Mechanisms of NO-resistant relaxation induced by acetylcholine in rabbit renal arteries

The effects of K+ channel blockers and P2Y receptor agonist/antagonist on the vasorelaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) were investigated in the rabbit renal artery. Acetylcholine (ACh, 1 nM-10 microM) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 microM) in a concentration-dependent manner. NG-nitro-L-arginine (L-NAME. 0.1 mM), an inhibitor of NO synthase, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was only partially inhibited by L-NAME whereas combined addition of L-NAME and 30 mM KCl completely inhibited the relaxation. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.1 microM) and apamin (1 microM), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 microM) in a concentration-dependent manner. Furthermore, ADPbetaS, a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue alone. In conclusion, ACh may activate the release of ATP from endothelial cells which in turn activates a P2Y receptor on the endothelial cells followed by a release of EDHF, resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. EY WORDS: ATP, K+ channel, rabbit renal artery.     

Mechanism of carbon monoxide-induced relaxation in the guinea pig ileal smooth muscle

The mechanism of carbon monoxide (CO)-induced relaxation were investigated in the guinea-pig ileum. CO (10%) inhibited the 40 mM KCl-induced contraction. This effect was antagonized by ODQ (1 microM), a soluble guanylate cyclase inhibitor. In contrast, CO did not inhibit the 40 mM KCl-induced increase in cytosolic Ca2+ level ([Ca2+]i). Cumulative addition of KCl induced a graded increase in both [Ca2+]i and muscle tension. In the presence of CO, the increase in muscle tension was attenuated whereas the increase in [Ca2+]i was only slightly decreased. Thus, the [Ca2+]i-tension relationship constructed by cumulative addition of KCl shifted downwards in the presence of CO. Using the patch clamp, CO was found to have little effect on the peak Ba currents (I(Ba)) when voltage was stepped from -60 mV to 0 mV. From these results, we conclude that CO inhibits contraction of guinea-pig ileum mainly by the decrease in the sensitivity of contractile elements to Ca2+ via a cyclic GMP-dependent pathway but not by the inhibition of L-type Ca2+ channel.     

Possible involvement of K+ channel opening to the interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.

We have previously shown that interleukin-1 beta relaxes vascular smooth muscle by the NO-dependent and independent mechanisms (Takizawa et al.: Eur. J. Pharmacol. 330: 143-150, 1997). In this study, we investigated the mechanism of NO-independent relaxation. Treatment of the rat aorta with interleukin-1 beta for 24 hr inhibited the high-K+ induced contraction by decreasing cytosolic Ca2+ level ([Ca2+]i). The relationship between [Ca2+]i and tension in intact muscle and the pCa-tension curves in permeabilized muscle suggested that Ca2+ sensitivity of contractile element was not changed after the interleukin-1 beta-treatment. After a treatment with interleukin-1 beta for 24 hr, contractile effects of phenylephrine (1 microM-10 microM) were markedly inhibited in the presence of L-NMMA (100 microM) applied to inhibit NO synthesis. A blocker of ATP-sensitive K+ channel, glibenclamide (1 microM), partially recovered the interleukin-1 beta-induced inhibition. In contrast, a blocker of Ca(2+)-activated K+ channel, charybdotoxin (0.1 microM), was ineffective. These results suggest that membrane hyperpolarization due to activation of ATP-sensitive K+ channels may partly be responsible for the NO-independent mechanism of interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.     

Effects of high-K+, Na+-deficient solution on contractility of the smooth muscles of the bovine trachea

A high-K+, Na+-deficient (I-154 K+) solution induced contraction followed by gradual relaxation of the smooth muscles of the bovine trachea, while hyperosmotic addition of 65 mM KCl induced a large sustained contraction. Exposure of the muscle to the I-154 K+ solution induced an increase in the ratio of cellular water content and a sustained increase in oxidized flavoprotein fluorescence or reduced pyridine nucleotide fluorescence. The I-154 K+ solution also induced a sustained increase in [Ca2+]i level. Decreases in developed tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl but not pyruvate. Substitution of KI for KCl in the I-154 K+ solution produced a greater inhibition of contraction, while substitution with K-propionate produced no inhibition of contraction. Moreover, decreases in developed tension and increases in cellular water content were both prevented by addition of 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), but not 10 microM bumetanide or 1 mM acetazolamide. In conclusion, I-154 K+ solution induced-relaxation in the bovine trachea may be due to swelling of smooth muscle cells and the mechanism of swelling is probably involved in DIDS-sensitive anion movement.     

