Bradykinin-induced relaxation of coronary microarteries: S-nitrosothiols as EDHF?

1. To investigate whether S-nitrosothiols, in addition to NO, mediate bradykinin-induced vasorelaxation, porcine coronary microarteries (PCMAs) were mounted in myographs. 2. Following preconstriction, concentration-response curves (CRCs) were constructed to bradykinin, the NO donors S-nitroso-N-penicillamine (SNAP) and diethylamine NONOate (DEA-NONOate) and the S-nitrosothiols L-S-nitrosocysteine (L-SNC) and D-SNC. All agonists relaxed PCMAs. L-SNC was approximately 5-fold more potent than D-SNC. 3. The guanylyl cyclase inhibitor ODQ and the NO scavenger hydroxocobalamin induced a larger shift of the bradykinin CRC than the NO synthase inhibitor L-NAME, although all three inhibitors equally suppressed bradykinin-induced cGMP responses. 4. Complete blockade of bradykinin-induced relaxation was obtained with L-NAME in the presence of the large- and intermediate-conductance Ca(2+)-activated K(+)-channel (BK(Ca), IK(Ca)) blocker charybdotoxin and the small-conductance Ca(2+)-activated K(+)-channel (SK(Ca)) channel blocker apamin, but not in the presence of L-NAME, apamin and the BK(Ca) channel blocker iberiotoxin. 5. Inhibitors of cytochrome P450 epoxygenase, cyclooxygenase, voltage-dependent K(+) channels and ATP-sensitive K(+) channels did not affect bradykinin-induced relaxation. 6. SNAP-, DEA-NONOate- and D-SNC-induced relaxations were mediated entirely by the NO-guanylyl cyclase pathway. L-SNC-induced relaxations were partially blocked by charybdotoxin+apamin, but not by iberiotoxin+apamin, and this blockade was abolished following endothelium removal. ODQ, but not hydroxocobalamin, prevented L-SNC-induced increases in cGMP, and both drugs shifted the L-SNC CRC 5-10-fold to the right. 7. L-SNC hyperpolarized intact and endothelium-denuded coronary arteries. 8. Our results support the concept that bradykinin-induced relaxation is mediated via de novo synthesized NO and a non-NO, endothelium-derived hyperpolarizing factor (EDHF). S-nitrosothiols, via stereoselective activation of endothelial IK(Ca) and SK(Ca) channels, and through direct effects on smooth muscle cells, may function as an EDHF in porcine coronary microarteries.     

The influence of hyperthyroidism on β-adrenoceptor-mediated relaxation of isolated small mesenteric arteries

We investigated the influence of hyperthyroidism on relaxant responses of small mesenteric resistance arteries to -adrenoceptor agonists and to compounds stimulating the corresponding second-messenger system. Hyperthyroidism was induced by feeding rats for 28 days with 5 mg/kg L-thyroxine (T4)-containing rat chow. This treatment produced a stable hyperthyroid state, as indicated by several biochemical/metabolic and haemodynamic parameters. Preparations of small mesenteric arteries were mounted in an isometric wire myograph. Subsequently, concentration-effect curves were determined for isoproterenol, noradrenaline and salbutamol as well as for forskolin, dibutyryl-cAMP and theophylline. We also determined concentration-effect curves to the -adrenoceptor agonists in the presence of ICI 118,551 and CGP 20712A (i.e., in the presence of a selective ₂- and ₁-adrenoceptor antagonist, respectively). Apparent pA₂-values were calculated to determine which -adrenoceptor subtype causes vasodilation. These experiments indicate that -adrenoceptor-mediated vasodilation involves both ₁- and ₂-adrenoceptors in mesenteric resistance vessels of both hyperthyroid and control rats. In our experiments hyperthyroidism has a sensitizing influence on vascular responses induced by the -adrenoceptor agonist isoproterenol and the selective ₂-adrenoceptor agonist salbutamol. Sensitization to isoproterenol was abolished in the presence of ICI 118,551, whereas it was emphasized in the presence of CGP 20712A. Although this was not fully supported by the results obtained with noradrenaline, these results indicate that the sensitization to -adrenoceptor agonists is probably limited to the ₂-adrenoceptor/G-protein complex and not associated with alterations of the corresponding second messenger system.     

