The GPR55 agonist lysophosphatidylinositol relaxes rat mesenteric resistance artery and induces Ca2+ release in rat mesenteric artery endothelial cells

Background and Purpose

Lysophosphatidylinositol (LPI), a lipid signalling molecule, activates GPR55 and elevates intracellular Ca²⁺. Here, we examine the actions of LPI in the rat resistance mesenteric artery and Ca²⁺ responses in endothelial cells isolated from the artery.

Experimental Approach

Vascular responses were studied using wire myographs. Single-cell fluorescence imaging was performed using a MetaFluor system. Hypotensive effects of LPI were assessed using a Biopac system.

Key Results

In isolated arteries, LPI-induced vasorelaxation was concentration- and endothelium-dependent and inhibited by CID 16020046, a GPR55 antagonist. The CB₁ receptor antagonist AM 251 had no effect, whereas rimonabant and O-1918 significantly potentiated LPI responses. Vasorelaxation was reduced by charybdotoxin and iberiotoxin, alone or combined. LPI decreased systemic arterial pressure. GPR55 is expressed in rat mesenteric artery. LPI caused biphasic elevations of endothelial cell intracellular Ca²⁺. Pretreatment with thapsigargin or 2-aminoethoxydiphenyl borate abolished both phases. The PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 attenuated the initial phase and enhanced the second phase, whereas the Rho-associated kinase inhibitor Y-27632 abolished the late phase but not the early phase.

Conclusions and Implications

LPI is an endothelium-dependent vasodilator in the rat small mesenteric artery and a hypotensive agent. The vascular response involves activation of Ca²⁺-sensitive K⁺ channels and is not mediated by CB₁ receptors, but unexpectedly enhanced by antagonists of the ‘endothelial anandamide’ receptor. In endothelial cells, LPI utilizes PLC-IP3 and perhaps ROCK-RhoA pathways to elevate intracellular Ca²⁺. Overall, these findings support an endothelial site of action for LPI and suggest a possible role for GPR55 in vasculature.     

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Aim:

Hydrogen peroxide (H₂O₂) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods:

Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP₂. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results:

H₂O₂ increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 μmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i that was reversed by removing extracellular Ca²⁺. H₂O₂ increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H₂O₂-induced membrane current increases and [Ca²⁺]_i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_i, which was abolished by the specific PLC blocker {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 but was not eliminated by removal of extracellular Ca²⁺. PLC activity was increased by 1 mmol/L H₂O₂ but not by 400 μmol/L H₂O₂.

Conclusion:

H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H₂O₂-induced sustained [Ca²⁺]_i elevation is mediated via a Ca²⁺ influx mechanism, under which H₂O₂ impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H₂O₂-induced transient elevation of [Ca²⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.     

Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinases

Background and purpose:

TRPC5 is a Ca²⁺-permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant-derived isoflavone, on TRPC5 activity, and the mechanism(s) involved.

Experimental approach:

Effects of genistein on TRPC5 channels were investigated in TRPC5-over-expressing human embryonic kidney 293 (HEK) cells and bovine aortic endothelial cells (BAECs) using fluorescent Ca²⁺ imaging and electrophysiological techniques.

Key results:

In TRPC5-over-expressing HEK cells, genistein stimulated TRPC5-mediated Ca²⁺ influx, concentration dependently (EC₅₀= 93 µM). Genistein and lanthanum activated TRPC5 channels synergistically. Effects of genistein on TRPC5 channels were mimicked by daidzein (100 µM), a genistein analogue inactive as a tyrosine kinase inhibitor, but not by known tyrosine kinase inhibitors herbimycin (2 µM), PP2 (20 µM) and lavendustin A (10 µM). Action of genistein on TRPC5 channels was not affected by an oestrogen receptor inhibitor ICI-182780 (50 µM) or a phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 µM), suggesting genistein did not act through oestrogen receptors or phospholipase C. In BAECs, genistein (100 µM) stimulated TRPC5-mediated Ca²⁺ influx. In patch clamp studies, both genistein (50 µM) and daidzein (50 µM) augmented TRPC5-mediated whole-cell cation current in TRPC5 over-expressing HEK cells. Genistein stimulated TRPC5 channel activity in excised inside-out membrane patch, suggesting that its action was relatively direct and did not require cytosolic factors.

Conclusions and implications:

The present study is the first to demonstrate stimulation of a TRP channel by isoflavones. Genistein is a lipophilic compound able to stimulate TRPC5 activity in TRPC5-over-expressing HEK cells and in native vascular endothelial cells.     

