Effects of trimebutine maleate on colonic motility through Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels and L-type Ca<Superscript>2+</Superscript> channels

The effects of trimebutine maleate (TM) on spontaneous contractions of colonic longitudinal muscle were investigated in guinea pigs. The contractile responses of smooth muscle strips were recorded by an isometric force transducer. Membrane and action potentials were detected by an intracellular microelectrode technique. The whole-cell patch clamp recording technique was used to record the changes in large conductance Ca²⁺-activated K⁺ (BK_ca) and L-type Ca²⁺ currents in colonic smooth muscle cells. At high concentrations (30, 100, and 300 μM), TM inhibited the amplitude of spontaneous contractions. At low concentrations (1 and 10 μM), TM attenuated the frequency and tone of smooth muscle strips, whereas TM had no influence on the amplitude of spontaneous contractions. TM depolarized the membrane potentials, but decreased the amplitude and frequency of action potentials at high concentrations. TM inhibited BK_ca and L-type Ca²⁺ currents in a dose-dependent manner. In the presence of the BK_ca channel opener, NS1619, TM also inhibited BK_ca currents. Bayk8644, a L-type Ca²⁺ channel opener, increased L-type Ca²⁺ currents. This augmentation was also attenuated by TM. These results suggest that TM attenuates intestinal motility through inhibition of L-type Ca²⁺ currents, and depolarizes membrane potentials by reducing BK_ca currents. Thus, TM may be a multiple-ion channel regulator in the gastrointestinal tract.     

The Influences of G Proteins, Ca2+, and K+ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

NO released by myenteric neurons controls the off contraction induced by electrical field stimulation (EFS) in distal esophageal smooth muscle, but in the presence of nitric oxide synthase (NOS) inhibitor, L-NAME, contraction by EFS occurs at the same time. The authors investigated the intracellular signaling pathways related with G protein and ionic channel EFS-induced contraction using cat esophageal muscles. EFS-induced contractions were significantly suppressed by tetrodotoxin (1 µM) and atropine (1 µM). Furthermore, nimodipine inhibited both on and off contractions by EFS in a concentration dependent meaner. The characteristics of ''on'' and ''off'' contraction and the effects of G-proteins, phospholipase, and K⁺ channel on EFS-induced contraction in smooth muscle were also investigated. Pertussis toxin (PTX, a G_i inactivator) attenuated both EFS-induced contractions. Cholera toxin (CTX, G_s inactivator) also decreased the amplitudes of EFS-induced off and on contractions. However, phospholipase inhibitors did not affect these contractions. Pinacidil (a K⁺ channel opener) decreased these contractions, and tetraethylammonium (TEA, K⁺_Ca channel blocker) increased them. These results suggest that EFS-induced on and off contractions can be mediated by the activations Gi or Gs proteins, and that L-type Ca²⁺ channel may be activated by G-protein α subunits. Furthermore, K⁺_Ca-channel involve in the depolarization of esophageal smooth muscle. Further studies are required to characterize the physiological regulation of Ca²⁺ channel and to investigate the effects of other K⁺ channels on EFS-induced on and off contractions.     

The effect of hypercholesterolemia on carbachol-induced contractions of the detrusor smooth muscle in rats: increased role of L-type Ca2+ channels

To investigate a possible relation between hypercholesterolemia and detrusor smooth muscle function, we studied the contractile response to potassium challenge, carbachol (CCh), and the components of CCh-induced contractile mechanism in high-cholesterol diet-fed rats. Adult male Sprague–Dawley rats were fed with standard (control group, N?=?17) or 4?% cholesterol diet (hypercholesterolemia group (HC), N?=?16) for 4?weeks. Spontaneous contractions of detrusor muscle strips and their responses to potassium chloride (KCl) or cumulative dose–contraction curves to CCh were recorded. The effects of muscarinic receptor antagonists (methoctramin and/or 4-diphenylacetoxy-N-methylpiperidine), L-type Ca⁺² channel blocker (nifedipine), and/or rho-kinase inhibitor Y-27632 were investigated. Blood cholesterol level was increased in the HC group with no sign of atherosclerosis. The KCl-induced detrusor smooth muscle contractions were higher in HC, whereas spontaneous and CCh-induced responses were similar in both groups. Preincubation with receptor antagonist for M₃ but not for M₂ attenuated contraction significantly, shifting the dose–response curve to the right. This response was similar in both groups. Among two effector mechanisms of M₃-mediated detrusor smooth muscle contraction, rho-kinase pathway was not affected by hypercholesterolemia, whereas blockade of L-type Ca⁺² channels potently reduced contractions. The results of this study point out a relation between hypercholesterolemia and contractile mechanism of detrusor smooth muscle likely to change urinary bladder function, via altering L-type Ca⁺² channels. Taken together with escalating incidence of hypercholesterolemia and lower urinary tract symptoms, it is a field which deserves to be investigated further.     