（—）Epicatechin induces and modulates endotheliumdependent relaxation in isolated rat mesenteric artery rings

AIM:The present study was aimed to examine the role of endothelial nitric oxide in the relaxant response to green tea (-)epicatechin and its modulation of endothelium-mediated relaxation in the isolated rat mesenteric artery rings.METHODS:Changes in the isometric tension were measured with Grass force-displacement transducers.RESULTS:The (-)epicatechin-induced relaxation was largely dependent on the presence of intact endothelium and was reversed by N^G-nitro-L-arginine methyl ester 10μmol/L or methylene blue 10μmol/L,the inhibitors of nitric oxidemediated relaxation.L-Arginine at 1mmol/L antagonized the effect of L-NAME or methylene blue.Pretreatment of endothelium-intact rings with (-)epicatechin 10μmol/L enhanced the relaxation induced by endothelium-dependent vasodilator,acetylcholine,while this concentration did not influence the endothelium-independent relaxation induced by sodium nitroprusside in the endothelium-denuded artery rings.CONCLUSION:The results indicate that the endothelium-dependent vasodilation by (-)epicatechin is mainly mediated through nitric oxide and low concentration of (-)epicatechin augments endothelium-dependent vasorelaxation in the rat mesenteric arteries.     

Acute potentiating effects of estrogens on endothe- lium-independent relaxation in porcine coronary artery rings

AIM: While the risk for coronary artery disease is reducedfollowing exposure to estrogens, the mechanisms of protectionhave yet to be fully elucidated. We recently found that short-term exposure to a physiological concentration (1 nmol·L~(-1)) of17β-estradiol potentiated endothelium-independent relaxation. Wetherefore sought to determine if estrone, another naturallyoccurring estrogen and diethylstilbestrol, a synthetic non-steroidalestrogen have similar enhancing effects. METHODS: Porcinecoronary artery rings were incubated with vehicle (ethanol), 17β-estradiol (1 nmol·L~(-1)), estrone (1 nmol·L~(-1)-1μmol·L~(-1)) or     

A role for cyclooxygenase in aging-related changes of β-adrenoceptor-mediated relaxation in rat aortas

β-Adrenoceptor-mediated vasorelaxation decreases with age in various vascular beds. The present study investigated the roles of cyclooxygenase (COX) on β-adrenoceptor vasorelaxation by isoprenaline in 8- and 54-week-old rat aortas. The vasorelaxation responses by isoprenaline (0.03–3 μM) were significantly reduced in 54-week-old aortas compared to 8-week. Addition of the non-selective COX inhibitors indomethacin (10 μM) or aspirin (10 μM) restored isoprenaline vasorelaxation of 54-week-old aortas to levels found in 8-week-old aortas. This suggests the involvement of COX prostanoids in the age-related reduction of β-adrenoceptor vasorelaxation. Immunohistochemistry revealed greater levels of COX-1 and COX-2 staining in 54-week-old aortas compared to 8-week with expression located mainly in medial smooth muscle. An age-linked increase in COX-1 and COX-2 protein was found in cremaster arterioles of 54-week-old rats (compared to 8-week) mainly in the endothelial layer. The age-related increase in COX-1 and COX-2 protein led to elevation of prostacyclin (measured as 6-keto prostaglandin ) and thromboxane A₂ (measured as thromboxane B₂) in 54-week compared to 8-week-old aortas. Endothelium removal in 54-week aortas markedly reduced the 6-keto prostaglandin level, thus suggesting an endothelial source for elevated prostacyclin. These findings in combination with the effects of COX inhibitors suggest that the age-related decrease in β-adrenoceptor vasorelaxation by isoprenaline is due to an age-linked increase in COX expression, which elevates production of COX-derived vasoactive prostanoids.     