Capsaicin-induced relaxation in rabbit coronary artery.

In the present study mechanism of inhibitory effects of capsaicin on the contractility of rabbit coronary artery were studied by measurement of isometric tension and intracellular Ca2+ concentration. Capsaicin (1 microM to 30 microM) relaxed the coronary artery pre-contracted with prostaglandin (PG) F2alpha (1 microM) in a concentration-dependent manner. The PGF2alpha-induced increase in intracellular Ca2+ concentration was also inhibited. The effects of capsaicin were readily reversed by washing capsaicin from the bath. Capsaicin-induced relaxation was not attenuated by pretreatment with capsazepine (1 microM), a blocker of vanilloid receptor or ruthenium red (1 microM), a blocker of non-selective cation channel. Previous exposure to a high concentration of capsaicin (100 microM) or repeated application of capsaicin did not eliminate the relaxation response to subsequent application of capsaicin. Increasing the external K+ concentration to 80 mM significantly attenuated the capsaicin-induced relaxation with simultaneous change in intracellular Ca2+ concentration. Pretreatment with iberiotoxin (100 nM), a blocker of Ca2+-activated K+ channel, only partially inhibited the capsaicin-induced relaxation. However, application of 4-aminopyridine (4-AP, 1 mM), a blocker of delayed rectifier K+ current significantly inhibited the capsaicin-induced relaxation with concomitant attenuation of the effect on intracellular Ca2+ concentration. These results indicate that capsaicin may have a direct relaxing effect on the smooth muscle contractility, and relaxation may be due to activation of the 4-AP-sensitive, delayed rectifier K+ channels in the rabbit coronary artery.     

Effects of substance P on nicotine-induced intracellular Ca2+ dynamics in bovine adrenal chromaffin cells

Substance P (SP) is colocalized with ACh in splanchnic nerves that innervate into adrenal medulla and the peptide has been shown to inhibit nicotinic agonists-induced catecholamine secretion. To elucidate the effects of SP on cytosolic Ca2+ dynamics, the present study was conducted using fura-2-loaded isolated bovine adrenal chromaffin cells. Stimulation of the cells with nicotine (10-100 microM) produced a rapid rise of cytosolic Ca2+ concentration ([Ca2+]i), the peak level of which increased in a dose-dependent manner, followed by a gradual decay. In the presence of 10 microM SP, the dose-response relationship of the peak levels shifted downward. Quantitative analyses implied that SP inhibits the nicotine-induced Ca2+ influx in a noncompetitive manner. Nicotinic acetylcholine receptor is composed of two major functional domains: an agonist-binding site and an ionophore or channel domain. Agonist binding activates ionophore/channel domain and causes mainly Na+ influx. This Na+ influx depolarizes the cell and activates voltage-dependent Ca2+ channels. Based on this fact, the present results indicate that SP dose not block nicotine binding sites but interferes with other sites of nicotinic receptor/channel molecule, most probably a channel domain. It was suggested that SP colocalized with ACh in splanchnic nerves functions as a physiological modulator of catecholamine secretion by non-competitively suppressing ACh-induced cytosolic Ca2+ dynamics in bovine adrenal chromaffin cells.     

Digoxin- and monensin-induced changes of intracellular Ca2+ concentration in isolated guinea-pig ventricular myocyte

This study was undertaken to determine the possible mechanisms of actions of monensin and digoxin by using isolated guinea-pig ventricular myocytes. Since Ca2+ is the major signal for triggering contraction of cardiac muscle, the objective of this study was to determine whether monensin and digoxin affect the [Ca2+]i of cardiac myocytes and if so is this effect due to an increase in [Na+]i. Three different concentrations of digoxin (0.3, 1 and 3 micromol/l) and three different concentrations of monensin (0.3, 1 and 3 micromol/l) were used. Each treatment was monitored for two hours by using computerized fluoroscopy. Both digoxin and monensin increased the [Ca2+]i and accelerated the onset time of [Ca2+]i increase in a dose-dependent manner. Normal myocytes (loaded with fura-2 for 30 min before the treatment) were also compared with 'weakened' myocytes (loaded with fura-2 for 3 h before the treatment to create a 'weakened' condition). It was found that although 0.3 micromol/l monensin and digoxin did not change the [Ca2+]i in normal myocytes, they increased the [Ca2 +]i in 'weakened' myocytes. Finally, a Na+-free medium was used to demonstrate the effect of [Na+]o on both monensin- and digoxin-induced increases in [Ca2+]i. It was found that digoxin did not increase the [Ca2+]i in the Na+-free medium. Although monensin increased the [Ca2+]i in the Na+-free solution, this increase was not as large as in the Na+-containing medium. The results of the study led to the conclusion that the positive inotropic effect of digoxin depends on [Na+]o. However, monensin increases [Ca2+]i in Na+-dependent and -independent ways. An addition conclusion was that 'weakened' myocytes are more sensitive to the monensin and digoxin treatment than normal myocytes.     

