Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

Temperature-dependent contractility of rat tunica dartos muscle: Contribution of cold, menthol-sensitive TRPM8.

Tunica dartos smooth muscle (TDSM) lies beneath the scrotal skin, and its contraction leads to scrotum wrinkling upon cooling. However, neither the nature of TDSM cold-sensitivity nor the underlying molecular sensors are well understood. Here we have investigated the role of cold/menthol-sensitive TRPM8 channel in TDSM temperature-dependent contractility. The contraction of isolated male rat TDSM strips was studied by tensiometry. TRPM8 expression was assayed by RT-PCR and fluorescence immunochemistry. Isolated TDSM strips responded to cooling from 33 °C to 20 °C by enhancement of basal tension, and increase of the amplitude and duration of electric field stimulated (EFS) contractions. The effects of cold on basal tension, but not on EFS-contractions, could be 80% inhibited by TRPM8 blockers, capsazepine and BCTC [N-(4­tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide], and could be partially mimicked by menthol. RT-PCR and immunolabeling showed TRPM8 mRNA and protein expression in TDSM cells with protein labelling being predominantly localized to intracellular compartments. Chemical castration of male rats consequent to the treatment with androgen receptor blocker, flutamide, led to the abrogation of cold effects on TDSM basal tension, but not on EFS-contractions, and to the disappearance of TRPM8 protein expression. We conclude that TRPM8 is involved in the maintenance of basal cold-induced TDSM tonus, but not in sympathetic nerve-mediated contractility, by acting as endoplasmic reticulum Ca2+ release channel whose expression in TDSM cells requires the presence of a functional androgen receptor. Thus, TRPM8 plays a crucial role in scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility.

Induction of A549 Nonsmall-Cell Lung Cancer Cells Proliferation by Photoreleased Nicotine.

Caged compounds comprise the group of artificially synthesized, light-sensitive molecules that enable in situ derivation of biologically active constituents capable of affecting cells, tissues and/or biological processes upon exposure to light. Ruthenium-bispyridine (RuBi) complexes are photolyzed by biologically harmless visible light. In the present study, we show that RuBi-caged nicotine can be used as a source of free nicotine to induce proliferation of A549 nonsmall-cell lung cancer (NSCLC) cells by acting on nicotinic acetylcholine receptors expressed in these cells. RuBi-nicotine was photolyzed using LED light source with the spectrum matching RuBi-absorption. Photorelease of free nicotine ([Nic]p/r ) was quantified by high-performance liquid chromatography (HPLC). 5-s-long light exposure of 10 µm of RuBi-nicotine generated 2 µm [Nic]p/r which enhanced A549 cell proliferation similarly to the 2 µm of plain nicotine during 72 h of cell culturing. Both RuBi-nicotine per se and its photolysis byproduct exerted no effect on A549 cells. We conclude that RuBi-nicotine can be a good source of free nicotine for inducing short- and long-term biological effects. Photolysis of RuBi-nicotine is quite effective, and can produce biologically relevant concentrations of nicotine at acceptable concentrations of the source material with the use of simple, inexpensive, and easily accessible light sources.

Impairment of urinary bladder mechanical properties in rat model of type 2 diabetes.

AIMS: The urinary bladder is a mechanosensitive organ that accumulates, stores, and expels considerable amounts of fluid. While the neuronal bladder control via the CNS is well defined, the data on the mechanisms of local mechanical sensitivity of the bladder wall are either insufficient or contradictory. Here we compared the mechanical properties of bladder wall of normal rats and rats with modeled type 2 diabetes (T2D). METHODS: T2D was modeled in 3-month-old Wistar male rats by combined administration of nicotinamide (230 mg/kg) and streptozotocin (65 mg/kg). Cystometry of isolated, denervated whole bladders and stress-strain tensiometry on detrusor smooth muscle (DSM) strips were used to assess the mechanical properties of bladder wall tissues from control and diabetic animals on 10th week after induction. RESULTS: The pressure-volume cystometrograms of both control and T2D bladders featured a quasi plateau between ascending sections. T2D cystometrograms revealed markedly elevated intravesicular pressure (~100% at 1 ml) and a shortened plateau, consistent with decreased bladder wall elasticity and reduced structural bladder capacity versus control. Experiments on urothelium-intact and urothelium-devoid DSM strips have shown that the decrease of bladder walls elasticity in T2D can be explained by the switch of stretched urothelium from inducing DSM relaxation to inducing DSM contraction due to a change in the prevalent release of contractile versus relaxing urothelial factor(s). CONCLUSIONS: The decreased elasticity of the bladder walls in T2D results from alterations in urothelium-dependent mechanosensory mechanisms. Elevated intravesical pressure in T2D may contribute to urge incontinence and/or symptoms of upper urinary tract damage.