Dual vascular actions of cardiotoxin from cobra venom: initial transient relaxation followed by contraction

AIM: To study the mechanisms of action of cardiotoxin(CTX), a basic polypeptides purified from cobra venom, on rat aortic muscle contractility. METHODS: Pharmacological approach of vascular contractility study. RESULTS: CTX (10μmol·L~(-1))-induced transient relax     

Reduction with age in the relaxation to β-adrenoceptor agonists and other vasodilators in rat aorta

Summary We have examined the relaxation produced by the -adrenoceptor agonists isoprenaline and procaterol, and by sodium nitroprusside, forskolin and isobutyl-methylxanthine in KCl (40 mmol/1)-contracted aortae from 6 week, 3 month and 6 month old Sprague-Dawley rats. The maximum relaxation to all agents was greatest in aortae from 6 week old animals. The relaxation to the -adrenoceptor agonists was virtually abolished, and the relaxation to the other agents significantly reduced, in aortae from 3 and 6 month old animals. There were no significant differences in the response to any agent between aortae from 3 and 6 month old animals, so that the changes seen occurred in maturation from 6 week to 3 month. The reduced vasodilator response does not occur at the level of the -adrenoceptor or adenylate cyclase, since the response to sodium nitroprusside was also affected, but is a general reduction in the ability of the aorta to relax. Send offprint requests to J. R. Docherty at the above address     

Characterization of β-adrenoceptor-mediated relaxation signals in isolated pulmonary artery of Dahl salt-sensitive hypertensive and normotensive rats

1?Relaxant responses to isoprenaline (ISO) were studied in the pulmonary arteries of normotensive and hypertensive Dahl salt-sensitive rats. Rats were fed either a high-salt (4.0%) or low-salt (0.14%) diet for 5 weeks. Animals fed a high-salt diet (167/123±2/2 mmHg) had a significantly higher blood pressure compared to those fed a low-salt diet (127/87 ± 2/2 mmHg). 2?Isoprenaline-elicited relaxations were not significantly different in tissues from hypertensive compared to normotensive animals. Responses to ISO were significantly attenuated in denuded tissues and substantially more so in hypertensive compared to normotensive animals. While relaxant responses to ISO were resistant to inhibition by Nω-nitro-L-arginine methyl ester, indomethacin, glibenclamide or a combination of barium chloride and ouabain, they were inhibited by Rp-cAMP, anandamide and acidic buffer. The inhibitory impact of anandamide and acidic buffer was significantly greater in tissues from hypertensive vs. normotensive rats. 3?The resting membrane potential (Em) of smooth muscle cells was -67.0±0.7 mV (n=43 cells) and -66.6±0.8 mV (n=55 cells) in pulmonary arteries from hypertensive and normotensive rats, respectively. Isoprenaline produced hyperpolarization of E(m) which was significant in the blood vessels of hypertensive (-71.6±0.8 mV; n=29 cells) but not normotensive (-68.1±0.7 mV; n=49 cells) rats. 4?The endothelium plays a critical role in β-adrenoceptor-mediated relaxation but nitric oxide is not the mediator for the response. It is possible that the greater hyperpolarization caused by ISO in blood vessels from hypertensive compared to normotensive rats is mediated by activation of TASK-1 channels.     

Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in mouse aortic smooth muscle cells

Background and purpose:

Reduction of intracellular calcium ([Ca²⁺]_i) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca²⁺ mobilization during SMC contraction influenced NO efficacy.

Experimental approach:

Isometric contractions by depolarization (high potassium, K⁺) or α-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca²⁺]_i (Fura-2) were measured in aortic segments from C57Bl6 mice.