Mechanisms responsible for reduced cardiotoxicity of mitoxantrone compared to doxorubicin examined in isolated guinea-pig heart preparations

We reported previously that doxorubicin, an anticancer agent that has an anthracycline structure, alters Ca2+ releasing and uptake mechanisms in the sarcoplasmic reticulum of myocardial cells. These effects of doxorubicin are apparently related to its cardiotoxicity. Mitoxantrone is a similar anticancer agent with an anthracenedion structure that has been shown to be significantly less cardiotoxic. In the present study, the effects of mitoxantrone on the functions of the sarcoplasmic reticulum were examined in isolated muscle preparations obtained from the guinea-pig heart. In electrically-stimulated left atrial muscle preparations, incubation in vitro for 4 hr with 30 or 100 microM mitoxantrone significantly prolonged the time to the peak of twitch tension, markedly increased the developed tension observed at lower stimulation frequencies, thereby attenuating the slope of positive force-frequency relationships, and increased the postrest contraction observed after a 60-sec quiescent period. In myocytes isolated from ventricular muscles, 30 microM mitoxantrone increased the peak and the size of intracellular Ca2+ concentrations ([Ca2+] i), and prolonged the time to peak [Ca2+]i. In skinned muscle fiber preparations obtained from the left ventricular muscle, 30 muM mitoxantrone significantly increased the caffeine-induced contraction without affecting the Ca2+ sensitivity of contractile proteins. These results suggest that mitoxantrone enhances Ca2+ release from the sarcoplasmic reticulum in isolated atrial muscle preparations obtained from the guinea-pig heart. Apparent enhancement of the sarcoplasmic reticulum functions, in contrast to anthracyclines that has been shown to suppress these functions, seems to explain the relative lack of marked cardiotoxicity of mitoxantrone.     

Alpha1-adrenoceptor-mediated negative inotropic effect caused by intracellular ionic activities in guinea-pig papillary muscle

In guinea-pig papillary muscle, phenylephrine (PE), an agonist of alpha1-adrenoceptor (alpha1-AR), led to a transient negative inotropic effect (NIE) and a subsequent sustained positive inotropic effect (PIE). To clarify the ionic mechanisms underlying the NIE, we measured the [Na+]i or [pH]i by ion-selective microelectrodes. PE produced a decrease in the intracellular Na+ concentration ([Na+]i) and an increase in intracellular pH ([pH]i). During the phase of NIE, PE produced only a (-) change of [Na+]i (Delta[Na+]i). With a decrease in extracellular Na+ or an increase in extracellular Ca2+, the PE-induced NIE was attenuated and PE produced (+)Delta[Na+]i. The PE-induced NIE and (-)Delta[Na+]i were definitely strengthened by lowering the bath temperature or increasing the stimulation frequency. 2-(2,6-di-methoxyphenoxyethyl)amino-methyl-1,4-benzidioxane HCl, an antagonist of alpha1A-AR, completely abolished the PE-induced NIE and (-)Delta[Na+]i. Phorbol 12,13-dibutyrate, an activator of protein kinase C (PKC), decreased the baseline [Na+]i and twitch force and increased the baseline [pH]i in mimicry of PE. Pretreatment with 1-5(isoquinolinesulphonyl)-2-methylpiperazine, an inhibitor of PKC, abolished the PE-induced NIE and (-)Delta[Na+]i. During pretreatment with benzamil, an inhibitor of Na+/Ca2+ exchange, we found that the PE-induced NIE and (-)Delta[Na+]i were reversibly abolished. Our results indicate the PE-induced NIE may be elicited upon the activation of Na+/Ca2+ exchange which can be attributed to the (-)Delta[Na+]i. (-)Delta[Na+]i is mediated through a PKC-dependent pathway via an activation of alpha1A-AR subtype and its effect could be strengthened remarkably at high [Na+]i and [Ca2+]i values.     