Alterations in detrusor contractility in rat model of bladder cancer.

Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with cancer are not known. Here by using tensiometry on detrusor smooth muscle (DSM) strips from normal rats and rats with bladder cancer induced by known urothelial carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), we show that bladder cancer is associated with considerable changes in DSM contractility. These changes include: (1) decrease in the amplitude and frequency of spontaneous contractions, consistent with the decline of luminal pressures during filling, and detrusor underactivity; (2) diminution of parasympathetic DSM stimulation mainly at the expense of m-cholinergic excitatory transmission, suggestive of difficulty in bladder emptying and weakening of urine stream; (3) strengthening of TRPV1-dependent afferent limb of micturition reflex and TRPV1-mediated local contractility, promoting urge incontinence; (4) attenuation of stretch-dependent, TRPV4-mediated spontaneous contractility leading to overflow incontinence. These changes are consistent with the symptomatic of bladder dysfunction in bladder cancer patients. Considering that BBN-induced urothelial lesions in rodents largely resemble human urothelial lesions at least in their morphology, our studies establish for the first time underlying reasons for bladder dysfunction in bladder cancer.

Beyond Neuronal Heat Sensing: Diversity of TRPV1 Heat-Capsaicin Receptor-Channel Functions.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel best known for its ability to be gated by the pungent constituent of red chili pepper, capsaicin, and related chemicals from the group of vanilloids as well as by noxious heat. As such, it is mostly expressed in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Its activation is also sensitized by the numerous endogenous inflammatory mediators and second messengers, making it an important determinant of nociceptive signaling. Except for such signaling, though, neuronal TRPV1 activation may influence various organ functions by promoting the release of bioactive neuropeptides from sensory fiber innervation organs. However, TRPV1 is also found outside the sensory nervous system in which its activation and function is not that straightforward. Thus, TRPV1 expression is detected in skeletal muscle; in some types of smooth muscle; in epithelial and immune cells; and in adipocytes, where it can be activated by the combination of dietary vanilloids, endovanilloids, and pro-inflammatory factors while the intracellular calcium signaling that this initiates can regulate processes as diverse as muscle constriction, cell differentiation, and carcinogenesis. The purpose of the present review is to provide a clear-cut distinction between neurogenic TRPV1 effects in various tissues consequent to its activation in sensory nerve endings and non-neurogenic TRPV1 effects due to its expression in cell types other than sensory neurons.

Impairment of cholinergic bladder contractility in rat model of type I diabetes complicated by cystitis: Contribution of neurotransmitter-degrading ectoenzymes.

Parasympathetic regulation of urinary bladder contractions primarily involves acetylcholine release and activation of detrusor smooth muscle (DSM) muscarinic acetylcholine (mACh) receptors. Co-release of ATP and activation of DSM purinergic P2X1-receptors may participate as well in some species. Both types of neuromuscular transmission (NMT) are impaired in diabetes, however, which factors may contribute to such impairment remains poorly understood. Here by using rats with streptozotocin(STZ)-induced type I diabetes (8th week after induction) we show that contribution of atropine-sensitive m-cholinergic component to the contractions of urothelium-denuded DSM strips evoked by electric field stimulation (EFS) greatly increased when diabetic bladders presented overt signs of accompanying cystitis. Modeling of hemorrhagic cystitis alone in control rats by cyclophosphamide injection only modestly increased m-cholinergic component of EFS-contractions. However, exposure of DSM strips from control animals to acetylcholinesterase (AChE) inhibitor, neostigmine (1-10 µM) largely reproduced alterations in EFS contractions observed in diabetic DSM complicated by cystitis. Ellman's assay revealed statistically significant 31% decrease of AChE activities in diabetic vs. control DSM. Changes in purinergic contractility of diabetic DSM were consistent with altered P2X1-receptor desensitization and re-sensitization. They could be mimicked by pharmacological inhibition of ATP-degrading ecto-ATPases with ARL 67156 (50 µM), pointing to compromised extracellular ATP clearance as underlying reason. We conclude that decreased AChE activities associated with diabetes and likely cystitis provide complementary factor to the described in literature altered expression of mACh receptor subtypes linked to diabetes as well as to cystitis to produce dramatic modification of cholinergic NMT.