Key results:

Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K⁺ solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca²⁺]_i, which was suppressed by sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K⁺-stimulated segments did not parallel a decline of [Ca²⁺]_i. However, stimulation (BAY K8644) of L-type Ca²⁺ influx diminished, while inhibition (nifedipine, 1–100 nM) augmented the relaxing capacity of NO.

Conclusions and implications:

In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca²⁺ re-uptake into the SR and was prominent when intracellular Ca²⁺ was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca²⁺]_i, presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca²⁺ influx, and L-type Ca²⁺ channel blockers increase the vasodilator efficacy of NO.     

Amplified NO/cGMP-mediated relaxation and ryanodine receptor-to-BKCa channel signalling in corpus cavernosum smooth muscle from phospholamban knockout mice

BACKGROUND AND PURPOSE

Relaxation of corpus cavernosum smooth muscle (CCSM) is induced by NO. NO promotes the formation of cGMP, which activates cGMP-dependent protein kinase I (PKGI). The large conductance calcium-activated potassium (BK_Ca) channel is regarded as a major target of NO/cGMP signalling; however, the mechanism of BK_Ca activation remains unclear. The aim of the present study was to determine whether sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ release from the SR via ryanodine receptors (RyRs) is important for BK_Ca channel activation in response to NO/cGMP.

EXPERIMENTAL APPROACH

In vitro myography was performed on CCSM strips from wild-type and PLB knockout (PLB^−/−) mice to evaluate contraction and relaxation in response to pharmacological agents and electrical field stimulation (EFS).

KEY RESULTS

In CCSM strips from PLB^−/− mice, a model of increased SR Ca²⁺ load, contractile force in response to EFS or phenylephrine (PE) was increased by nearly 100%. EFS of strips precontracted with PE induced transient relaxation in CCSM, an effect that was significantly larger in PLB^−/− strips. Likewise, the relaxation of PE-induced contraction in response to SNP and cGMP was greater in PLB^−/−, as demonstrated by a shift in the concentration–response curve towards lower concentrations. Blocking RyRs and BK_Ca channels diminished the induced relaxations and eliminated the difference between wild-type and PLB^−/−.

CONCLUSIONS AND IMPLICATIONS

NO/cGMP activates BK_Ca channels through RyR-mediated Ca²⁺ release. This signalling pathway is responsible for approximately 40% of the NO/cGMP effects and is amplified by increased SR Ca²⁺ concentrations.     

Vascular actions of calcimimetics: role of Ca²(+) -sensing receptors versus Ca²(+) influx through L-type Ca²(+) channels

BACKGROUND AND PURPOSE

The calcimimetic, (R)-N-(3-(3-(trifluoromethyl)phenyl)propyl)-1-(1-napthyl)ethylamine hydrochloride (cinacalcet), which activates Ca²⁺-sensing receptors (CaR) in parathyroid glands, is used to treat hyperparathyroidism. Interestingly, CaR in perivascular nerves or endothelial cells is also thought to modulate vascular tone. This study aims to characterize the vascular actions of calcimimetics.

EXPERIMENTAL APPROACH

In rat isolated small mesenteric arteries, the relaxant responses to the calcimimetics, cinacalcet and (R)-2-[[[1-(1-naphthyl)ethyl]amino]methyl]-1H-indole hydrochloride (calindol) were characterized, with particular emphasis on the role of CaR, endothelium, perivascular nerves, K⁺ channels and Ca²⁺ channels. Effects of L-ornithine, which activates a Ca²⁺-sensitive receptor related to CaR (GPRC6A), were also tested.