Photodynamic stimulation causes sustained increase in intracellular calcium concentration in cells of small cell lung carcinoma

Photodynamic agents, due to their selective uptake by tumor cells and photon-dependent selective activation, have immense implications for cancer treatment. The present study provided direct evidence that the photon activation of chloro-aluminum phthalocyanine sulphonate (A1PcS4) in the presence of extracellular Ca2+ caused a rapid increase followed by a sustained increase in intracellular concentration of calcium ion ([Ca2+]i) in a small cell lung carcinoma (SCLC) cell line, SBC-3. The [Ca2+]i increase by photodynamic stimulation was completely inhibited by the removal of extracellular Ca2+ and reintroduction of extracellular Ca2+ immediately led to a rapid elevation of [Ca2+]i. However, the increase was not inhibited by application of Ni2+, nifedipine, or SK&F 96365, a receptor-mediated and voltage-dependent Ca2+ entry blocker. The photosensitizer A1PcS4 alone or light alone (4 min) had no effect on [Ca2+]i. Cytotoxicity examination by trypan blue exclusion test, however, suggested photodynamic stimulation-induced cell injury which was observed in both the presence and the absence of extracellular Ca2+. These results indicate that [Ca2+]i increase may not be mandatory for photodynamic stimulation-induced cell injury. Whether [Ca2+]i increase can accelerate, at least in part, cell death under the physiological condition, whether the mechanism(s) of cell death can be different in the presence and the absence of extracellular Ca2+, and whether [Ca2+]i increase can be totally unrelated to cell death await further work.     

Homeostasis of intracellular Ca2+ in equine chondrocytes: response to hypotonic shock

REASONS FOR PERFORMING STUDY: Ca2+ homeostasis in articular chondrocytes affects synthesis and degradation of the cartilage matrix, as well as other cellular functions, thereby contributing to joint integrity. Although it will be affected by mechanical loading, the sensitivity of intracellular Ca2+ concentration ([Ca2+]i) in equine articular chondrocytes to many stimuli remains unknown. HYPOTHESIS: An improved understanding of Ca2+ homeostasis in equine articular chondrocytes, and how it is altered during joint loading and pathology, will be important in understanding how joints respond to mechanical loads. METHODS: [Ca2+]i was determined using the fluorophore fura-2. We examined the effects of hypotonic shock, a perturbation experienced in vivo during mechanical loading cycles. We used inhibitors of Ca2+ transporters to ascertain the important factors in Ca2+ homeostasis. RESULTS: Under isotonic conditions, [Ca2+]i was 148 +/- 23 nmol/l, increasing by 216 +/- 66 nmol/l in response to reduction in extracellular osmolality of 50%. Resting [Ca2+]i, and the increase following hypotonic shock, were decreased by Ca2+ removal; they were both elevated when extracellular [Ca2+] ([Ca2+]o) was raised or following Na+ removal. The hypotonicity-induced rise in [Ca2+]i was inhibited by exposure of cells to gadolinium (Gd3+; 10 micromol/l), an inhibitor of mechanosensitive channels. [Ca2+]i was also elevated following treatment of cells with thapsigargin (10 micromol/l), an inhibitor of the Ca2+ pump of intracellular stores. CONCLUSIONS: A model is presented which interprets these findings in relation to Ca2+ homeostasis in equine articular chondrocytes, including the presence of mechanosensitive channels allowing Ca2+ entry, a Na+/Ca2+ exchanger for removal of intracellular Ca2+ and intracellular stores sensitive to thapsigargin. POTENTIAL RELEVANCE: A more complete understanding of Ca2+ homeostasis in equine chondrocytes may allow development of future therapeutic regimes to ameliorate joint disease.     

Photodynamic action inhibits compound 48/80-induced exocytosis in rat peritoneal mast cells.