Actions of manganese ions in contraction of smooth muscle

• 1.1. Manganese ions (Mn²⁺) have been used in the last few decades as one of a number of inorganic Ca²⁺ channel blockers to investigate Ca²⁺ channels in excitable smooth muscle cells.
• 2.2. It has been recently reported that in addition to its inhibitory effects on the Ca²⁺ channels, Mn²⁺ in millimolar concentrations also produces contraction in the state of cell membrane depolarization.
• 3.3. Mn²⁺ has been shown to be able to permeate the cells via voltage-operated L-type Ca²⁺ channels in the membranes and directly activates contractile proteins in smooth muscles.
• 4.4. Intracellular sites of action have been proposed for Mn²⁺ in smooth muscles.

     

The involvement of protein kinase C and tyrosine kinase in vanadate-induced contraction

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM LaCl₃ and significantly inhibited by 10 μM verapamil and 1 μM nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular Ca²⁺ concentration, and the influx of extracellular Ca²⁺ was mediated through voltage-dependent Ca²⁺ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin (1 μM) and sodium nitroprusside (1 μM) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosine kinase are involved in the vanadate-induced contraction which required the influx of extracellular Ca²⁺ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.     

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

To determine which types of voltage-dependent Ca²⁺ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca²⁺ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na⁺ channel activator veratridine. Contractions caused by either 45.9mMKCl or veratridine (10μM) were inhibited by spantide (10μM), a tachykinin receptor antagonist, and capsaicin (10μM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca²⁺ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. ω-Conotoxin GVIA (1μM), an N-type Ca²⁺ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K8644, an L-type Ca²⁺ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9mMKCl, and enhanced contractions induced by 15.9mMKCl and veratridine (2μM). ω-Agatoxin IVA (0.3μM), a P-type voltage-dependent Ca²⁺ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca²⁺ channel blockers or by Bay K8644. These results suggest that, in the rabbit iris sphincter muscle, L- and N-type voltage-dependent Ca²⁺ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine. Received: 21 May 1996 / Accepted: 14 January 1997     

MLCK and PKC Involvements via Gi and Rho A Protein in Contraction by the Electrical Field Stimulation in Feline Esophageal Smooth Muscle

We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electrical field stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C (PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractions were recorded using an isometric force transducer. On-contraction occurred in the presence of N^G-nitro-L-arginine methyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatory composition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in Ca²⁺-free buffer but reappeared in normal Ca²⁺-containing buffer indicating that the contraction was Ca²⁺ dependent. 4-aminopyridine (4-AP), voltage-dependent K⁺ channel blocker, significantly enhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a G_i inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoA protein may be related with Ca²⁺ and K⁺ channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction, and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergic contractions activated directly by low-frequency EFS may be mediated by Ca²⁺, and G proteins, such as Gi and rhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smooth muscle.     

Rhynchophylline-induced vasodilation in human mesenteric artery is mainly due to blockage of L-type calcium channels in vascular smooth muscle cells

Rhynchophylline (Rhy) is a pharmacologically active substance isolated from Uncaria rhynchophylla which has been used to treat cardiovascular diseases and has drawn considerable attention in recent years for its antihypertensive activities. We investigated the actions of Rhy on endothelium-denuded human mesenteric artery by tension measurement and its actions on high conductance Ca²⁺-activated K⁺ channels (BK_Ca) currents and calcium currents (I_Ca) in freshly isolated smooth muscle cells using perforated patch clamp technique. Intracellular Ca²⁺ level was measured in Fura-2-loaded cells. Rhy inhibited both the KCl and BayK-evoked mesenteric artery constrictions in a dose-dependent manner. K⁺ channel blockers (TEA, glibenclamide, IbTX, and 4-AP) did not affect the vasorelaxing effect of Rhy. Rhy inhibited L-type voltage-gated Ca²⁺ current (I_Ca,L) but had no significant effect on macroscopic BK_Ca current. Rhy preincubation markedly reduced the elevation of [Ca²⁺]_i level induced by KCl depolarization. Caffeine-stimulated [Ca²⁺]_i elevation was also decreased to some extent by pretreatment with Rhy for 20 min. Our results show that Rhy relaxes smooth muscles of human mesenteric resistance arteries, mainly due to inhibition of Ca²⁺ influx by blockage of L-type Ca²⁺ channels and thereby the decrease in [Ca²⁺]_i.     