Noradrenaline stimulates cell proliferation by suppressing potassium channels via Gi/o-protein-coupled α1B-adrenoceptors in human osteoblasts

Background and Purpose

Recent studies demonstrated that the sympathetic nervous system regulates bone metabolism via β₂-adrenoceptors. Although α-adrenoceptors are also expressed in osteogenic cells, their functions in bone metabolism have been less studied. We previously demonstrated that noradrenaline suppressed potassium currents via α_1B-adrenoceptors in the human osteoblast SaM-1 cell line. The aim of this study was to investigate the signal transduction pathway and the physiological role of noradrenaline in human osteoblasts in more detail.

Experimental Approach

To investigate signal transduction through α_1B-adrenoceptors, we used whole-cell patch clamp recording and Ca fluorescence imaging. Potassium channels regulate membrane potential and cell proliferation activity in non-excitable cells, so we evaluated cell proliferation activity by BrdU incorporation and WST assay.

Key Results

In SaM-1 cells, bath-applied noradrenaline elevated intracellular Ca²⁺ concentration and this effect was abolished by both chloroethylclonidine, an α_1B-adrenoceptor antagonist, and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, a PLC inhibitor. However, the inhibitory effect of noradrenaline on whole-cell current was unaffected by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122. In contrast, in cells pretreated with either Pertussis toxin, a G_i/o-protein-coupled receptor inhibitor, or gallein, a Gβγ-protein inhibitor, the inhibitory effect of noradrenaline on whole-cell current was significantly suppressed. Noradrenaline-induced enhancement of cell proliferation was inhibited by CsCl, a non-selective potassium channel blocker, gallein and H89, a PKA inhibitor, but not by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122.

Conclusions and Implications

Noradrenaline facilitated cell proliferation by regulation of potassium currents in human osteoblasts via G_i/o-protein-coupled α_1B-adrenoceptors, not via coupling to Gq-proteins.     

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca²⁺ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and the increase of [Ca²⁺]_i by OxLDL or by LPC was inhibited by La³⁺ or heparin. LPC failed to increase [Ca²⁺]_i in the presence of an antioxidant tempol. In addition, store-operated Ca²⁺ entry (SOC), which was evoked by intracellular Ca²⁺ store depletion in Ca²⁺-free solution using the sarcoplasmic reticulum Ca²⁺ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca²⁺]_i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La³⁺ or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca²⁺ to 1 µM activated large-conductance Ca²⁺-activated K⁺ (BK_Ca) current spontaneously, and this activated BK_Ca current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca²⁺-permeable Ca²⁺-activated NSC current and BK_Ca current simultaneously, thereby increasing SOC.     

Inositol trisphosphate-dependent Ca2+ stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland

Background and purpose

Changes in smooth muscle tone of the prostate gland are involved in aetiology of symptomatic prostatic hyperplasia, however the control mechanisms of prostatic smooth muscle are not well understood. Here, we have examined the role of internal Ca²⁺ compartments in regulating slow wave activity in the guinea pig prostate.

Experimental approach

Standard intracellular membrane potential recording techniques were used.

Key results

The majority (89%) of impaled cells displayed ‘slow wave’ activity. Cyclopiazonic acid (10 µmol·L⁻¹) transiently depolarized (3–9 mV) the membrane potential of the prostatic stroma and transiently increased slow wave frequency. Thereafter, slow wave frequency slowly decreased over 20–30 min. Ryanodine transiently increased slow wave frequency, although after 30 min exposure slow wave frequency and time course returned to near control values. Caffeine (1 mmol·L⁻¹) reduced slow wave frequency, accompanied by membrane depolarization of about 8 mV. Blockade of inositol trisphosphate receptor (IP₃R)-mediated Ca²⁺ release with 2-aminoethoxy-diphenylborate (60 µmol·L⁻¹) or Xestospongin C (3 µmol·L⁻¹) or inhibiting phospholipase C and IP₃ formation using {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (5 µmol·L⁻¹) or neomycin (1 and 4 mmol·L⁻¹) reduced slow wave frequency, amplitude and duration. The mitochondrial uncouplers, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (1–10 µmol·L⁻¹), carbonyl cyanide m-chlorophenylhydrazone (1–3 µmol·L⁻¹) or rotenone (10 µmol·L⁻¹), depolarized the membrane (8–10 mV) before abolishing electrical activity.