Photostimulation of sulfonated aluminum phthalocyanine (SALPC)-loaded mast cells (20,000 lux, 2 min) itself caused neither exocytosis nor [Ca2+]i increase in isolated rat peritoneal mast cells. This result is incompatible with that reported in other cell types such as pancreatic acinar cells. Stimulation with 50 micrograms/ml compound 48/80, a direct G-protein activator, induced massive exocytosis which was easily detectable under conventional microscope. The fluorescent granules stained with sulforhodamine B were found to be numerous on the perimetry of mast cells, confirming occurrence of exocytosis. The stimulation also increased [Ca2+]i and cell volume before initiation of exocytosis. Pretreatment of the cells with photodynamic action with 5 microM SALPC inhibited the compound 48/80-induced exocytosis, but the [Ca2+]i increase and the increase of cell volume were unaffected. NaN3 at 0.5 mM could relieve the photodynamic action-induced inhibition of exocytosis. These results indicate that, unlikely to other secretory or contractile cells, photodynamic action with SALPC does not directly affect exocytotic machinery but modulates some functional proteins involved in signal transduction process which may be posterior to G-protein activation in mast cells. Singlet oxygen may be involved in the photodynamic action-induced modulation. A possible target protein can be a protein in the cell membrane which binds with a protein of a granular membrane during the course of exocytosis.     

Cdc42 contributes to phorbol ester-induced Ca2+-independent contraction of pulmonary artery smooth muscle

We determined the contribution of the Rho family of low molecular GTP-binding proteins to phorbol ester-induced contraction in swine pulmonary artery smooth muscle. In Ca2+-free medium containing 1 mM EGTA, 12-deoxyphorbol 13-isobutyrate (DPB, 1 microM), a protein kinase C (PKC) activator, elicited sustained contractions, which were not inhibited by treatment with verapamil, a voltage-dependent Ca2+ channel antagonist, and Y27632, a Rho-associated kinase inhibitor. Immunoblot analysis showed three PKC isoforms (alpha, epsilon, and zeta) and two Rho GTPases (RhoA and Cdc42) in both cytosolic and the membrane fractions from quiescent strips. DPB (1 microM) significantly induced PKCalpha and epsilon to translocate from the cytosolic to the membrane fraction in Ca2+-free medium. DPB also elicited the translocation of Cdc42, but not RhoA to the membrane fraction. Similarly, in the experiment for measurement of Rho GTPase activity by pull-down assay, DPB (1 microM) significantly increased the activity of Cdc42 in Ca2+-free medium. Norepinephrine (NE, 10 microM) stimulated the redistribution of RhoA from the cytosolic to the membrane fraction in swine pulmonary artery smooth muscle. In contrast, NE did not alter the subcellular distributions of Cdc42 and the PKC isoforms. These results indicate that phorbol ester evokes PKC-mediated Ca2+-independent contraction via a Rho GTPase pathway, especially Cdc42, in smooth muscle from swine pulmonary arteries.     

Effects of induction of capacitative calcium entry on equine laminar microvessels

OBJECTIVE: To determine the effects of induction of capacitative Ca2+ entry on tone in equine laminar arteries and veins. SAMPLE POPULATION: Laminar arteries and veins from 6 adult mixed-breed horses. PROCEDURE: Arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Capacitative Ca2+ entry was induced by incubating the vessels with the specific Ca2+-ATPase inhibitor thapsigargin (100nM) in a Ca2+-free physiologic salt solution. Capacitative Ca2+ entry-associated contractile responses were determined by the subsequent addition of 2mM Ca2+ to the solution bathing the vessels; in some experiments, either the voltage-gated Ca2+ blocker diltiazem (10microM) or the putative capacitative Ca2+ entry inhibitor trifluoromethylphenylimidazole (300microM) was added to the bathing solution 15 minutes prior to a second 2mM Ca2+ exposure. The Sr2+ permeability of the capacitative Ca2+ entry pathway in laminar vessels was assessed by exposing the vessels to 4mM Sr2+ after induction of capacitative Ca2+ entry with thapsigargin. RESULTS: Induction of capacitative Ca2+ entry elicited robust contractile responses in laminar veins but did not increase tone in laminar arteries. In laminar veins, capacitative Ca2+ entry-induced contractile responses were unaffected by preincubation with diltiazem, attenuated by trifluoromethylphenylimidazole, and were impermeable to Sr+. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that induction of capacitative Ca2+ entry elicits vasoconstriction in equine laminar veins but not in laminar arteries and should therefore be considered a potential mechanism by which selective venoconstriction occurs in horses during the development of acute laminitis.     