Calcium-antagonizing activity of S-petasin, a hypotensive sesquiterpene from Petasites formosanus, on inotropic and chronotropic responses in isolated rat atria and cardiac myocytes

Petasites formosanus, an indigenous species of Petasites, has been used to treat cardiovascular diseases such as hypertension for years. We have suggested recently that S-petasin, a major sesquiterpene from P. formosanus, inhibits vascular smooth muscle contraction through inhibition of voltage-dependent Ca²⁺ channels, a phenomenon possibly responsible for the hypotensive effect of P. formosanus. This study was designed to examine the chronotropic and inotropic actions of S-petasin in the heart in vivo and in vitro. Administration of S-petasin (0.1–1.5 mg/kg i.v.) in anesthetized rats reduced heart rate dose-dependently. This response was consistent with significant suppression of both contractile amplitude and spontaneous firing rate of isolated atria, responses that were not antagonized by atropine (1 µM). Mechanical evaluation in isolated ventricular myocytes showed that S-petasin (0.1 to 100 µM) depressed peak myocyte contraction and intracellular Ca²⁺ transients concentration-dependently. The duration of myocyte contraction was not affected. Whole-cell voltage clamp analysis revealed that S-petasin inhibited the L-type Ca²⁺ current (I_Ca,L) concentration-dependently and shifted the steady-state inactivation curve of I_Ca,L to more negative potentials. However, a receptor-binding assay failed to identify any significant interaction between S-petasin (0.1–300 µM) and the dihydropyridine binding sites of L-type voltage-dependent Ca²⁺ channels. Taken together, these data show that the negative chronotropic and inotropic properties of S-petasin that can be ascribed mainly to I_Ca,L inhibition, but not to blockade of dihydropyridine binding sites of L-type Ca²⁺ channel or to muscarinic receptor activation.     

PROBING EXCITATION-CONTRACTION COUPLING IN TRACHEALIS SMOOTH MUSCLE WITH THE MYCOTOXIN CYCLOPIAZONIC ACID

1. Muscarinic stimulation-induced tonic contraction of airway smooth muscle is independent of membrane potential. This contraction is not sensitive to inhibition by voltage-operated Ca²⁺ channel blockers or by K⁺ channel openers. 2. Cyclopiazonic acid (CPA) inhibits Ca²⁺ loading of internal stores but does not affect maximal tonic contraction induced by acetylcholine (ACh) in steady state conditions. 3. After depletion of internal Ca²⁺ stores with CPA, AChinduced tonic contraction becomes dependent upon values of membrane potential. The contraction is then sensitive to voltage-operated Ca²⁺ channel blockers and to K⁺ channel openers. 4. Treatment of trachealis muscle with CPA potentiates the M₂-mediated component of ACh stimulation, but this potentiation is not entirely responsible for the switch in excitation-contraction (E-C) coupling. 5. It is proposed that depletion of internal Ca²⁺ stores with CPA and promotion of M₂-stimulation can lead to a switch in E-C coupling in trachealis smooth muscle from pharmaco- to electromechanical mode, perhaps by targeting a plasma membrane K⁺ channel.     