A-Kinase-Anchoring Protein (AKAP150) is expressed in Astrocytes and Upregulated in Response to Ischemia.

A-kinase-anchoring proteins, AKAPs, are scaffolding proteins that associate with kinases and phosphatases, and direct them to a specific submembrane site to coordinate signaling events. AKAP150, a rodent ortholog of human AKAP79, has been extensively studied in neurons, but very little is known about the localization and function of AKAP150 in astrocytes, the major cell type in brain. Thus, in this study, we assessed the localization of AKAP150 in astrocytes and elucidated its role during physiological and ischemic conditions. Herein, we demonstrate that AKAP150 is localized in astrocytes and is up-regulated during ischemia both in vitro and in vivo. Knock-down of AKAP150 by RNAi depolarizes the astrocytic membrane potential and substantially reduces by 80% the ability of astrocytes to take up extracellular potassium during ischemic conditions. Therefore, upregulation of AKAP150 during ischemia preserves potassium conductance and the associated hyperpolarized membrane potential of astrocytes; properties of astrocytes needed to maintain extracellular brain homeostasis. Taken together, these data suggest that AKAP150 may play a pivotal role in the neuroprotective mechanism of astrocytes during pathological conditions.

TRPV1 alterations in urinary bladder dysfunction in a rat model of STZ-induced diabetes.

AIMS: More than half of diabetic patients experience voiding disorder termed diabetic urinary bladder dysfunction (DBD). Here we have investigated how the alterations in transient receptor potential vanilloid 1 (TRPV1) ion channel expressed in bladder-innervating afferents may contribute to DBD pathogenesis. MAIN METHODS: The rat model of streptozotocin (STZ)-induced diabetes was used. The functional profile of TRPV1 in retrogradely labeled afferent, bladder-innervating dorsal root ganglia (DRG) neurons was examined using patch clamp. The level of TRPV1 transcripts in DRG was assessed with qRT-PCR. TRPV1-dependent component of detrusor smooth muscle (DSM) contractions was studied with muscle strip tensiometry. KEY FINDINGS: TRPV1-mediated current (ITRPV1) was increased in diabetic animals vs. controls by 42%. The expression of Trpv1 gene was found to be 63% higher in STZ-treated rats compared to controls, consistent with the respective electrophysiological data. Surprisingly, capsaicin-induced contractions of DSM were found to be 3-to-10-fold weaker in diabetic group depending on concentration of the agonist (100nM to 10µM). SIGNIFICANCE: Our findings suggest the dual role of TRPV1 in DBD. On the one hand, the increase of its functional expression may enhance micturition reflex arc functioning. On the other hand, at the local level, the decrease of TRPV1-dependent contractions may contribute to organ decompensation.

TRPA1-dependent regulation of bladder detrusor smooth muscle contractility in normal and type I diabetic rats.

TRPA1 is a Ca(2+)-permeable cation channel that is activated by painful low temperatures (<17°C), irritating chemicals, reactive metabolites and mediators of inflammation. In the bladder TRPA1 is predominantly expressed in sensory afferent nerve endings, where it mediates sensory transduction. The contractile effect of its activation on detrusor smooth muscle (DSM) is explained by the release from sensory afferents of inflammatory factors - tachykinins and prostaglandins, which cause smooth muscle cell contraction. Diabetes is a systemic disease, with common complications being diabetic cystopathies and urinary incontinence. However, data on how diabetes affects bladder contractility associated with TRPA1 activation are not available. In this study, by using a rat model with streptozotocin-induced type I diabetes, contractility measurements of DSM strips in response to TRPA1-activating and modulating pharmacological agents and assessment of TRPA1 mRNA expression in bladder-innervating dorsal root ganglia, we have shown that diabetes enhances the TRPA1-dependent mechanism involved in bladder DSM contractility. This is not due to changes in TRPA1 expression, but mainly due to the general inflammatory reaction caused by diabetes. The latter leads to an increase in cyclooxygenase-2-dependent prostaglandin synthesis through the mechanisms associated with substance P activity. This results in the enhanced functional coupling between the tachykinin and prostanoid systems, and the concomitant increase of their impact on DSM contractility in response to TRPA1 activation.

Myorelaxant action of fluorine-containing pinacidil analog, flocalin, in bladder smooth muscle is mediated by inhibition of L-type calcium channels rather than activation of KATP channels.