KEY RESULTS

Cinacalcet induced endothelium-independent relaxation (pEC₅₀ 5.58 ± 0.07, E_max 97 ± 6%) that was insensitive to sensory nerve desensitization by capsaicin or blockade of large-conductance Ca²⁺-activated K⁺ channels by iberiotoxin. Calindol, another calcimimetic, caused more potent relaxation (pEC₅₀ 6.10 ± 0.10, E_max 101 ± 6%), which was attenuated by endothelial removal or capsaicin, but not iberiotoxin. The negative modulator of CaR, calhex 231 or changes in [Ca²⁺]_o had negligible effect on relaxation to both calcimimetics. The calcimimetics relaxed vessels precontracted with high [K⁺]_o and inhibited Ca²⁺ influx in endothelium-denuded vessels stimulated by methoxamine, but not ionomycin. They also inhibited contractions to the L-type Ca²⁺ channel activator, BayK8644. L-ornithine induced small relaxation alone and had no effect on the responses to calcimimetics.

CONCLUSION AND IMPLICATIONS

Cinacalcet and calindol are potent arterial relaxants. Under the experimental conditions used, they predominantly act by inhibiting Ca²⁺ influx through L-type Ca²⁺ channels into vascular smooth muscle, whereas Ca²⁺-sensitive receptors (CaR or GPRC6A) play a minor role.     

The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium-dependent NO signalling

BACKGROUND AND PURPOSE

Obestatin is a recently discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation.

EXPERIMENTAL APPROACH

Cumulative relaxation responses to obestatin peptides were assessed in rat isolated aorta and mesenteric artery (n≥ 8) in the presence and absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC).

KEY RESULTS

Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1–23) were greater than those to obestatin(1–10) and obestatin(11–23). Obestatin(1–23)-induced relaxation was attenuated by endothelial denudation, l-NAME (NOS inhibitor), high extracellular K⁺, GDP-β-S (G-protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK_Ca blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked GPCR, PI3K/PKB, Ca²⁺-dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K⁺. Supporting data from BAEC indicated that nitrite production, intracellular Ca²⁺ and PKB phosphorylation were increased after exposure to obestatin(1–23). Relaxations to obestatin(1–23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarizing factors (EDHFs) and combined SK_Ca/IK_Ca blockade, suggesting that EDHF-mediated pathways were not involved.

CONCLUSIONS AND IMPLICATIONS

Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterized by endothelial dysfunction and cardiovascular complications.     

Insulin resistance in penile arteries from a rat model of metabolic syndrome

BACKGROUND AND PURPOSE

Metabolic and cardiovascular abnormalities accompanying metabolic syndrome, such as obesity, insulin resistance and hypertension, are all associated with endothelial dysfunction and are independent risk factors for erectile dysfunction. The purpose of the present study was to investigate the vascular effects of insulin in penile arteries and whether these effects are impaired in a rat model of insulin resistance and metabolic syndrome.

EXPERIMENTAL APPROACH

Penile arteries from obese Zucker rats (OZR) and their counterpart, lean Zucker rats (LZR), were mounted on microvascular myographs and the effects of insulin were assessed in the absence and presence of endothelium and of specific inhibitors of nitric oxide (NO) synthesis, phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK). Insulin-induced changes in intracellular Ca²⁺ concentration [Ca²⁺]_i were also examined.

KEY RESULTS

OZR exhibited mild hyperglycaemia, hypercholesterolemia, hypertryglyceridemia and hyperinsulinemia. Insulin induced endothelium- and NO-dependent relaxations in LZR that were impaired in OZR. Inhibition of PI3K reduced relaxation induced by insulin and by the β-adrenoceptor agonist isoprenaline, mainly in arteries from LZR. Antagonism of endothelin 1 (ET-1) receptors did not alter insulin-induced relaxation in either LZR or OZR, but MAPK blockade increased the responses in OZR. Insulin decreased [Ca²⁺]_i, a response impaired in OZR.

CONCLUSIONS AND IMPLICATIONS

Insulin-induced relaxation was impaired in penile arteries of OZR due to altered NO release through the PI3K pathway and unmasking of a MAPK-mediated vasoconstriction. This vascular insulin resistance is likely to contribute to the endothelial dysfunction and erectile dysfunction associated with insulin resistant states.     