Conclusion and implications

These results suggest that slow wave activity was dependent on the cyclical release of Ca²⁺ from IP₃-controlled internal stores and mitochondria. This implies that intracellular compartments were essential in the initiation and/or maintenance of the regenerative contractile activity in the guinea pig prostate gland.     

The GPR 55 agonist,L-α-lysophosphatidylinositol,mediates ovarian carcinoma cell-induced angiogenesis

Background and Purpose

Highly vascularized ovarian carcinoma secretes the putative endocannabinoid and GPR55 agonist, L-α-lysophosphatidylinositol (LPI), into the circulation. We aimed to assess the involvement of this agonist and its receptor in ovarian cancer angiogenesis.

Experimental Approach

Secretion of LPI by three ovarian cancer cell lines (OVCAR-3, OVCAR-5 and COV-362) was tested by mass spectrometry. Involvement of cancer cell-derived LPI on angiogenesis was tested in the in vivo chicken chorioallantoic membrane (CAM) assay along with the assessment of the effect of LPI on proliferation, network formation, and migration of neonatal and adult human endothelial colony-forming cells (ECFCs). Engagement of GPR55 was verified by using its pharmacological inhibitor CID16020046 and diminution of GPR55 expression by four different target-specific siRNAs. To study underlying signal transduction, Western blot analysis was performed.

Key Results

Ovarian carcinoma cell-derived LPI stimulated angiogenesis in the CAM assay. Applied LPI stimulated proliferation, network formation, and migration of neonatal ECFCs in vitro and angiogenesis in the in vivo CAM. The pharmacological GPR55 inhibitor CID16020046 inhibited LPI-stimulated ECFC proliferation, network formation and migration in vitro as well as ovarian carcinoma cell- and LPI-induced angiogenesis in vivo. Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis. These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.

Conclusions and Implications

We conclude that inhibiting the pro-angiogenic LPI/GPR55 pathway appears a promising target against angiogenesis in ovarian carcinoma.     

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.     

A role for L-��-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells

Background and purpose:

Increased circulating levels of L-α-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.

Experimental approach:

Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex® invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves.

Key results:

GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [³⁵S]GTPγS to cell membranes (pEC₅₀ 6.47 ± 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces.

Conclusions and implications:

LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Role of mitochondria in contraction and pacemaking in the mouse uterus

BACKGROUND AND PURPOSE

Uterine spontaneous contraction and pacemaking are poorly understood. This study investigates the role of the mitochondrial Ca²⁺ store in uterine activity.

EXPERIMENTAL APPROACH

We investigated the effects of mitochondrial and sarco-endoplasmic reticulum (SER) inhibitors on contraction, membrane potential (Vm) and cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in longitudinal smooth muscle of the mouse uterus.

KEY RESULTS

The mitochondrial agents rotenone, carbonylcyanide-3-chlorophenylhydrazone (CCCP), 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one ({"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157) and kaempferol decreased the force of contractions. The ATP synthase inhibitor oligomycin had no significant effect. The effects of these agents were compared with those of SER inhibitors cyclopiazonic acid (CPA), 2-amino ethoxyphenylborate (2-APB) and caffeine. All agents, except CPA and oligomycin, decreased contractile force. CPA and CCCP transiently increased contraction frequency, which returned to control levels, whereas rotenone, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157, kaempferol and 2-APB decreased frequency and caffeine had no significant effect. Application of the mitochondrial agents when CPA functionally inhibited stores did not change contraction frequency but, with the exception of kaempferol, decreased force. CCCP caused depolarization and maintained increase in [Ca²⁺]_c or depolarization/transient hyperpolarization and transient increase in [Ca²⁺]_c for oestrus and di-oestrus tissues respectively. Rotenone caused hyperpolarization and maintained increase in [Ca²⁺]_c. {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 and kaempferol caused hyperpolarization but no measurable change in [Ca²⁺]_c. Application of a range of K⁺ channel blockers indicated a role of Ca²⁺-activated K⁺ (K_Ca) channels in the CCCP- and {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157-induced actions.

CONCLUSIONS AND IMPLICATIONS

Mitochondria have a modulatory role on uterine contractions, with mitochondrial inhibition reducing contraction amplitude and pacemaker frequency by changes in Vm, [Ca²⁺]_c and/or Ca²⁺ influx.     

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder

Background and Purpose

In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca²⁺ transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.