Contractile Action of Mn2+ via Ca2+ Channels Activated by Bay K 8644 in Guinea-pig Taenia Coli

Mn²⁺ has been shown to inhibit K+-induced contraction of smooth-muscle, to induce contraction of smooth-muscle in Ca²⁺-free, K+ medium and to activate the contractile proteins of skinned fibres of smooth muscle cells. Further work has suggested that Mn²⁺ penetrates the cytoplasm through voltage-dependent Ca²⁺ channels when the cell membranes of smooth muscles are depolarized with K+. We have investigated whether in Ca²⁺-free medium, Mn²⁺ enters the cytoplasm through Ca²⁺ channels and induces contraction of guinea-pig taenia coli in the presence of Bay K 8644, a dihydropyridine Ca²⁺-channel agonist which prolongs the open state of the voltage-dependent Ca²⁺ channels in smooth-muscle cells. In Ca²⁺-free medium the application of 5 mM Mn²⁺ in the presence of Bay K 8644 caused contraction of and concomitant increase in Mn²⁺ uptake in guinea-pig taenia coli smooth muscle. In the presence of Bay K 8644 nifedipine, a dihydropyridine Ca²⁺ channel antagonist, dose-dependently inhibited both manganese uptake and the contraction induced by Mn²⁺. These results suggest that Mn²⁺ enters the cytoplasm through dihydropyridine-sensitive, voltage-dependent Ca²⁺ channels activated by Bay K 8644 and then induces contraction in taenia coli.     

Relaxant effect induced by wogonin from Scutellaria baicalensis on rat isolated uterine smooth muscle

Context: Wogonin is a flavone derivative isolated from Scutellaria baicalensis Georgi (Labiatae) root, which is a traditional Chinese drug used as an anti-inflammatory and for management of dysmenorrhea.

Objective: The effect of wogonin on the uterus has not yet been examined. We investigated the relaxant effects of wogonin on contractile activity of isolated uterine strips of rats.

Materials and methods: The effect of wogonin on spontaneous uterine contraction, and uterine contraction induced by agonists, K⁺-depolarization and oxytocin in Ca²⁺-free solution was observed. To clarify the type of potassium channel, we tested the effects of 4-aminopyridine, tetraethylammonium and glibenclamide.

Results: Wogonin reduced the contractile amplitude of uterine strip smooth muscle of rats in a dose-dependent manner. The concentration of wogonin for reducing the contraction amplitude by 50% (IC₅₀) on spontaneous contractions was 60.5 μM. Wogonin also inhibited the contraction induced by three agonists (oxytocin, prostaglandin F_2α and acetylcholine). For the uterine strips pretreated with oxytocin in Ca²⁺-free solution or K⁺-depolarization, wogonin showed relaxant effect on the induced uterine contractions. In addition, whereas the inhibitive effect of wogonin on the contraction of uterine smooth muscle in rats could be partly blocked by 4-aminopyridine and tetraethylammonium, it was not influenced by glibenclamide.

Discussion and conclusion: Wogonin significantly inhibited the contraction of rat uterine smooth muscle probably through the inhibition of the inflow of extracellular calcium into cells via cell membrane, and intracellular release of calcium ions. In addition, the relaxant effect induced by wogonin might be due in part to the opening of voltage-dependent and large conductance Ca²⁺-activated K⁺ channels.     

High K+-induced contraction requires depolarization-induced Ca2+ release from internal stores in rat gut smooth muscle

Aim:

Depolarization-induced contraction of smooth muscle is thought to be mediated by Ca²⁺ influx through voltage-gated L-type Ca²⁺ channels. We describe a novel contraction mechanism that is independent of Ca²⁺ entry.

Methods:

Pharmacological experiments were carried out on isolated rat gut longitudinal smooth muscle preparations, measuring isometric contraction strength upon high K⁺-induced depolarization.

Results:

Treatment with verapamil, which presumably leads to a conformational change in the channel, completely abolished K⁺-induced contraction, while residual contraction still occurred when Ca²⁺ entry was blocked with Cd²⁺. These results were further confirmed by measuring intracellular Ca²⁺ transients using Fura-2. Co-application of Cd²⁺ and the ryanodine receptor blocker DHBP further reduced contraction, albeit incompletely. Additional blockage of either phospholipase C (U 73122) or inositol 1,4,5-trisphophate (IP₃) receptors (2-APB) abolished most contractions, while sole application of these blockers and Cd²⁺ (without parallel ryanodine receptor manipulation) also resulted in incomplete contraction block.

Conclusion:

We conclude that there are parallel mechanisms of depolarization-induced smooth muscle contraction via (a) Ca²⁺ entry and (b) Ca²⁺ entry-independent, depolarization-induced Ca²⁺-release through ryanodine receptors and IP₃, with the latter being dependent on phospholipase C activation.     