The effect of progesterone on oxytocin-stimulated intracellular Ca2+ mobilisation and prostaglandin secretion in porcine endometrium

We have studied in the porcine endometrium the expression of oxytocin receptor (OTR) mRNA and the effect of progesterone (P4) on oxytocin/oxytocin receptor (OT/OTR) function concerning intracellular Ca2+ mobilisation ([Ca2+]i), prostaglandin F2alpha (PGF2alpha) and E2 (PGE2; PG) secretion. Tissue was taken from cyclic and early pregnant pigs (days 14-16). A higher expression of OTR mRNA (P < 0.05) was observed in the endometrium of cyclic than pregnant pigs. The stimulatory (P < 0.05) effect of OT (10(-7) M) on [Ca2+]i mobilisation was noticed within 15-60 s and 30-60 s in endometrial stromal cells of cyclic and pregnant pigs, respectively. In the presence of P4 (10(-5) M) basal and OT-stimulated [Ca2+]i concentrations decreased in stromal cells during luteolysis and pregnancy. In stromal cells P4 delayed mobilisation of [Ca2+]i in response to OT by 15 s during luteolysis and had no effect during pregnancy. In cyclic and pregnant epithelial cells OT stimulated mobilisation of [Ca2+]i in 45 s and 60 s, respectively. Oxytocin increased (P < 0.05) PGF2alpha secretion during luteolysis and pregnancy and PGE2 during luteolysis from endometrial slices. Progesterone did not inhibit this stimulatory effect. During luteolysis OT increased (P < 0.05) PGF2alpha in epithelial and stromal cells and PGE2 secretion in epithelial cells. In the presence of P4 this effect of OT was reduced only in stromal cyclic cells (6 h culture). The presence of P4 decreased the effect of OT on [Ca2+]i mobilisation only in stromal cells. We found that, in most conditions, P4 did not inhibit the OT-stimulated secretion of PG in the porcine endometrium.     

Characterization of muscarinic receptor subtype mediating contraction and relaxation in equine coronary artery in vitro

In coronary arterial rings isolated from horses, 10-^-8-10^-6 mol/l acetylcholine (ACh) induced concentration-dependent contractions which were potentiated by the removal of endothelium and by pretreatment with I,-nitro-arginine (LNAG) or methylene blue (MB). Relatively lower concentrations of Ach 10-¹⁴-10-⁸ mol/l) induced relaxation when the coronary rings were contracted by phenylephrine (PE). ACh-induced contractions in the coronary rings without endothelium were competitively inhibited by each muscarinic subtype selective antagonist in the following order of potency: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine ≥ parafluoro-hexahydrosiladiphenidol (pFHHSiD) > methoctramine. ACh-induced relaxation in the rings with endothelium was inhibited by LNAG or MB, and by each selective antagonist in the following order of potency: 4-DAMP < pFHHSiD ≥ pirenzepine ≥ methoctramine. These results suggest that the ACh-induced contraction and relaxation in equine coronary arteries are mediated mainly by an M₃-receptor located on the smooth muscle cells and endothelial cells, respectively, and that the stimulation of the M₃-receptor on the endothelial cells liberates nitric oxide.     

Endothelium-dependent relaxation of canine uterine artery in response to acetylcholine: the possible involvement of alternative pathways

The effect of acetylcholine on the isolated, pre-contracted, uterine artery of non-pregnant dog was investigated. Acetylcholine-induced concentration-dependent relaxation of isolated canine uterine artery with endothelium (pEC50 = 6.48 +/-0.01, n = 37) and was without effect on arterial segments denuded of endothelium. Indomethacin, 4-aminopyridine (10-5 m) and pre-contraction with K+-rich Krebs-Ringer bicarbonate solution had no effect on acetylcholine-induced relaxation. NG-nitro-l-arginine (l-NOARG) (10-5 m) inhibited relaxation evoked by acetylcholine. Indomethacin applied with l-NOARG led to further inhibition of acetylcholine-induced relaxation. In the presence of both l-NOARG and indomethacin, 4-aminopiridine did not provoke further inhibition of acetylcholine-induced relaxation of canine uterine artery. It is concluded that the acetylcholine-induced relaxation of canine uterine artery is probably mediated by endothelial production of nitric oxide (NO). However, if NO-synthase is inhibited, acetylcholine-induced vasorelaxation may be, in part, mediated through activation of cyclooxygenase pathway.     