N-arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium-activated potassium channels

Background and purpose:

N-arachidonoyl glycine (NAGly) is an endogenous lipid that is structurally similar to the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). While NAGly does not activate cannabinoid receptors, it exerts cannabimimetic effects in pain regulation. Here, we have determined if NAGly, like anandamide, modulates vascular tone.

Experimental approach:

In rat isolated small mesenteric arteries, the relaxant responses to NAGly were characterized. Effects of N-arachidonoyl serine and N-arachidonoyl γ-aminobutyric acid were also examined.

Key results:

In endothelium-intact arteries, NAGly-induced relaxation (pEC_50%= 5.7 ± 0.2; relaxation at 30 µM = 98 ± 1%) was attenuated by l-NAME (a nitric oxide synthase inhibitor) or iberiotoxin [selective blocker of large conductance Ca²⁺-activated K⁺ channels (BK_Ca)], and abolished by high extracellular K⁺ concentration. Endothelial removal reduced the potency of NAGly, and the resultant relaxation was inhibited by iberiotoxin, but not l-NAME. NAGly responses were sensitive to the novel cannabinoid receptor antagonist O-1918 independently of endothelial integrity, whereas pertussis toxin, which uncouples G_i/o proteins, attenuated NAGly relaxation only in endothelium-intact arteries. Treatments with antagonists for CB₁, CB₂ and TRPV1 receptors, or inhibitors of fatty acid amide hydrolase and COX had no effect. The two other arachidonoyl amino acids also induced iberiotoxin- and L-NAME-sensitive relaxations.

Conclusion and implications:

NAGly acts as a vasorelaxant predominantly via activation of BK_Ca in rat small mesenteric arteries. We suggest that NAGly activates an unknown G_i/o-coupled receptor, stimulating endothelial release of nitric oxide which in turn activates BK_Ca in the smooth muscle. In addition, NAGly might also activate BK_Ca through G_i/o- and nitric oxide-independent mechanisms.     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Doxorubicin-induced vasomotion and [Ca^2＋]i elevation n vascular smooth muscle cells from C57BL/6 mice

Aim:

To explore the action of doxorubicin on vascular smooth muscle cells.

Methods:

Isometric tension of denuded or intact thoracic aortic vessels was recorded and [Ca²⁺]_i in isolated aortic smooth muscle cells was measured by using Fluo-3.

Results:

Doxorubicin induced phasic and tonic contractions in denuded vessels and increased levels of [Ca²⁺]_i in single muscle cells. Treatment with 10 μmol/L ryanodine had no effect on basal tension, but it did abolish doxorubicin-induced phasic contraction. Treatment with 10 mmol/L caffeine induced a transient phasic contraction only, and the effect was not significantly altered by ryanodine, the omission of extracellular Ca²⁺ or both. Phenylephrine induced rhythmic contraction (RC) in intact vessels. Treatment with 100 μmol/L doxorubicin enhanced RC amplitude, but 1 mmol/L doxorubicin abolished RC, with an increase in maximal tension. Caffeine at 100 μmol/L increased the frequency of the RC only. In the presence of 100 μmol/L caffeine, however, 100 μmol/L doxorubicin abolished the RC and decreased its maximal tension. Treatment with 10 μmol/L ryanodine abolished the RC, with an increase in the maximal tension. In Ca²⁺-free solution, doxorubicin induced a transient [Ca²⁺]_i increase that could be abolished by ryanodine pretreatment in single muscle cells. The doxorubicin-induced increase in [Ca²⁺]_i was suppressed by nifedipine and potentiated by ryanodine and charybdotoxin.

Conclusion:

Doxorubicin not only releases Ca²⁺ from the sarcoplasmic reticulum but also promotes the entry of extracellular Ca²⁺ into vascular smooth muscle cells.     

Calcium signalling mediated through ��7 and non-��7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release

BACKGROUND AND PURPOSE

Ca²⁺ signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the α7 nAChR, in bovine chromaffin cells are still matters of debate.

