channel activity in a CGRP receptor and cyclic adenosine monophosphate-dependent way. Collectively, these data indicate that methanandamide relaxes and hyperpolarizes intact mesenteric vessels by releasing CGRP from perivascular nerves.

TMEM16A channels generate Ca2+-activated Cl− currents in cerebral artery smooth muscle cells

Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca(2+)-activated Cl(-) (Cl(Ca)) channels in native cells. The molecular identity of proteins that generate Cl(Ca) currents in smooth muscle cells (SMCs) of resistance-size arteries is unclear. Similarly, whether cerebral artery SMCs generate Cl(Ca) currents is controversial. Here, using molecular biology and patch-clamp electrophysiology, we examined TMEM16A channel expression and characterized Cl(-) currents in arterial SMCs of resistance-size rat cerebral arteries. RT-PCR amplified transcripts for TMEM16A but not TMEM16B-TMEM16H, TMEM16J, or TMEM16K family members in isolated pure cerebral artery SMCs. Western blot analysis using an antibody that recognized recombinant (r)TMEM16A channels detected TMEM16A protein in cerebral artery lysates. Arterial surface biotinylation and immunofluorescence indicated that TMEM16A channels are located primarily within the arterial SMC plasma membrane. Whole cell Cl(Ca) currents in arterial SMCs displayed properties similar to those generated by rTMEM16A channels, including Ca(2+) dependence, current-voltage relationship linearization by an elevation in intracellular Ca(2+) concentration, a Nerstian shift in reversal potential induced by reducing the extracellular Cl(-) concentration, and a negative reversal potential shift when substituting extracellular I(-) for Cl(-). A pore-targeting TMEM16A antibody similarly inhibited both arterial SMC Cl(Ca) and rTMEM16A currents. TMEM16A knockdown using small interfering RNA also inhibited arterial SMC Cl(Ca) currents. In summary, these data indicate that TMEM16A channels are expressed, insert into the plasma membrane, and generate Cl(Ca) currents in cerebral artery SMCs.

P2X Receptor Currents in Smooth Muscle Cells Contribute to Nerve Mediated Contractions of Rabbit Urethral Smooth Muscle

Adenosine triphosphate is capable of relaxing and contracting urethral smooth muscle. The mechanisms responsible for the relaxing effects of adenosine triphosphate have been well studied but those involved in the contractile response are still unclear. We investigated the contributions of interstitial cells of Cajal and smooth muscle cells to nerve mediated, adenosine triphosphate dependent contractions of urethral smooth muscle. Tension recordings were made from strips of rabbit urethral smooth muscle. Recordings were made of membrane potential and ionic currents from freshly isolated smooth muscle cells and interstitial cells of Cajal using the patch clamp technique. Stimulating intramural nerves in urethral smooth muscle yielded contractions that were inhibited by the broad spectrum P2 receptor inhibitor pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and the P2X receptor agonist α,β-methylene adenosine triphosphate but not by the P2Y receptor antagonist MRS2500. When studied under voltage clamp at a holding potential of -60 mV, interstitial cells of Cajal showed spontaneous transient inward currents that were increased in frequency by adenosine triphosphate but not by α,β-methylene adenosine triphosphate. In contrast, smooth muscle cells were quiescent but responded to adenosine triphosphate and α,β-methylene adenosine triphosphate by producing a single transient inward current. Currents evoked by adenosine triphosphate in smooth muscle cells were inhibited by α,β-methylene adenosine triphosphate, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and suramin, and by a decrease in extracellular Na+ from 130 to 13 mM. Stimulating purinergic nerves in rabbit urethral smooth muscle induces contractions via the activation of P2X receptors on smooth muscle cells.

TMEM16A channels generate Ca2+‐activated Cl− currents in cerebral artery smooth muscle cells

Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that display electrophysiological properties similar to Ca2+‐activated chloride (ClCa) channels in native cells. The molecular identity of proteins that generate ClCa currents in smooth muscle cells of resistance‐size arteries is unclear. Here, using molecular biology and patch‐clamp electrophysiology we examined TMEM16A channel expression and characterized Cl− currents in arterial smooth muscle cells of resistance‐size rat cerebral arteries. RT‐PCR amplified transcripts for TMEM16A, but not TMEM16B‐K family members, in isolated pure cerebral artery smooth muscle cells. Western blotting of cerebral artery lysate using an antibody raised against TMEM16A revealed a band consistent with the predicted molecular weight of TMEM16A. Surface biotinylation and immunofluorescence indicated that TMEM16A channels are located predominantly within the arterial smooth muscle cell plasma membrane. ClCa currents recorded in arterial smooth muscle cells displayed properties similar to those of currents generated by recombinant TMEM16A channels expressed in HEK293 cells. A TMEM16A antibody and TMEM16A knockdown using siRNA both attenuated arterial smooth muscle cell ClCa currents. In conclusion, our data indicate that TMEM16A channels generate ClCa currents in cerebral artery smooth muscle cells.