Nitric oxide stimulates IP3 production via a cGMP/ PKG-dependent pathway in rat pancreatic acinar cells

In an attempt to explore the functioning of nitric oxide (NO) in pancreatic exocrine cells, we have recently obtained several lines of circumstantial evidence indicating that one of molecular targets of NO is phospholipase C (PLC), the activation of which leads to an increase in the cytosolic Ca2+ concentration ([Ca2+]i) via inositol 1, 4, 5-trisphosphate, IP3. However, whether IP3 is actually produced by NO has not yet been substantiated. The present study was therefore designed to directly measure the intracellular IP3, concentration ([IP3]i) for better understanding of the underlying mechanisms with the help of pharmacological tools. [IP3]i was measured using a fluorescence polarization technique (HitHunter). We obtained the following results: 1) varying concentrations of an NO donor, sodium nitroprusside (SNP), elevated [IP3]i, 2) this elevation was completely inhibited in the presence of the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (ODQ), 3) varying concentrations of the cGMP analogue, 8-Br-cGMP, also increased [IP3]i, 4) the cGMP analogue-induced IP3 production was abolished by pretreatment with either a PLC inhibitor, U73122, or a G-protein inhibitor, GP2A, and 5) KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase G (PKG), also abolished the IP3 production induced by 8-Br-cGMP. These results suggest that the NO-induced [Ca2+]i increase is triggered by an increase in [IP3]i located downstream from intracellular cGMP elevation. In this intracellular pathway, each sGC, cGMP-dependent PKG, G-protein and PLC were suggested to be involved. The present work provides new insights into the intracellular signaling accelerated by NO. NO triggers a [Ca2+]I increase via cGMP and IP3 in pancreatic acinar cells.     

Tamoxifen induces vasorelaxation via inhibition of mitogen-activated protein kinase in rat aortic smooth muscle

The contribution of the mitogen-activated protein kinase (MAPK) pathway to the relaxation induced by tamoxifen, a synthetic non-steroidal anti-estrogen, was examined in rat vascular smooth muscle. Tamoxifen (0.1-300 microM) inhibited the contraction induced by endothelin-1 (ET-1, 3 nM) in aortic smooth muscle in a concentration-dependent manner. The inhibitory effect of tamoxifen was not attenuated by 10 microM ICI 182,780, a selective antagonist of estrogen receptors. In the Ca(2+) channel inhibitor verapamil (1 microM)-pretreated strips, tamoxifen also inhibited the contraction induced by ET-1. Both PD098059 and SB203580, inhibitors of MAPK/extracellular signal-regulated kinase (ERK) kinase and p38 MAPK, respectively, inhibited ET-1-induced contraction in aortic smooth muscle. In Western blot analysis with anti-phosphorylated MAPK antibodies, ET-1 (3 nM) enhanced activities of both ERK1/2 and p38 MAPK in aortic muscle strips, which were not attenuated by the treatment with 4 mM EGTA. Tamoxifen (100 microM) inhibited the activities of ERK1/2 and p38 MAPK induced by ET-1 without significant changes in the expression of these kinases. These results suggest that tamoxifen induces relaxation of rat vascular smooth muscle, and that this is, at least in part, mediated by the inhibition of the Ca(2+)-independent MAPK pathway.     

Short chain fatty acids stimulate feline colonic smooth muscle contraction

The effect of short chain fatty acids (SCFA) on feline colonic smooth muscle contraction was evaluated in vitro. Colonic tissue was obtained from seven healthy male and female adult cats and seven healthy male and female kittens. Longitudinal and circular colonic smooth muscle strips from proximal and distal colon were incubated with SCFA (acetate, butyrate and propionate; 1-100mM). SCFA-induced contractions were compared to responses obtained using maximal concentrations (10(-4)M) of acetylcholine (ACh). The calcium dependence of the SCFA response was investigated by incubating with nifedipine (1 microM) or verapamil (1 microM). Acetate, butyrate and propionate elicited isometric stress responses (0.25-1.98 x 10(4)N/m(2)) in longitudinal, but not circular, smooth muscle from both the proximal and distal colon of adult cats. Maximal responses were attained at 50 and 100mM SCFA. Maximal butyrate and propionate responses were 29 and 19% of the maximal ACh response (10(-4)M), respectively. Acetate was least effective in stimulating contractile responses. Nifedipine and verapamil abolished all responses. Contractile responses in kittens were similar to those observed in adult cats, but were smaller in amplitude.Results of these studies have shown that SCFA stimulate longitudinal colonic smooth muscle contractions in kittens and adult cats in vitro. These SCFA-induced contractions involve activation of calcium influx. These in vitro findings may account for some of the effects of dietary fiber on feline colonic motility in vivo.