Experimental Approach

The morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca²⁺ dynamics were visualized using Fluo-4 fluorescence Ca²⁺ imaging.

Key Results

A network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca²⁺ transients, which were accompanied by phasic venular constrictions. Nicardipine (1 μM) disrupted the synchrony of spontaneous Ca²⁺ transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca²⁺ transients were suppressed by cyclopiazonic acid (10 μM), 2-aminoethoxydiphenyl borate (10 μM), U-73122 (1 μM), oligomycin (1 μM) and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (10 μM), but unaffected by ryanodine (100 μM) or YM-244769 (1 μM), suggesting that pericyte Ca²⁺ transients rely on Ca²⁺ release from the endoplasmic reticulum via the InsP₃ receptor and also require Ca²⁺ influx through store-operated Ca²⁺ channels.

Conclusions and Implications

The pericytes in mouse bladder can generate spontaneous Ca²⁺ transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.     

The integrin α2β1 agonist,aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production

Background and purpose:

During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin α2β1 agonist.

Experimental approach:

Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques.

Key results:

Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin α2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-β. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by α2β1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-β mAb, or a phosphoinositide-3 kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Aggretin stimulated the phosphorylation of PDGFR-β, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and PD98059.

Conclusions and implications:

Aggretin induces VSMC proliferation and migration mainly through binding to integrin α2β1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.     

Contractile effect of tachykinins on rabbit small intestine

Aim:

To study the role of the tachykinin receptors in spontaneous contractions of longitudinal and circular smooth muscle from rabbit small intestine and to determine the mechanism of action of Substance P (SP).

Methods:

Rabbit duodenum, jejunum and ileum segments were prepared. The spontaneous contractions of longitudinal and circular smooth muscle were recorded using a computer via an isometric force transducer. The specific agonists and antagonists of tachykinin receptors were added into the organ bath.

Results:

The agonists of tachykinin NK1 receptor (SP and [Sar9] SP), NK2 receptor (NKA and (β-Ala8)-NKA), and NK3 receptor (NKB and Senktide) all induced contractions in the small intestine. The contractions were diminished by NK1 receptor antagonist L-733,060, NK2 receptor antagonist GR-94800, and NK3 receptor antagonist SB 218795. Contractions caused by SP were also reduced by atropine, verapamil, PKC inhibitor staurosporine, and PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusion:

Ttachykinin NK1, NK2, and NK3 receptors mediate the contractions of the smooth muscle in rabbit intestine. Furthermore, SP acts directly on smooth muscle cells through the tachykinin NK1 receptor.     

The phospholipase C inhibitor U-73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

Background and purpose:

The sarcoplasmic reticulum (SR) releases Ca²⁺ via inositol 1,4,5-trisphosphate receptors (IP₃R) in response to IP₃-generating agonists. Ca²⁺ release subsequently propagates as Ca²⁺ waves. To clarify the role of IP₃ production in wave generation, the contribution of a key enzyme in the production of IP₃ was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122.

Experimental approach:

Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) measured using fluo-3. SR Ca²⁺ release was evoked either by activation of IP₃Rs (by carbachol or photolysis of caged IP₃) or ryanodine receptors (RyRs; by caffeine).

Key results:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto transients. The drug also inhibited [Ca²⁺]_cyto increases, evoked by direct IP₃R activation (by photolysis of caged IP₃) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca²⁺]_cyto and slowed the rate of Ca²⁺ removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP₃R- or RyR-mediated Ca²⁺ release.

Conclusions and implications:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto increases. However, the drug also reduced Ca²⁺ release when evoked by direct activation of IP₃R or RyR, slowed Ca²⁺ removal and increased steady-state [Ca²⁺]_cyto. These results suggest U-73122 reduces IP₃-evoked Ca²⁺ transients by inhibiting the SR Ca²⁺ pump to deplete the SR of Ca²⁺ rather than by inhibiting PI-PLC.     

Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2+ waves in interstitial cells of Cajal-like cells of the rabbit urethra

BACKGROUND AND PURPOSE

Although spontaneous Ca²⁺ waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca²⁺ release, the interactions among mitochondrial Ca²⁺ buffering, cellular energetics and ER Ca²⁺ release in determining the spatiotemporal dynamics of intracellular Ca²⁺ remain to be elucidated.

EXPERIMENTAL APPROACH

Spontaneous Ca²⁺ transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca²⁺ imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red.

