Propionate-induced relaxation in rat mesenteric arteries: a role for endothelium-derived hyperpolarising factor.

Short chain fatty acids, including propionate, are generated in the caecum and large intestine, and when absorbed may elicit localised increases in intestinal blood flow. We sought to assess the mechanism by which propionate caused vasorelaxation. Propionate-mediated relaxation of noradrenaline-preconstricted rat mesenteric small arteries (RMSAs, i.d. 200-300 microm) was studied using small vessel myography. Propionate (1-30 mM) produced a concentration-dependent relaxation. Relaxation induced by 10 mM propionate (the approximate EC50) was almost abolished by endothelial denudation, although a marked relaxation to a very high concentration of propionate (50 mM) persisted in the absence of the endothelium. In endothelium-intact RMSAs, relaxation to 10 mM propionate was almost abolished by elevating [K+]o to 25 mM, but was unaffected by 100 microM N(omega)-nitro-L-arginine methyl ester (L-NAME) (68 +/- 4 vs. 66 +/- 3% in controls, n = 35), or by 1 microM indomethacin (60 +/- 4 vs. 61 +/- 7 % in controls, n = 15). In the presence of L-NAME, relaxation to 10 mM propionate was significantly and markedly (i.e. > 50 %) inhibited by 50 microM Ba2+ and by the combination of 100 nM charybdotoxin and 100 nM apamin. A similar effect on propionate-mediated relaxation was also exerted by 100 microM ouabain, and by the combination of 50 microM barium with ouabain. Relaxation was also significantly and markedly inhibited by pre-treatment of RMSAs with 100 nM thapsigargin or 10 microM cyclopiazonic acid (CPA). The results demonstrate that 10 mM propionate relaxes RMSAs via endothelium-derived hyperpolarising factor (EDHF). The observation that relaxation by propionate is inhibited by thapsigargin and CPA suggests that this action of propionate involves the release of endothelial cell Ca2+ stores.

Hypoxia induces the release of a pulmonary-selective, Ca(2+)-sensitising, vasoconstrictor from the perfused rat lung.

OBJECTIVE: Sustained hypoxic pulmonary vasoconstriction is dependent upon the presence of an intact endothelium, strongly suggesting that an endothelium-derived constrictor factor is involved in this response. In the present study we have attempted to determine whether hypoxia induces the release of a vasoconstrictor(s) from the lung, and whether this vasoconstrictor shares mechanistic features with the hypoxic constrictor response. METHODS: The salt-perfused rat lung, coupled with a simple solid-phase extraction process, and a rat intrapulmonary artery functional bioassay were utilised in this study. RESULTS: Hypoxic, but not normoxic, perfusion of the isolated lung of the rat induced the release of a vasoconstrictor(s) which appeared to be selective for pulmonary over mesenteric arteries of the rat. The vasoconstriction observed was unaffected by inhibition of voltage-gated Ca(2+) channels, and was not associated with a rise in intracellular [Ca(2+)], suggesting Ca(2+)-sensitisation of the contractile apparatus. The vasoconstriction was also unaffected by the protein kinase C (PKC) inhibitor Ro-31-8220, or the endothelin-1 antagonists BQ123/BQ788 but was markedly potentiated in the presence of prostaglandin F(2alpha). CONCLUSION: We conclude that hypoxic perfusion of the rat lung results in the release of a vasoconstrictor(s) which shares some of the facets of the sustained hypoxic constriction of isolated intrapulmonary arteries of the rat, since it involves PKC-independent Ca(2+) sensitisation, is independent of voltage-gated Ca(2+) entry, and is potentiated by the presence of preconstriction.

Modulation of potassium current characteristics in human myometrial smooth muscle by 17beta-estradiol and progesterone.

The K(+) channel currents are important modulators of smooth muscle membrane potential and excitability. We assessed whether voltage-gated K(+) currents from human myometrium are regulated by placental steroid hormones during pregnancy and labor. Pregnant human myometrial cells were isolated from samples obtained at cesarean section. Primary cultured cells were treated with 100 nM 17beta-estradiol, 1 microM progesterone, or both hormones in combination for 24 h. Acute effects of the two hormones were also determined. The K(+) currents were recorded using the standard whole-cell, patch-clamp technique. Primary cultures possessed both delayed rectifier (I(KV)) and A-like (I(KA)) voltage-gated K(+) currents. The 24-h 17beta-estradiol treatment caused a hyperpolarizing shift in the steady-state inactivation of both I(KV) and I(KA). Progesterone treatment also shifted the inactivation of I(KA) and increased I(KV) amplitude by 60%-110%. Conversely, the combined treatment had no effect on these currents. Neither 17beta-estradiol (0.1-1 microM) nor progesterone (1-5 microM) had any effect on the K(+) current when applied acutely. These results show that 17beta-estradiol should inhibit myometrial K(+) channel activity, whereas progesterone is likely to have the opposite effect. These results are consistent with the respective procontractile and proquiescence roles for 17beta-estradiol and progesterone in human uterus during pregnancy.

Inhibition of sustained hypoxic vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat.

We have examined the effects of Y-27632, a specific inhibitor of Rho-activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y-27632 (100 nM - 3 microM) was found to cause a concentration-dependent inhibition of acute sustained HPV in rat IPA. Application of Y-27632 (10-600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration-dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y-27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho-associated kinase may be a pivotal step in the generation of sustained HPV.

Mechanism of effect of extracellular pH on L-type Ca(2+) channel currents in human mesenteric arterial cells.

Extracellular pH (pH(o)) influences vasoconstriction partly by modulating Ca(2+) influx through voltage-gated Ca(2+) channels in the vasculature. The mechanism of this effect of pH(o) is, however, controversial. Using the whole cell voltage-clamp technique, we examined the influence of pH(o) on L-type Ca(2+) channel currents in isolated human mesenteric arterial myocytes. Acidification to pH 6.2 and alkalinization to 8.2 from 7.2 decreased by approximately 50% and increased by 25-30%, respectively, the peak amplitude of Ca(2+) and Ba(2+) currents (1.5 and 10 mM), with an apparent pK(a) of 6.8. Activation and inactivation of Ca(2+) and Ba(2+) currents were shifted toward positive membrane voltages during acidification and in the opposite direction during alkalinization. The relationship between the current amplitude and shifts in the gating parameters in solutions of different pH(o) conformed closely to that predicted by the Gouy-Chapman model, in which the divalent cation concentration at the outer surface of the membrane varies with the extent to which protons neutralize the membrane surface potential.

Endothelium-dependent relaxation of rat aorta and main pulmonary artery by the phytoestrogens genistein and daidzein.

OBJECTIVE: The dietary phytoestrogens genistein and daidzein have been shown to relax agonist-preconstricted arteries in vitro; the mechanisms of relaxation remain incompletely understood. This study aimed to determine whether the relaxation of phenylephrine (PE)-constricted rat aorta and main pulmonary artery by genistein and daidzein was endothelium-dependent. METHODS: Effects of endothelial-denudation, and pretreatment with with 100 microM L-N(G)-nitroarginine methyl ester (L-NAME) and/or 10 microM indomethacin on relaxation of PE (1 microM)-preconstricted contractures by genistein (1-100 microM) and daidzein (3-100 microM) were assessed by measuring isometric force development by rat arterial rings. The effect of L-NAME on relaxation to 17beta-estradiol (10 microM) was also measured in aorta. RESULTS AND CONCLUSIONS: Genistein and daidzein caused concentration-dependent relaxation of aorta rings preconstricted with PE (1 microM). The IC50 values were 5.7 microM (n=8, 95% confidence limits 4.3-7.7 microM) and 36.7 microM (n=12, 95% confidence limits 25.7-44.1 microM), respectively. Removal of the endothelium and pretreatment with L-NAME (100 microM) significantly inhibited relaxation at 3, 10 and 30 microM genistein and 10 and 30 microM daidzein. The contracture evoked in rat aorta by depolarization with 75 mM K+ solution was similarly relaxed by genistein in a partially endothelium-dependent manner. 17Beta-estradiol (10 microM) caused a 48.7+/-5.0% (n=11) relaxation of the PE contracture, which was significantly reduced to 25.1+/-5.3% (n=7) by L-NAME. Relaxations brought about by 17beta-estradiol, genistein, and daidzein were not significantly affected by the genomic estrogen receptor antagonist ICI 182,780 (10 microM). Similar endothelium-dependent effects of genistein were observed in the main pulmonary artery. The results show that the relaxation of these rat arteries by concentrations of genistein and daidzein which overlap those present in human plasma after ingestion of soybean-containing meals is largely endothelium dependent.

Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat.

It has been proposed that hypoxic pulmonary vasoconstriction (HPV) is mediated via K+ channel inhibition and Ca2+ influx through voltage-gated channels. HPV depends strongly on the degree of preconstriction, and we therefore examined the effect of Ca2+ channel blockade on tension and intracellular [Ca2+] ([Ca2+]i) during HPV in rat intrapulmonary arteries (IPAs), whilst maintaining preconstriction constant. We also investigated the role of intracellular Ca2+ stores. HPV demonstrated a transient constriction (phase I) superimposed on a sustained constriction (phase II). Nifedipine (1 microM) partially inhibited phase I, but did not affect phase II. In arteries exposed to 80 mM K+ and nifedipine or diltiazem the rises in tension and [Ca2+]i were blunted during phase I, but were unaffected during phase II. At low concentrations (< 3 microM), La3+ almost abolished the phase I constriction and rise in [Ca2+]i, but had no effect on phase II, or constriction in response to 80 mM K+. Phase II was inhibited by higher concentrations of La3+ (IC50 approximately 50 microM). IPA treated with thapsigargin (1 microM) in Ca2+-free solution to deplete Ca2+ stores showed sustained constriction upon re-exposure to Ca2+ and an increase in the rate of Mn2+ influx, suggesting capacitative Ca2+ entry. The concentration dependency of the block of constriction by La3+ was similar to that for phase I of HPV. Pretreatment of IPA with 30 microM CPA reduced phase I by > 80 %, but had no significant effect on phase II. We conclude that depolarization-mediated Ca2+ influx plays at best a minor role in the transient phase I constriction of HPV, and is not involved in the sustained phase II constriction. Instead, phase I appears to be mainly dependent on capacitative Ca2+ entry related to release of thapsigargin-sensitive Ca2+ stores, whereas phase II is supported by Ca2+ entry via a separate voltage-independent pathway.

Differential block by troglitazone and rosiglitazone of glibenclamide-sensitive K(+) current in rat aorta myocytes.

Thiazolidinediones are insulin-sensitising agents effective in controlling type II diabetes. These compounds also cause vasodilation. We evaluated the effects of the thiazolidinediones troglitazone and rosiglitazone on the glibenclamide-sensitive K(+) current in freshly isolated rat aorta myocytes. Troglitazone inhibited this current in a concentration-dependent manner (IC(50) approximately 1 microM). Rosiglitazone had a similar, but much less potent (IC(50) approximately 20 microM) action. Block of the glibenclamide-sensitive K(+) channels, in particular by troglitazone, may potentially affect the response of arteries to hypoxia and to certain endogenous and exogenous vasodilators.

Calcium antagonistic properties of the cyclooxygenase-2 inhibitor nimesulide in human myometrial myocytes.

The non-steroidal anti-inflammatory drug nimesulide is a selective inhibitor of cyclooxygenase-2 which relaxes spontaneously contracting human myometrium in vivo and is potentially a useful tocolytic drug. Part of the relaxant action of nimesulide may be via block of myometrial Ca2+ channels. Here, we describe the Ca2+ channel blocking properties of nimesulide in freshly dispersed human term-pregnant myometrial smooth muscle cells (HMSMCs). Both L- and T-components of the whole cell Ca2+ channel current were inhibited by 100 microM nimesulide (38+/-3 and 35+/-1% block, respectively). At physiological pH inside and outside the cell (pHo/pHi = 7.4/7.2), this block did not depend on the holding or test potential, although a degree of use-dependence was observed during high frequency stimulation at a higher concentration of drug (300 microM). At pHo/pHi = 6.8, under which condition the concentration of the non-ionized form of the drug is increased 3 fold compared to pH 7.4, nimesulide blocked the L-type current more potently (58+/-3% inhibition at 100 microM, P<0.01) compared to physiological pH. Nimesulide caused a 7 mV leftward shift in the availability curve of the current at pH 6.8, suggesting that the affinity of the drug for the inactivated channel is approximately 4 fold higher than its affinity for the closed channel. We speculate that acidification and depolarization of the myometrium during the intense and prolonged contractions of labour might increase the potency of nimesulide as a Ca2+ channel antagonist, promoting its action as a tocolytic agent.

Voltage-gated K+ currents in freshly isolated myocytes of the pregnant human myometrium.

1. Voltage-gated K+ currents in human myometrium are not well characterized, and were therefore investigated, using the whole-cell patch clamp technique, in freshly isolated myometrial smooth muscle cells from pregnant women at term. 2. Three types of voltage-gated K+ currents were identified. IK1 was a 4-aminopyridine-insensitive current with a negative half-inactivation (V0.5 = -61 to -67 mV) and negative activation characteristics (threshold between -60 and -40 mV) and slow kinetics. IK2 was a 4-aminopyridine-sensitive current (half-maximal block at approximately 1 mM) with relatively positive half-inactivation (V0.5 = -30 mV) and activation characteristics (threshold between -40 and -30 mV) and faster kinetics. IK,A was a 4-aminopyridine-sensitive current with a negative inactivation and very fast inactivation kinetics. 3. Both IK1 and IK2 were sensitive to high concentrations of tetraethylammonium (half-maximal block at approximately 3 mM) and low concentrations of clofilium (half-maximal block by 3-10 microM). 4. IK1 and IK2 were unevenly distributed between myometrial cells, most cells possessing either IK1 (30 cells) or IK2 (24 cells) as the predominant current. 5. The characteristics of these currents suggest a possible function in the control of membrane potentials and smooth muscle quiescence in the pregnant human myometrium.

Mechanisms of hypoxic pulmonary vasoconstriction: can anyone be right?

Despite extensive studies over many years. there is still no real consensus regarding the mechanisms responsible for hypoxic pulmonary vasoconstriction (HPV). This is partially related to extensive variation between preparations, species, and the length of the hypoxic challenge, but also to an apparent abundance of potential mechanisms. Whereas there is good evidence that hypoxia causes inhibition of K channels in pulmonary artery smooth muscle, with subsequent depolarisation and Ca2+ influx through voltage-activated Ca2+ channels, there is also strong support for a critical role for Ca2+ release from intracellular stores. Moreover other studies suggest that the endothelium provides an essential component of the overall response. We suggest in this review that sustained HPV, as seen in the intact animal, is multi-factorial in origin and requires activation of more than one process for the full response to develop. Fundamental issues that remain unresolved are outlined.

Differential effects of insulin-sensitizers troglitazone and rosiglitazone on ion currents in rat vascular myocytes.

Insulin-sensitizing thiazolidinediones such as troglitazone and pioglitazone have been shown to lower blood pressure in vivo and cause vasorelaxation in vitro. Rosiglitazone (BRL 49653) is a novel thiazolidinedione which has been reported not to cause vasoleraxation. We therefore compared the effects of troglitazone and rosiglitazone on Ca2+ and K+ currents in rat aorta and pulmonary artery smooth muscle cells. Currents were recorded with the conventional whole cell patch clamp technique. Both drugs reduced the voltage-gated (L-type) Ca2+ current in rat aorta cells, with half-maximal current inhibition by troglitazone and rosiglitazone at 2 and 10 microM, respectively. Troglitazone, 2 microM and rosiglitazone, 20 microM caused a similar hyperpolarizing shift of 12 mV in the potential-dependence of Ca2+ current availability. Troglitazone (20 microM) produced a marked block of the tetraethylammonium- and paxilline-sensitive Ca2+ activated K+ current, while rosiglitazone (20 microM and 60 microM) slightly enhanced this current. Rat pulmonary artery smooth muscle cells have a prominent delayed rectifier K+ current. Troglitazone produced a potent block of this current (half-maximal inhibition at <1 microM), while rosiglitazone caused a smaller inhibition at 10 and 60 microM. These results show that troglitazone has relatively potent blocking effects on a wide variety of ion currents in vascular smooth muscle cells. Rosiglitazone exerts less potent, but similar effects on the Ca2+ current and delayed rectifier K+ current, but it enhances the Ca2+ activated K+ current. reserved.

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta.

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.

pH-dependent block of the L-type Ca2+ channel current by diltiazem in human mesenteric arterial myocytes.

Inhibition of the L-type Ca2+ channel current (IBa) by diltiazem was characterised in human mesenteric arterial myocytes. External (pHo) and internal (pHi) pH was varied to alter the proportion of drug in charged and neutral forms. Diltiazem (20 microM) reduced IBa amplitude by approximately half at pHo 7.2 and 9.2 at holding potential -60 mV. The IBa decay was increased by diltiazem at pHo = 9.2 (97% uncharged), but not at 7.2. The IC50 for inhibition of IBa by diltiazem at holding potential -60 mV was decreased from 51 to 20 microM at pHo 7.2 and 9.2, respectively. At holding potential of -90 mV, but not -60 mV, tonic block increased and use-dependent block decreased as pHo was raised from 6.2 to 9.2. Diltiazem also caused a hyperpolarizing shift in IBa availability at alkaline pHo. The results suggest that raising pH promotes Ca2+ channel blockade by increasing the proportion of uncharged diltiazem.

Effect of nimesulide and indomethacin on contractility and the Ca2+ channel current in myometrial smooth muscle from pregnant women.

The non-steroidal anti-inflammatory drug (NSAID) indomethacin inhibits both constitutive and inducible forms of cyclo-oxygenase (COX-1 and COX-2, respectively), while nimesulide is a selective COX-2 inhibitor. Uterine COX-2 is upregulated before and during term and pre-term labour, and prostaglandins play a crucial role in parturition. We therefore evaluated the effects of these drugs on myometrial contractility and the voltage-gated Ca2+ channel current in tissue strips and isolated human myometrial smooth muscle cells (HMSMC) from myometrial biopsies taken with informed consent from women undergoing caesarean section at term (not in labour). Nimesulide and indomethacin caused almost complete inhibition of spontaneous myometrial contractions at concentrations of 100 and 300 microM, respectively. The Ca2+ channel current was inhibited in a concentration-dependent manner by both drugs, with a 40% reduction of the current at 100 microM nimesulide and 300 microM indomethacin. Nimesulide also accelerated the decay of the Ca2+ channel current. The inhibition of the Ca2+ channel current by 100 microM nimesulide and 300 microM indomethacin was unaffected by the presence of either PGF2alpha or PGE2 (30 microM), and was of similar magnitude whether 10 mM Ba2+ or 1.5 mM Ca2+ was used as the charge carrier. The concentrations of indomethacin and nimesulide required to suppress spontaneous contractility in human pregnant myometrium were much higher than those necessary to inhibit prostaglandin production. The results suggest that both nimesulide and indomethacin inhibit myometrial contractility via mechanisms independent of cyclo-oxygenase inhibition. Blockade of the Ca2+ current may contribute to this effect.

Modulation of arachidonic acid release and membrane fluidity by albumin in vascular smooth muscle and endothelial cells.

Albumin is the major plasma protein circulating in blood. Albumin potently decreases capillary permeability, although the mechanisms are not understood completely. Albumin also effectively binds arachidonic acid (AA), which increases capillary permeability. To investigate the interactions of BSA and AA with the cell membrane, the effect of these substances on [3H]AA release and membrane fluidity was studied in vascular myocytes and endothelial cells. BSA (0.2 and 1 mg . mL-1) stimulated a significant release of [3H]AA from both intact rat aorta and cultured smooth muscle cells. This effect was not mimicked by gamma-globulin or myoglobin (both 1 mg . mL-1) in intact tissue. BSA, but not gamma-globulin and myoglobin, decreased the membrane fluidity (assessed as changes in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3, 5-hexatriene) in a concentration-dependent manner with a half-maximum concentration between 0.007 and 0.4 mg . mL-1 in both freshly isolated and cultured rat aortic myocytes and human umbilical vein endothelial cells. AA (1 to 200 micromol/L) caused the opposite effect, increasing membrane fluidity and antagonizing the effect of BSA. BSA modified at its arginine residues, which are thought to be important in AA binding, did not stimulate [3H]AA release and was significantly less potent than native BSA in altering the membrane fluidity. The effect of BSA can be explained by a high-affinity binding of AA to the protein and extraction of AA from the cell membrane. The interaction between BSA and AA could play a role in the regulation of vascular permeability.

Effects of the 5-lipoxygenase activating protein inhibitor MK886 on voltage-gated and Ca2+-activated K+ currents in rat arterial myocytes.

1. The effects on the voltage-gated (IK) and Ca2+ activated (I(K,Ca)) K+ currents in rat arterial myocytes of the 5-lipoxygenase activating protein (FLAP) inhibitor MK886, and its inactive analogue L583,916 were evaluated. 2. In rat pulmonary arterial myocytes (RPAMs), MK886 caused a concentration-dependent reduction of the IK, with little obvious change in the kinetics of the current. Half maximal current block was observed at 75 nM MK886. 3. MK886 application led to a concentration-dependent increase in the amplitude of the TEA-sensitive I(K,Ca) current and single channel activity in RPAMs in whole cell and inside-out configurations, respectively. The threshold concentration for this effect was approximately 300 nM and a maximal 4-5 fold increase was observed at 10 microM MK886. MK886 also increased I(K,Ca) in rat mesenteric arterial myocytes (RMAMs). 4. L538,916, an analogue of MK886 which does not block FLAP, had no effect on either IK or I(K,Ca) at a concentration of 10 microM. 5. Leukotriene C4 (100 nM) had no effect on either IK or I(K,Ca) in RPAMs. MK886 produced its usual increase in I(K,Ca) and also blocked IK, in the presence of leukotriene C4. Similarly, leukotriene E4 (100 nM) did not alter the amplitude of IK. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 microM) did not affect IK in RPAMs, and did not affect the response to MK886. 6. Arachidonic acid (10 microM) enhanced I(K,Ca) in both RPAMs and RMAMs. 7. The results show that MK886 markedly affects both IK and I(K,Ca) in a manner similar to that of arachidonic acid and independent of the endogenous production of leukotrienes. It is therefore possible that MK886, which is thought to compete with arachidonic acid for its binding to FLAP, may similarly occupy arachidonic acid binding sites on these K+ channels, and mimic its effects. Alternatively, MK886 might act via non-selective effects on other arachidonic acid metabolites which could modify K+ channel function.

Modulatory effects of arachidonic acid on the delayed rectifier K+ current in rat pulmonary arterial myocytes. Structural aspects and involvement of protein kinase C.

The effect of arachidonic acid (AA) on the delayed rectifier K+ current (IK) was evaluated in rat pulmonary myocytes by using the whole-cell patch-clamp technique. Externally applied AA (50 mumol/L) caused a membrane depolarization, averaging 16 mV in six cells. AA (1 to 50 mumol/L) caused a dual effect on IK. First, AA accelerated the rate of IK activation, increasing current amplitude at the beginning of voltage step. Second, AA caused a marked acceleration of current decay, thereby reducing IK amplitude measured toward the end of the depolarizing steps. These effects were not prevented by indomethacin or nordihydroguaiaretic acid, blockers of cyclooxygenase and lipoxygenase, respectively. AA did not affect the voltage dependence of current activation or inactivation. The magnitude of the inhibitory effect on IK was correlated with the number of double bonds but was independent of tail length in fatty acids containing between 14 and 22 carbons. Linoleic acid (18:2, cis-9,12) inhibited IK much more than did its trans-stereo-isomer, linolelaidic acid. Arachidonyl alcohol, which is uncharged, and arachidonyl coenzyme A, which does not 'flip' across the cell membrane, were less effective than AA in inhibiting IK; this effect of fatty acids may therefore require passage across the cell membrane. The enhancement of early IK was mimicked by the protein kinase C (PKC) stimulator 1-oleoyl-2-acetyl-sn-glycerol (10 mumol/L), was suppressed by ATP removal from the pipette solution, and was blocked by PKC inhibitors chelerythrine (10 mumol/L) and staurosporine (100 nmol/L). This effect may therefore require PKC-dependent phosphorylation.

Mechanism of lactate-induced relaxation of isolated rat mesenteric resistance arteries.

1. The effects of the sodium salt of the weak acid lactate on tension and intracellular pH (pH1) were studied in rat mesenteric small arteries mounted on a wire myograph. Sodium lactate was substituted iso-osmotically for sodium chloride. 2. At a concentration of 50 mM, both L- and D-stereoisomers of lactate markedly relaxed arteries preconstricted with noradrenaline (NA) within 10 min. The concentration-response relationship for L-lactate showed that the NA contracture was relaxed by 50% at approximately 26 mM. L-Lactate did not, however, relax arteries preconstricted with high-K+(45 mM) solution. 3. L-Lactate did not alter extracellular pH (pHo) but caused a small but significant decrease in pH1, measured using the pH-sensitive fluorochrome, 2',7'-bis(carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). Relaxation to L-lactate was unaffected when this change in pHi was offset by the simultaneous addition of NH4Cl to the solution. 4. Sodium pyruvate (50 mM) caused a significant intracellular acidosis but did not relax arteries preconstricted with NA. 5. L-Lactate-induced relaxations were unaffected by removal of the endothelium or when the synthesis of nitric oxide (NO) was inhibited by 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME). 6. The potassium channel blockers glibenclamide (10 microM), 4-aminopyridine (3 mM) and tetraethylammonium chloride (10 mM) did not affect L-lactate-induced relaxation in arteries preconstricted with NA. Inhibition of guanylate cyclase with Methylene Blue, or cyclooxgenase with indomethacin, also did not affect relaxation to L-lactate. 7. The Rp stereoisomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), an analogue of cAMP which inhibits competitively stimulation of protein kinase A, reduced significantly L-lactate-induced relaxation at a concentration of 25 microM. Rp-cAMPS also significantly reduced forskolin-induced relaxation of the NA contracture. 8. It is concluded that L-lactate-induced relaxation in this vascular bed is pHi-1 endothelium-, and nitric oxide-independent. It is not mediated by inhibition of voltage-gated Ca2+ channels, opening of K+ channels, prostacylin or cyclic GMP. cAMP may however play a role in L-lactate-induced relaxation.

Mechanism of butyrate-induced vasorelaxation of rat mesenteric resistance artery.

1. The vasorelaxant effect of the sodium salt of the short chain fatty acid, butyrate, on preconstricted rat small mesenteric arteries (mean inner diameter approximately 300 microns) was characterized. Isometric force development was measured with a myograph, and intracellular pH (pHi) was simultaneously monitored, in arteries loaded with the fluorescent dye BCECF in its acetomethoxy form. Sodium butyrate (substituted isosmotically for NaCl) was applied to arteries after noradrenaline (NA) or high K+ contractures were established. 2. Arteries preconstricted with a concentration of NA inducing an approximately half maximal contraction were relaxed by 91.5 +/- 6.3% by 50 mmol l-1 butyrate. This concentration of butyrate did not, however, cause a significant relaxation of contractures to a maximal (5 mumol l-1) NA concentration, and also failed to relax significantly contractures stimulated by high (45 and 90 mmol l-1) K+ solutions. Contractures elicited with a combination of NA (at a submaximal concentration) and 45 mmol l-1 K+ were, however, markedly relaxed by butyrate. 3. Investigation of the concentration-dependency of the butyrate-induced relaxation of the half maximal NA response revealed an EC50 for butyrate of approximately 22 mmol l-1. 4. Sodium butyrate (50 mmol l-1) caused pHi to decrease from 7.25 +/- 0.02 to 6.89 +/- 0.08 (n = 4, P < 0.001). However, the vasorelaxant effect of butyrate on the submaximal NA contracture was not significantly modified when this fall in intracellular pH was prevented by the simultaneous application of NH4Cl. 5. Butyrate-induced relaxation was also unaffected by endothelial denudation and inhibition of NO synthase with N omega-nitro-L-arginine methyl ester (100 mumol l-1). 6. The relaxation of the NA contracture by 50 mmol l-1 sodium butyrate was abolished in arteries pretreated with the cyclic AMP antagonist Rp-cAMPS (25 mumol l-1). 7. We conclude that the butyrate-induced relaxation of the NA contracture is independent of intracellular acidification. The ability of Rp-cAMPS to abolish the butyrate relaxation indicates that stimulation of the cyclic AMP second messenger system may play an important role in mediating this effect.