Flocalin (FLO) is a new ATP-sensitive K(+) (KATP) channel opener (KCO) derived from pinacidil (PIN) by adding fluorine group to the drug's structure. FLO acts as a potent cardioprotector against ischemia-reperfusion damage in isolated heart and whole animal models primarily via activating cardiac-specific Kir6.2/SUR2A KATP channels. Given that FLO also confers relaxation on several types of smooth muscles and can partially inhibit L-type Ca(2+) channels, in this study, we asked what is the mechanism of FLO action in bladder detrusor smooth muscle (DSM). The actions of FLO and PIN on contractility of rat and guinea pig DSM strips and membrane currents of isolated DSM cells were compared by tensiometry and patch clamp. Kir6 and SUR subunit expression in rat DSM was assayed by reverse transcription PCR (RT-PCR). In contrast to PIN (10 µM), FLO (10 µM) did not produce glibenclamide-sensitive DSM strips' relaxation and inhibition of spontaneous and electrically evoked contractions. However, FLO, but not PIN, inhibited contractions evoked by high K(+) depolarization. FLO (40 µM) did not change the level of isolated DSM cell's background K(+) current, but suppressed by 20 % L-type Ca(2+) current. Determining various Kir6 and SUR messenger RNA (mRNA) expressions in rat DSM by RT-PCR indicated that dominant KATP channel in rat DSM is of vascular type involving association of Kir6.1 and SUR2B subunits. Myorelaxant effects of FLO in bladder DSM are explained by partial blockade of L-type Ca(2+) channel-mediated Ca(2+) influx rather than by hyperpolarization associated with increased K(+) permeability. Thus, insertion of fluorine group in PIN's structure made the drug more discriminative between Kir6.2/SUR2A cardiac- and Kir6.1/SUR2B vascular-type KATP channels and rendered it partial L-type Ca(2+) channel-blocking potency.

Compensatory reduction of Cav3.1 expression in thalamocortical neurons of juvenile rats of WAG/Rij model of absence epilepsy.

Absence seizures are the non-convulsive form of generalized epilepsy critically dependent on T-type calcium channels (Cav3) in thalamic neurons. In humans, absences accompany only childhood or adolescent epileptic syndromes--though in its polygenic rat models WAG/Rij and GAERS the opposite developmental pattern is observed. Hereby we address this issue by transcriptional and functional study of thalamic Cav3 in juvenile (i.e., free of seizures) rats of the absence-prone WAG/Rij strain and their coevals of the maternal Wistar strain. First, we measured the low voltage-activated (LVA) Ca(2+) current in freshly isolated thalamocortical neurons from laterodorsal nucleus of thalamus. The difference between current densities in control (12.9 ± 1.8pA/pF) and absence epilepsy (7.9 ± 1.8pA/pF) groups reached ∼ 39%. Second, we assessed the contribution of different T-channel isoforms into the reduction of Cav3-mediated current in WAG/Rij juveniles by means of RT PCR. The expression of all three LVA calcium channels was revealed with the prevalence of G and I isoforms. The expression level of G isoform (Cav3.1) was 35% smaller in WAG/Rij strain if compared to the control animals while that of H and I isoforms (Cav3.2 and Cav3.3, respectively) remained stable. The weakened expression of Cav3.1 in juveniles of WAG/Rij rats could represent a compensatory mechanism determining the pattern of the age dependency in the disease manifestation by this model of absence epilepsy.

Effects of cannabidiol on contractions and calcium signaling in rat ventricular myocytes.

Cannabidiol (CBD), a major nonpsychotropic cannabinoid found in Cannabis plant, has been shown to influence cardiovascular functions under various physiological and pathological conditions. In the present study, the effects of CBD on contractility and electrophysiological properties of rat ventricular myocytes were investigated. Video edge detection was used to measure myocyte shortening. Intracellular Ca(2+) was measured in cells loaded with the Ca(2+) sensitive fluorescent indicator fura-2 AM. Whole-cell patch clamp was used to measure action potential and Ca(2+) currents. Radioligand binding was employed to study pharmacological characteristics of CBD binding. CBD (1µM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca(2+) transients. However, the amplitudes of caffeine-evoked Ca(2+) transients and the rate of recovery of electrically evoked Ca(2+) transients following caffeine application were not altered. CBD (1µM) significantly decreased the duration of APs. Further studies on L-type Ca(2+) channels indicated that CBD inhibits these channels with IC50 of 0.1µM in a voltage-independent manner. Radioligand studies indicated that the specific binding of [(3)H]Isradipine, was not altered significantly by CBD. The results suggest that CBD depresses myocyte contractility by suppressing L-type Ca(2+) channels at a site different than dihydropyridine binding site and inhibits excitation-contraction coupling in cardiomyocytes.