KEY RESULTS

Spontaneous Ca²⁺ waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca²⁺ dynamics were characterized by a gradual rise in basal Ca²⁺ that preceded each regenerative Ca²⁺ transient. Caffeine evoked oscillatory Ca²⁺ waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca²⁺ wave generation with a rise in basal Ca²⁺, and subsequent caffeine evoked a single rudimentary Ca²⁺ transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca²⁺ and abolished Ca²⁺ waves. However, caffeine still induced oscillatory Ca²⁺ transients. Mitochondrial Ca²⁺ uptake inhibition with RU360 attenuated Ca²⁺ wave amplitudes, while mitochondrial Ca²⁺ efflux inhibition with {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 suppressed the initial Ca²⁺ rise to reduce Ca²⁺ wave frequency.

CONCLUSIONS AND IMPLICATIONS

Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca²⁺ wave generation and may regulate ER Ca²⁺ release frequency by buffering Ca²⁺ within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca²⁺ buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.     

Mechanisms of U46619-induced contraction of rat pulmonary arteries in the presence and absence of the endothelium

Background and purpose:

Thromboxane A₂ and endothelial dysfunction are implicated in the development of pulmonary hypertension. The receptor-transduction pathway for U46619 (9,11-dideoxy-9α, 11α-methanoepoxy prostaglandin F_2α)-induced contraction was examined in endothelium-intact (E+) and denuded (E−) rat pulmonary artery rings.

Experimental approach:

Artery rings were mounted on a wire myograph under a tension of 7–7.5 mN at 37°C and gassed with 95% O₂/5% CO₂. Isometric recording was made by using Powerlab data collection and Chart 5 software.

Key results:

Both E+ and E− contractile responses were sensitive to Rho-kinase inhibition and the chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid]. The E+ response was sensitive to the store-operated calcium channel blockers SKF-96365 {1-[B-[3-(4-methoxyphenyl)propoxy]-4-methoxy-phenethyl]-1H-imidazole hydrochloride} and 2-APB (2-amino ethoxy diphenylborate) (75–100 µmol·L⁻¹). The E− response was sensitive to 2-APB (10–30 µmol·L⁻¹), a putative IP₃ receptor antagonist, and the calcium and chloride channel blockers nifedipine, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulphonic acid) and niflumic acid but was insensitive to SKF-96365. Inhibiting K_V with 4-AP in E+ rings exposed a contraction sensitive to nifedipine, DIDS and niflumic acid, whereas inhibiting BK_Ca exposed a contraction sensitive to mibefradil, DIDS and niflumic acid. This indicates that removal of the endothelium allows the TP receptor to inhibit K_V, which may involve coupling to phospholipase C, because inhibition of phospholipase C with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-y]amino]hexyl]– 1H-pyrrole-2,5-dione) switched the E− pathway to the E+ pathway.

Conclusions and implications:

The results from this study indicate that distinct transduction pathways can be employed by the TP receptor to produce contraction and that the endothelium is able to influence the coupling of the TP receptor.British Journal of Pharmacology (2009) 157, 581–596; doi:10.1111/j.1476-5381.2008.00084.x; published online 22 April 2009This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle

BACKGROUND AND PURPOSE

The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca²⁺ sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels.

EXPERIMENTAL APPROACH

We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques.

KEY RESULTS

The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca²⁺ sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents.

CONCLUSIONS AND IMPLICATIONS

The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca²⁺ sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca²⁺ sensitivity of the contractile apparatus.     

Biphasic effects of sodium danshensu on vesse function in isolated rat aorta

Aim:

To investigate the effects of sodium danshensu on vessel function in isolated rat aortic ring.

Methods:

Thoracic aortae from normal rats were isolated and equilibrated in organ bath with Krebs-Henseleit buffer and ring tension was recorded. Effects of sodium danshensu on basal tonus of the vessel and its effects on vessel contraction and relaxation with or without endothelium were observed.

Results:

In thoracic arteries under basal tonus, sodium danshensu (0.3–3 g/L) produced a dose-dependent transient contraction. In phenylephrine-precontracted thoracic arteries with or without endothelium, low concentration (0.1–0.3 g/L) of sodium danshensu produced a weak contraction, while high concentrations (1–3 g/L) produced a pronounced vasodilator after a transient vasocontraction. Pre-incubation with sodium danshensu could inhibit vessel contraction induced by phenylephrine and potassium chloride in a concentration-dependent way. Sodium danshensu inhibited phenylephrine- and CaCl₂-induced vasoconstriction in Ca²⁺-free medium. Pre-incubation with tetraethylammonium, a non-selective K⁺ channel blocker, and apamin, a small-conductance calcium-activated K⁺ channel blocker partially antagonized the relaxation response induced by sodium danshensu. However, iberiotoxin (big-conductance calcium-sensitive K⁺ channel blocker), barium chloride (inward rectifier K⁺ channel blocker), and glibencalmide (ATP-sensitive K⁺ channel blocker) had no influence on the vasodialtion effect of sodium danshensu.

Conclusion:

Sodium danshensu showed a biphasic effects on vessel tension. While low dosage of sodium danshensu produced small contraction possibly through transient enhancement of Ca²⁺ influx, high dosage produced significant vasodilation mainly through promoting the opening of non-selective K⁺ channels and small-conductance calcium-sensitive K⁺ channels in the vascular smooth muscle cells.     

Effect of intravenous anesthetic propofol on synaptic vesicle exocytosis at the frog neuromuscular junction

Aim:

To investigate the presynaptic effects of propofol, a short-acting intravenous anesthetic, in the frog neuromuscular junction.

Methods:

Frog cutaneous pectoris nerve muscle preparations were prepared. A fluorescent tool (FM1-43) was used to visualize the effect of propofol on synaptic vesicle exocytosos in the frog neuromuscular junction.

Results:

Low concentrations of propofol, ranging from 10 to 25 μmol/L, enhanced spontaneous vesicle exocytosis monitored by FM1-43 in a Ca²⁺-dependent and Na⁺-independent fashion. Higher concentrations of propofol (50, 100, and 200 μmol/L) had no effect on spontaneous exocytosis. By contrast, higher concentrations of propofol inhibited the Na⁺-dependent exocytosis evoked by 4-aminopyridine but did not affect the Na⁺-independent exocytosis evoked by KCl. This action was similar and non-additive with that observed by tetrodotoxin, a Na⁺ channel blocker.

Conclusion:

Our data suggest that propofol has a dose-dependent presynaptic effect at the neuromuscular transmission which may help to understand some of the clinical effects of this agent on neuromuscular function.     

Evaluation of the insulin releasing and antihyperglycaemic activities of GPR55 lipid agonists using clonal beta-cells,isolated pancreatic islets and mice

Background and Purpose

G-protein coupled receptor (GPR)55 is a novel lipid sensing receptor activated by both cannabinoid endogenous ligands (endocannabinoids) and other non-cannabinoid lipid transmitters. This study assessed the effects of various GPR55 agonists on glucose homeostasis.

Experimental Approach

Insulin secretion and changes in intracellular Ca²⁺ and cAMP in response to glucose and a range of GPR55 agonists [endogenous ligands (OEA, PEA), chemically synthetic cannabidiol (CBD) analogues (Abn-CBD, 0–1602), an analogue of rimonabant (AM-251) and antagonist (CBD)] were investigated in clonal BRIN-BD11 cells and mouse pancreatic islets. Cytotoxicity was assessed by LDH release, cellular localization by double-staining immunohistochemistry and in vivo effects assessed in mice.

Key Results

The most potent and selective GPR55 agonist was the synthetic CBD analogue, Abn-CBD (pEC₅₀ 10.33), maximum stimulation of 67% at 10⁻⁴ mol·L⁻¹ (P < 0.001) in BRIN-BD11 cells. AM-251 (pEC₅₀ 7.0), OEA (pEC₅₀ 7.0), 0–1602 (pEC₅₀ 7.3) and PEA (pEC₅₀ 6.0) stimulated insulin secretion. Results were corroborated by islet studies, with no cytotoxic effects. Concentration-dependent insulin secretion by GPR55 agonists was glucose-sensitive and accompanied by elevations of [Ca²⁺]_i (P < 0.01–P < 0.001) and cAMP (P < 0.05–P < 0.01). GPR55 agonists exhibited insulinotropic and glucose lowering activity in vivo. GPR55 was expressed on BRIN-BD11 cells and confined to islet beta cells with no distribution on alpha cells.

Conclusion and Implications

These results demonstrate GPR55 is distributed in pancreatic beta cells and is a strong activator of insulin secretion, with glucose-lowering effects in vivo. Development of agents agonizing the GPR55 receptor may have therapeutic potential in the treatment of type 2 diabetes.