Phorbol ester-induced contractions of mouse detrusor muscle are inhibited by nifedipine

The effects of phorbol esters on contractions of detrusor strips isolated from mouse urinary bladder were studied. β-Phorbol-12,13-dibutyrate (β-PDBu, 10 nM) significantly enhances both the neurogenic and myogenic detrusor contractions to a similar extent. By contrast, an inactive isoform of protein kinase C (PKC) stimulation, α- phorbol-12,13-dibutyrate (100 nM) has no such enhancing effect on the muscle contraction. The effect of β-PDBu was dependent on the extracellular Ca²⁺ concentration. Nifedipine (0.3 μM, a L-type Ca²⁺ channel blocker), staurosporine (1 μM) and bisindolylmaleimide I ( μM, a selective PKC inhibitor) but not ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) abolished the enhancing effect of β-PDBu. In other words, β-PDBu failed to augment the nifedipine-insensitive component of the muscle contraction. Moreover, β-PDBu not only enhances the muscle response induced by exogenous agonists (acetylcholine or ATP) and KCl but also increases the resting tone of detrusor muscle, an effect which is also inhibited by nifedipine and bisindolylmaleimide I. From these findings, it is concluded that the enhancing effect of β-PDBu is due to activation of the L-type Ca²⁺ channel through phosphorylation by protein kinase C. This allows more Ca²⁺ influx from the extracellular medium, leading to an increase in the contractions of the mouse detrusor muscle. Received: 29 October 1997 / Accepted; 17 February 1998     

Inhibitory effects of protein kinase inhibitors and cytoskeletal inhibitors on Ca2+-free contraction of rat uterus

In rat uterine smooth muscle, sustained Ca²⁺-free contraction was observed by oxytocin in Ca²⁺-free solution. This Ca²⁺-free contraction was effectively inhibited by protein kinase inhibitors and cytoskeletal inhibitors but myosin-light chain kinase (MLCK) inhibitors were not so effective. Simultaneous addition of a protein kinase inhibitor and a cytoskeletal inhibitor caused synergestic inhibition. These results suggest that the mechanism for Ca²⁺-free contraction involves some protein kinase and cytoskeletal elements rather than MLCK.     

Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Although extracellular Ca²⁺ entry through the voltage-dependent Ca²⁺ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca²⁺ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca²⁺ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca²⁺]_i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca²⁺ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (<1 µM) of nifedipine, a L-type Ca²⁺ channel blocker, produced a decrease in the amplitude of the spontaneous Ca²⁺ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (α1G, α1H) of the T-type Ca²⁺ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca²⁺ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca²⁺ transients consistent with the reduction of the frequency. It is concluded that T-type Ca²⁺ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.     

Phospholipase C mediated Ca signals in murine urinary bladder smooth muscle

Muscarinic stimulation of urinary bladder induces contraction via an increase in intracellular Ca²⁺ concentration that results from Ca²⁺ influx through Ca²⁺ channels and/or IP₃-mediated Ca²⁺ release controlled by phospholipase C (PLC) signalling. The significance of PLC/IP₃ signalling in this cascade has recently been questioned because PLC inhibitors were without effect on carbachol-induced contractions in detrusor muscle strips. However, PLC/IP₃-mediated Ca²⁺ release was clearly observed in recordings of Ca²⁺ signals in isolated myocytes. Therefore, we investigated the presence of PLC/IP₃-dependent Ca²⁺ release by directly monitoring Ca²⁺ signals in intact detrusor muscle strips. Concomitant Ca²⁺ signals from Ca²⁺ channel activity were eliminated by the Ca²⁺ channel antagonist isradipine (3 µM) or by the use of muscles from Ca_v1.2 channel-deficient (SMACKO) mice. In absence of Ca²⁺ channel activity, carbachol elicited contractions and Ca²⁺ signals in muscles from wild type and SMACKO mice that were inhibited by the PLC inhibitor U73122 (10 µM). The results show that PLC/IP₃-dependent Ca²⁺ release is activated by stimulation with carbachol in urinary bladder smooth muscle but has a minor contribution to overall carbachol-induced Ca²⁺ signals.     

Electrophysiological effects of anandamide on rat myocardium

Background and purpose:

The endocannabinoid, anandamide, has anti-arrhythmic effects. The aim of the present study was to explore the electrophysiological effects of anandamide on rat myocardium.

Experimental approach:

Evoked action potentials (APs) were recorded using intracellular recording technique in rat cardiac papillary muscles. In addition, L-type Ca²⁺ current was measured and analysed using whole-cell patch-clamp recording technique in isolated rat cardiac ventricular myocytes.

Key results:

In cardiac papillary muscles, anandamide (1, 10, 100 nM) decreased AP duration in a concentration-dependent manner. Furthermore, 100 nM anandamide decreased AP amplitude, overshoot and V_max in partially depolarized papillary muscles. These effects were abolished by AM251 (100 nM), a selective antagonist for CB₁ receptors, but not AM630 (100 nM), a CB₂ receptor antagonist. Furthermore, an agonist of L-type Ca²⁺ channels, Bay K 8644 (0.5 µM), a K⁺ channel blocker tetraethylammonium chloride (20 mM) and the nitric oxide synthase inhibitor l-NAME (1 mM) had no effect on anandamide-induced decrease in AP duration. In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased L-type Ca²⁺ current concentration-dependently, and shifted the current–voltage relationship curve of the Ca²⁺ current. Anandamide (100 nM) shifted the steady-state inactivation curve to the left and the recovery curve to the right. Blockade of CB₁ receptors with AM251 (100 nM), but not CB₂ receptors with AM630 (100 nM), eliminated the effect of anandamide on L-type Ca²⁺ currents.

Conclusions and implications:

These data suggest that anandamide suppressed AP and L-type Ca²⁺ current in cardiac myocytes through CB₁ receptors.     

Chemerin-induced arterial contraction is Gi- and calcium-dependent

Chemerin is an adipokine associated with increased blood pressure, and may link obesity with hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via calcium flux in smooth muscle cells. Isometric contraction of rat aortic rings was performed in parallel with calcium kinetics of rat aortic smooth muscle cells to assess the possible signaling pathway. Chemerin-9 (nonapeptide of the chemerin S¹⁵⁷ isoform) caused a concentration-dependent contraction of isolated aorta (EC₅₀ 100 nM) and elicited a concentration-dependent intracellular calcium response (EC₅₀ 10 nM). Pertussis toxin (G_i inhibitor), verapamil (L-type Ca^2 + channel inhibitor), PP1 (Src inhibitor), and Y27632 (Rho kinase inhibitor) reduced both calcium influx and isometric contraction to chemerin-9 but PD098059 (Erk MAPK inhibitor) and U73122 (PLC inhibitor) had little to no effect on either measure of chemerin signaling. Although our primary aim was to examine chemerin signaling, we also highlight differences in the mechanisms of chemerin-9 and recombinant chemerin S¹⁵⁷. These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through G_i proteins to activate L-type Ca^2 + channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us closer to targeting chemerin as a form of therapy.     

The effect of Mn2+, Zn2+, Ba2+ and Ca2+ on spontaneous motility in sheep duodenum in vitro

• 1.1. The effects of several ions, Mn²⁺, Zn²⁺, Ba²⁺ and Ca²⁺, on spontaneous motility were investigated in longitudinal smooth muscle strips from sheep duodenum, in vitro
• 2.2. Mn²⁺ (0.5–1.5 mM) and Zn²⁺ (0.5–5 mM) inhibited both the amplitude and frequency of motility in Krebs solution and in Ca²⁺-free medium.
• 3.3. Ba²⁺(0.5–5 mM) evoked three types of contractile responses: (i) an increase in the frequency and a reduction of the amplitude of spontaneous contractions; (ii) a slight increase in muscle tone of the phasic contractions; and (iii) a rapid initial phasic contraction followed by slowly fading contraction. Ca²⁺ induced two kinds of responses in spontaneous motility: (i) a fast phasic contraction, followed by an increase in the amplitude and frequency of phasic contractions with no changes in its tone; and (ii) an increase in the amplitude of contractions.
• 4.4. The Ba²⁺-induced contractions were inhibited by EDTA, verapamil and diltiazem, but were not modified by sodium nitroprusside. The Ca²⁺-induced contractions were reduced by verapamil and diltiazem.
• 5.5. Our results show that Mn²⁺ and Zn²⁺ behave as inhibitors of sheep duodenum motility. In contrast, Ba²⁺ and Ca²⁺ stimulate motility. It is suggested that Ba²⁺ can penetrate the cells through voltage-dependent Ca²⁺ channels and behave as a partial substitute for Ca²⁺.