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes.

BACKGROUND AND PURPOSE: The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. EXPERIMENTAL APPROACH: Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. KEY RESULTS: In the presence of 1-10 µM AEA, suppression of both Na(+) and L-type Ca(2+) channels was observed. Inhibition of Na(+) channels was voltage and Pertussis toxin (PTX) - independent. Radioligand-binding studies indicated that specific binding of [(3) H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 µM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd . Further studies on L-type Ca(2+) channels indicated that AEA potently inhibited these channels (IC50 0.1 µM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba(2+) currents. MetAEA also inhibited Na(+) and L-type Ca(2+) currents. Radioligand studies indicated that specific binding of [(3) H]isradipine, was inhibited significantly by metAEA. (10 µM), changing Bmax but not Kd . CONCLUSION AND IMPLICATIONS: Results indicate that AEA inhibited the function of voltage-dependent Na(+) and L-type Ca(2+) channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation.

Effects of endogenous cannabinoid anandamide on cardiac Na⁺/Ca²âº exchanger.

Endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) has been shown to cause negative inotropic and antiarrhythmic effects in ventricular myocytes. In this study, using whole-cell patch clamp technique, we have investigated the effects of AEA on cardiac Na(+)/Ca(2+) exchanger (NCX1)-mediated currents. AEA suppressed NCX1 with an IC50 value of 4.7 µM. Both inward and outward components of exchanger currents were suppressed by AEA equally. AEA inhibition was mimicked by the metabolically stable analogue, methanandamide (metAEA, 10 µM) while it was not influenced by inhibition of fatty acid amide hydrolase with 1 µM URB597 incubation. The effect of AEA, was not altered in the presence of cannabinoid receptor 1 and 2 antagonists AM251 (1 µM) and AM630 (1 µM), respectively. In addition, inhibition by AEA remained unchanged after pertussis toxin (PTX, 2 µg/ml) treatment or following the inclusion of GDP-ß-S (1 mM) in pipette solution. Currents mediated by NCX1 expressed in HEK-293 cells were also inhibited by 10 µM AEA a partially reversible manner. Confocal microscopy images indicated that the intensity of YFP-NCX1 expression on cell surface was not altered by AEA. Collectively, the results indicate that AEA directly inhibits the function of NCX1 in rat ventricular myocytes and in HEK-293 cells expressing NCX1.

Models of calcium permeation through T-type channels.

Ca(2+) entry is indispensable part of intracellular Ca(2+) signaling, which is vital for most of cellular functions. Low voltage-activated (LVA or T-type) calcium channels belong to the family of voltage-gated calcium channels (VGCCs) which provide Ca(2+) entry in response to membrane depolarization. VGCCs are generally characterized by exceptional Ca(2+) selectivity combined with high permeation rate, thought to be determined by the presence in their selectivity filter of a versatile Ca(2+) binding site formed by four glutamate residues (EEEE motif). The subfamily of LVA channels includes three members, Cav3.1, Cav3.2 and Cav3.3. They all possess two aspartates instead of glutamates (i.e., EEDD motif) in their selectivity filter and are the least Ca(2+)-selective of all VGCCs. They also have the lowest conductance, weakly discriminate Ca(2+), Sr(2+) and Ba(2+) and demonstrate channel-specific sensitivity to divalent metal blockers, such as Ni(2+). The available data suggest that EEDD binding site of LVA channels is more rigid compared to EEEE one, and their selectivity permeation and block are determined by two supplementary low-affinity intrapore Ca(2+) binding sites located above and below EEDD locus. In addition, LVA channels have extracellular metal binding site that allosterically regulates channel's gating, permeation and block depending on trace metals concentration.

Effects of endogenous cannabinoid anandamide on excitation-contraction coupling in rat ventricular myocytes.

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 µM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca2+ -uptake and Ca2+ -ATPase activity in a biphasic manner. [3H]-ryanodine binding and passive Ca2+ release from SR vesicles were not altered by 10 µM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 µM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 µg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3 µM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3 µM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.

Frequency dependence of excitation-contraction of multicellular smooth muscle preparations: the relevance to bipolar electrosurgery.

BACKGROUND: Bipolar electrosurgical tissue welding uses forceps-like electrodes for grasping the tissues and delivering high-frequency electric current (HFEC) to produce local heat, desiccation, and protein denaturation, resulting in the fusion of the contacting tissues. Although in this technique no electric current is flowing through the whole body to cause electric injury, depending on the frequency of applied energy, it may produce local excitation of intramural nerves, which can propagate beyond the surgical site potentially causing harmful effects. MATERIALS AND METHODS: The effects of varying frequency of HFEC on tissue excitability in bipolar electrosurgical modality were studied in vitro using electric field stimulation (EFS) method on multicellular smooth muscle strips of rat vas deferens. Contractile response to 5-s-long sine wave EFS train was taken as the measure of excitation of intramural nerves. RESULTS: EFS-induced contraction consisted of phasic and tonic components. The amplitude of both components decreased with increasing frequency, with tonic component disappearing at about 10 kHz and phasic component at about 50 kHz. Because components of EFS-induced contraction depend on different neurotransmitters, this indicates that various neurotransmitter systems are characterized by distinct frequency dependence, but above 50 kHz they all become inactivated. Bipolar electrosurgical sealing of porcine gut showed no difference in the structure of seal area at HFEC of 67 and 533 kHz. CONCLUSIONS: EFS frequency of 50 kHz represents the upper limit for excitation. HFEC above 50 kHz is safe to use for bipolar electrosurgical tissue welding without concerns of excitation propagating beyond the surgical site.

Complex modulation of Ca(v)3.1 T-type calcium channel by nickel.

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni(2+)-binding site with critical histidine-191 (H191) within the extracellular IS3-IS4 domain of the most Ni(2+)-sensitive Cav3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni(2+) effect and its dependence on PDC remain uncertain. Here we compared Ni(2+) action on the wild-type "Ni(2+)-insensitive" Cav3.1w/t channel and Cav3.1Q172H mutant having glutamine (Q) equivalent to H191 of Cav3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni(2+) blockade of Ca(2+), Sr(2+), or Ba(2+) currents was assessed by electrophysiology. Inhibition of Cav3.1w/t by Ni(2+) conformed to two sites binding. Ni(2+) binding with high-affinity site (IC50 = 0.03-3 µM depending on PDC) produced maximal inhibition of 20-30% and was voltage-dependent, consistent with its location within the channel's pore. Most of the inhibition (70-80%) was produced by Ni(2+) binding with low-affinity site (IC50 = 240-700 µM). Q172H-mutation mainly affected low-affinity binding (IC50 = 120-160 µM). The IC50 of Ni(2+) binding with both sites in the Cav3.1w/t and Cav3.1Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca(2+), Sr(2+), or Ba(2+) with Ni(2+). We conclude that differential Ni(2+)-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni(2+), may be occupied by PDC, influencing in turn the channel's permeation.

Effects of fluorine-containing opener of ATP-sensitive potassium channels, pinacidil-derivative flocalin, on cardiac voltage-gated sodium and calcium channels.

Fluorine-containing pinacidil-derivative flocalin is an effective adenosine triphosphate-sensitive potassium (K(ATP))-channel opener with pronounced vasodilatory, cardioprotective effects and low general toxicity. By activating cardiac K(ATP) channels, flocalin hyperpolarizes cardiac myocytes, decreases their excitability, reduces Ca(2+) entry, and inhibits Ca(2+)-dependent signalling processes. Since our previous studies indicated that the drug also influences the rate of rise and amplitude of the cardiomyocyte's action potential, here we have investigated its possible actions on depolarizing inward currents through voltage-gated sodium (VGSC) and L-type calcium (VGCC) channels. Experiments were conducted on cultured cardiac myocytes prepared from the whole hearts of neonatal rats and maintained in culture for 1-3 days using whole-cell patch-clamp technique with no distinction of myocyte's type. Flocalin concentration dependently inhibited the Na(+) inward current through VGSCs with IC(50) = 17.4 µM and a maximal extent of 0.54, slowed down its inactivation kinetics, and hyperpolarized steady-state inactivation by 5.6 mV. The drug also inhibited calcium current through L-type VGCCs with IC(50) = 24.1 µM and a maximal block of 0.38, without affecting its inactivation but producing 5.3-mV hyperpolarization shifting of steady-state activation. Inhibition of both depolarizing currents by flocalin in addition to its ability to open K(ATP) channels enhances the suppressive action of the drug on cardiac excitability and broadens its pharmacological effects. Since, according to our previous data, cardiac K(ATP)-channel opening by flocalin occurs with ЕC(50) = 8 µM, the possibility of partial blockade of VGSC and L-type VGCCs should be considered when determining the therapeutic concentrations of the compound during its use as a cardioprotector.