EXPERIMENTAL APPROACH

We have used chromaffin cell cultures loaded with Fluo-4 or transfected with aequorins directed to the cytosol or mitochondria, several nAChR agonists (nicotine, 5-iodo-A-85380, PNU282987 and choline), and the α7 nAChR allosteric modulator PNU120596.

KEY RESULTS

Minimal [Ca²⁺]_c transients, induced by low concentrations of selective α7 nAChR agonists and nicotine, were markedly increased by the α7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the α7 nAChR antagonist α-bungarotoxin (α7-modulated-response). Conversely, high concentrations of the α7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca²⁺]_c transients, that were blocked by mecamylamine but were unaffected by α-bungarotoxin (non-α7 response). [Ca²⁺]_c increases mediated by α7 nAChR were related to Ca²⁺ entry through non-L-type Ca²⁺ channels, whereas non-α7 nAChR-mediated signals were related to L-type Ca²⁺ channels; Ca²⁺-induced Ca²⁺-release contributed to both responses. Mitochondrial involvement in the control of [Ca²⁺]_c transients, mediated by either receptor, was minimal. Catecholamine release coupled to α7 nAChRs was more efficient in terms of catecholamine released/[Ca²⁺]_c.

CONCLUSIONS AND IMPLICATIONS

[Ca²⁺]_c and catecholamine release mediated by α7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the α7 nAChR response was lost and the non-α7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of α7 nAChRs.     

5-hydroxytryptamine induced relaxation in the pig urinary bladder neck

Background and purpose

5-Hydroxytryptamine (5-HT) is one of the inhibitory mediators in the urinary bladder outlet region. Here we investigated mechanisms involved in 5-HT-induced relaxations of the pig bladder neck.

Experimental approach

Urothelium-denuded strips of pig bladder were mounted in organ baths for isometric force recordings of responses to 5-HT and electrical field stimulation (EFS).

Key results

After phenylephrine-induced contraction, 5-HT and 5-HT receptor agonists concentration-dependently relaxed the preparations, with the potency order: 5-carboxamidotryptamine (5-CT) > 5-HT = RS67333 > (±)-8-hydroxy-2-dipropylaminotetralinhydrobromide > m-chlorophenylbiguanide > α-methyl-5-HT > ergotamine. 5-HT and 5-CT relaxations were reduced by the 5-HT₇ receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulphonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride and potentiated by (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide dihydrochloride (WAY 100135) and cyanopindolol, 5-HT_1A and 5-HT_1A/1B receptor antagonists respectively. Inhibitors of 5-HT_1B/1D, 5-HT₂, 5-HT_2B/2C, 5-HT₃, 5-HT₄, 5-HT_5A and 5-HT₆ receptors failed to modify 5-HT responses. Blockade of monoamine oxidase A/B, noradrenergic neurotransmission, α-adrenoceptors, muscarinic and purinergic receptors, nitric oxide synthase, guanylate cyclase and prostanoid synthesis did not alter relaxations to 5-HT. Inhibitors of Ca²⁺-activated K⁺ and ATP-dependent K⁺ channels failed to modify 5-HT responses but blockade of neuronal voltage-gated Na⁺-, Ca²⁺-and voltage-gated K⁺ (K_v)-channels potentiated these relaxations. Adenylyl cyclase activation and cAMP-dependent protein kinase (PKA) inhibition potentiated and reduced, respectively, 5-HT-induced responses. Under non-adrenergic, non-cholinergic, non-nitrergic conditions, EFS induced neurogenic, frequency-dependent, relaxations which were resistant to WAY 100135 and cyanopindolol.

Conclusions and implications

5-HT relaxed the pig urinary bladder neck through muscle 5-HT₇ receptors linked to the cAMP-PKA pathway. Prejunctional 5-HT_1A receptors and K_v channels modulated 5-HT-induced relaxations whereas postjunctional K⁺ channels were not involved in such responses. 5-HT₇ receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency.