The actions of the cannabinoid receptor antagonist,SR 141716A,in the rat isolated mesenteric artery

1. The actions of the cannabinoid receptor antagonist, SR 141716A, were examined in rat isolated mesenteric arteries. At concentrations greater than 3 μM, it caused concentration-dependent, but endothelium-independent, relaxations of both methoxamine- and 60 mM KCl-precontracted vessels.
2. SR 141716A (at 10 μM, but not at 1 μM) inhibited contractions to Ca²⁺ in methoxamine-stimulated mesenteric arteries previously depleted of intracellular Ca²⁺ stores. Neither concentration affected the phasic contractions induced by methoxamine in the absence of extracellular Ca²⁺.
3. SR 141716A (10 μM) caused a 130 fold rightward shift in the concentration-response curve to levcromakalim, a K⁺ channel activator, but had no effect at 1 μM.
4. SR 141716A (10 μM) attenuated relaxations to NS 1619 (which activates large conductance, Ca²⁺-activated K⁺ channels; BK_Ca). The inhibitory effect of SR 141716A on NS 1619 was not significantly different from, and was not additive with, that caused by a selective BK_Ca inhibitor, iberiotoxin (100 nM). SR 141716A (1 μM) did not effect NS 1619 relaxation.
5. SR 141716A (10 μM) had no effect on relaxations to the nitric oxide donor S-nitroso-N-acetylpenicillamine, or relaxations to carbachol in the presence of 25 mM KCl.
6. The results show that, at concentrations of 10 μM and above, SR 141716A causes endothelium-independent vasorelaxation by inhibition of Ca²⁺ entry. It also inhibits relaxations mediated by K⁺ channel activation. This suggests that such concentrations of SR 141716A are not appropriate for investigation of cannabinoid receptor-dependent processes.

     

Sex differences in the relative contributions of nitric oxide and EDHF to agonist-stimulated endothelium-dependent relaxations in the rat isolated mesenteric arterial bed

1. We have used the isolated, buffer-perfused, superior mesenteric arterial bed of male and female rats to assess the relative contributions of nitric oxide (NO) and the endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxations to carbachol.
2. Carbachol caused dose-related relaxations of methoxamine-induced tone in mesenteric vascular beds from male rats described by an ED_50(M) of 0.43±0.15 nmol and a maximum relaxation (R_max(M) of 89.6±1.2% (n=28) which were not significantly different from those observed in mesenteries from female rats (ED_50(F)=0.72±0.19 nmol and R_max(F)=90.7±0.9%; n=22).
3. In the males, the addition of 100 μM N^G-nitro-L-arginine methyl ester (L-NAME) caused the dose-response curve to carbachol to be significantly (P<0.001) shifted to the right 15 fold (ED_50(M)=6.45±3.53 nmol) and significantly (P<0.01) reduced R_max(M) (79.7±2.8%, n=13). By contrast, L-NAME had no effect on vasorelaxation to carbachol in mesenteries from female rats (ED_50(F)=0.89±0.19 nmol, R_max(F)=86.9±2.3%, n=9).
4. Raising tone with 60 mM KCl significantly reduced the maximum relaxation to carbachol in mesenteries from male rats 2 fold (R_max(M)=40.3±9.2%, n=4; P<0.001) and female rats by 1.5 fold (R_max(F)=55.3±3.3%, n=6; P<0.001), compared with methoxamine-induced tone. The potency of carbachol was also significantly reduced 1.2 fold in preparations from males (ED_50(M)=0.87±0.26 nmol; P<0.01) but not the females (ED_50(F)=4.04±1.46 nmol). In the presence of both 60 mM KCl and L-NAME, the vasorelaxation to carbachol was completely abolished in mesenteries from both groups.
5. The cannabinoid receptor antagonist SR141716A (1 μM), which is also a putative EDHF antagonist, had no significant effect on the responses to carbachol in mesenteries from males or females (ED_50(M)=1.41±0.74 nmol, R_max(M)=89.4±2.5%, n=7; ED_50(F)=2.17±0.95 nmol, R_max(F)=89.9±1.8%, n=9). In mesenteries from male rats, in the presence of 100 μM L-NAME, SR141716A significantly (P<0.05) shifted the dose-response curve to carbachol 8 fold further to the right than that seen in the presence of L-NAME alone (ED_50(M)=53.8±36.8 nmol) without affecting R_max(M) (72.4±4.8%, n=10). In mesenteries from female rats, the combined presence of L-NAME and SR141716A, significantly (P<0.01) shifted the dose-response curve to carbachol 7.5 fold, (ED_50(F)=6.66±2.46 nmol), as compared to L-NAME alone and significantly (P<0.001) decreased R_max(F) (70.1±5.5%, n=8).
6. Vasorelaxations to the nitric oxide donor sodium nitroprusside (SNP), to the endogenous cannabinoid, anandamide (a putative EDHF) and to the ATP-sensitive potassium channel activator, levcromakalim, did not differ significantly between male and female mesenteric vascular beds.
7. The continuous presence of sodium nitroprusside (SNP; 20–60 nM) had no effect on vasorelaxation to carbachol in mesenteries from either males or females. In the presence of L-NAME, SNP significantly (P<0.05) reduced the potency of carbachol 6 fold, without affecting the maximal relaxation in mesenteries from male rats (ED_50(M)=40.9±19.6 nmol, R_max(M)=79.4±2.5%, n=11). Similarly in mesenteries from female rats, the ED_50(F) was also significantly (P<0.01) increased 7 fold (6.24±2.02 nmol), while the R_max(F) was unaffected (81.9±11.0%; n=4).
8. The results of the present investigation demonstrate that the relative contributions of agonist-stimulated NO and EDHF to endothelium-dependent relaxations in the rat isolated mesenteric arterial bed, differ between males and females. Specifically, although both NO and EDHF appear to contribute towards endothelium-dependent relaxations in males and females, blockade of NO synthesis alone has no effect in the female. This suggests that EDHF is functionally more important in females; one possible explanation for this is that in the absence of NO, the recently identified ability of EDHF to compensate for the loss of NO, is functionally more important in females than males.

     

Combination of Ca2+ -activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery

BACKGROUND AND PURPOSE: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation. EXPERIMENTAL APPROACH: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured. KEY RESULTS: A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ -activated K channels abolished acetylcholine (10 microM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 microM), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA. CONCLUSIONS AND IMPLICATIONS: Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.     

The actions of some cannabinoid receptor ligands in the rat isolated mesenteric artery

1. The actions of a number of cannabinoid receptor ligands were investigated using the myograph-mounted rat isolated mesenteric artery. Anandamide, CP 55,940, HU-210, palmitoylethanolamide and WIN 55,212-2 all caused concentration-dependent relaxations of methoxamine-precontracted vessels which were not affected by removal of the endothelium.
2. Precontracting vessels with 60 mM KCl instead of methoxamine greatly reduced the vasorelaxant effects of anandamide and palmitoylethanolamide. High K⁺ solution caused a modest decrease in the relaxant potency of CP 55,940 and HU-210, and had no effect on relaxations induced by WIN 55,212-2.
3. Relaxations of methoxamine-induced tone by anandamide, CP 55,940 and HU-210, but not palmitoylethanolamide and WIN 55,212-2, were attenuated by the cannabinoid receptor antagonist, SR 141716A. Relaxation of vessels contracted with 60 mM KCl by CP 55,940 was also sensitive to SR 141716A.
4. Anandamide and CP 55,940 caused small but concentration-dependent contractions in resting vessels in the absence of extracellular calcium. These were not sensitive to SR 141716A. Palmitoylethanolamide and WIN 55,212-2 produced smaller contractions only at higher concentrations.
5. Anandamide and CP 55,940, but not palmitoylethanolamide and WIN 55,212-2, caused concentration-dependent inhibition of the phasic contractions induced by methoxamine in calcium-free conditions, but only anandamide caused inhibition of contractions to caffeine under such conditions. These inhibitory effects were not antagonised by SR 141716A.
6. The present study provides the first detailed investigation of the actions of cannabinoid agonists on vascular smooth muscle. Our results show that these compounds exert both receptor-dependent and -independent effects on agonist-induced calcium mobilization in the rat isolated mesenteric artery.

     

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques.
2. NDGA (5–50 μM) produced a concentration-dependent inhibition of whole-cell K⁺ currents at all activating test potentials (holding potential −70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 μM), was without effect on whole-cell K⁺ currents in carotid body type I cells.
3. NDGA (5–50 μM) also inhibited whole-cell Ca²⁺ channel currents (recorded with Ba²⁺ as charge carrier) in a concentration-dependent manner.
4. Isolation of voltage-gated K⁺ channels by use of high [Mg²⁺] (6 mM), low [Ca²⁺] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K⁺ current by NDGA (50 μM).
5. In excised, outside-out patches NDGA (20–50 μM) increased large conductance, Ca²⁺ activated K⁺ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM).
6. It is concluded that NDGA activates maxi-K⁺ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K⁺ and Ca²⁺ channels. The inhibition of whole cell K⁺ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K⁺ current and indirect inhibition of maxi-K⁺ channel activity through blockade of Ca²⁺ channels.

     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed.
2. NO (10⁻⁷ M) induced only contraction while 10⁻⁶ M NO induced contraction followed by relaxation. Methylene blue (up to 10⁻⁴ M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10⁻⁶ M NO. Administration of 8-bromo-cyclic GMP (10⁻⁶–10⁻⁴ M) only induced relaxation.
3. Sodium nitroprusside (SNP; 10⁻⁷–10⁻⁵ M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10⁻⁸ M NO and 10⁻⁶ M SNP; it was increased by a factor of 2 during contraction with 10⁻⁷ M NO, and by a factor of 12 during relaxation with 3×10⁻⁶ M NO.
4. The NO-induced contractions were not affected by ryanodine (3×10⁻⁵ M) but were concentration-dependently reduced by nifedipine (10⁻⁸–10⁻⁷ M) and apamin (3×10⁻⁹–3×10⁻⁸ M).
5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca²⁺ influx through L-type Ca²⁺ channels, that might be activated in response to the closure of Ca²⁺-dependent K⁺ channels.

     

Regulation of apical and basolateral K+ conductances in the rat colon

1. Apical administration of an ionophore, nystatin, and basolateral depolarization by K⁺ were used to investigate the regulation of apical and basolateral electrogenic transport pathways for K⁺ in the rat proximal and distal colon.
2. Administration of nystatin (100 μg ml⁻¹ at the mucosal side), in the presence of Na⁺ and in the presence of a serosally directed K⁺ gradient, stimulate a large increase in short-circuit current (I_SC) and tissue conductance in both colonic segments. This response was composed of a pump current generated by the Na⁺-K⁺-ATPase and of a current across a quinine-sensitive basolateral K⁺ conductance.
3. The pump current, measured as Na⁺-dependent or scilliroside-sensitive current in the absence of a K⁺ gradient, was significantly greater in the distal than in the proximal colon. The pump current was unaltered by pretreatment of the tissue with forskolin (5×10⁻⁶ mol l⁻¹).
4. The current across the basolateral K⁺ conductance, measured as current in the presence of a serosally directed K⁺ gradient either in the absence of Na⁺ or in the presence of scilliroside, was increased by the cholinoreceptor agonist, carbachol (5×10⁻⁵ mol l⁻¹), but inhibited by forskolin (5×10⁻⁶ mol l⁻¹).
5. Basolateral K⁺ depolarization induced a negative I_SC in both colonic segments, which was inhibited by the K⁺ channel blocker quinine (10⁻³ mol l⁻¹ at the mucosal side), but was resistant to tetraethylammonium (5×10⁻³ mol l⁻¹ at the mucosal side). This K⁺ current across an apical K⁺ conductance was stimulated in both colonic segments by carbachol, whereas forskolin had no effect, although control experiments revealed that forskolin was still able to open an apical Cl⁻ conductance under these conditions.
6. These results demonstrate that an increase in intracellular Ca²⁺ concentration induced by carbachol causes an increase in the basolateral and the apical K⁺ conductance, thereby inducing K⁺ secretion in parallel with an indirect support for Cl⁻ secretion due to the hyperpolarization of the cell membrane. In contrast, the dominating effect of an increase in the intracellular cyclic AMP concentration is inhibition of a basolateral K⁺ conductance; a mechanism which might contribute to the inhibition of K⁺ absorption.

     

NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery

1. The nature and cellular mechanisms that are responsible for endothelium-dependent relaxations resistant to indomethacin and N^G-nitro-L-arginine methyl ester (L-NAME) were investigated in phenylephrine (PE) precontracted isolated carotid arteries from the rabbit.
2. In the presence of the cyclo-oxygenase inhibitor, indomethacin (10 μM), acetylcholine (ACh) induced a concentration- and endothelium-dependent relaxation of PE-induced tone which was more potent than the calcium ionophore A23187 with pD₂ values of 7.03±0.12 (n=8) and 6.37±0.12 (n=6), respectively. The ACh-induced response was abolished by removal of the endothelium, but was not altered when indomethacin was omitted (pD₂ value 7.00±0.10 and maximal relaxation 99±3%, n=6). Bradykinin and histamine (0.01–100 μM) had no effect either upon resting or PE-induced tone (n=5).
3. In the presence of indomethacin plus the NO synthase inhibitor, L-NAME (30 μM), the response to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 was abolished. However, the response to ACh was not abolished, although it was significantly inhibited with the pD₂ value and the maximal relaxation decreasing to 6.48±0.10 and 67±3%, respectively (for both P<0.01, n=8). The L-NAME/indomethacin insensitive vasorelaxation to ACh was completely abolished by preconstriction of the tissues with potassium chloride (40 mM, n=8).
4. The Ca²⁺-activated K⁺ (K_Ca) channel blockers, tetrabutylammonium (TBA, 1 mM, n=5) and charybdotoxin (CTX, 0.1 μM, n=5), completely inhibited the nitric oxide (NO) and prostacyclin (PGI₂)-independent relaxation response to ACh. However, iberiotoxin (ITX, 0.1 M, n=8) or apamin (1–3 μM, n=6) only partially inhibited the relaxation.
5. Inhibitors of the cytochrome P450 mono-oxygenase, SKF-525A (1–10 μM, n=6), clotrimazole (1 μM, n=5) and 17-octadecynoic acid (17-ODYA, 3 μM, n=7) also reduced the NO/PGI₂-independent relaxation response to ACh.
6. In endothelium-denuded rings of rabbit carotid arteries, the relaxation response to exogenous NO was not altered by either K_Ca channel blockade with apamin (1 μM, n=5) or CTX (0.1 μM, n=5), or by the cytochrome P450 mono-oxygenase blockers SKF-525A (10 μM, n=4) and clotrimazole (10 μM, n=5). However, the NO-induced response was shifted to the right by LY83583 (10 μM, n=4), a guanylyl cyclase inhibitor, with the pD₂ value decreasing from 6.95±0.14 to 6.04±0.09 (P<0.01).
7. ACh (0.01–100 μM) induced a concentration-dependent relaxation of PE-induced tone in endothelium-denuded arterial segments sandwiched with endothelium-intact donor segments. This relaxation to ACh was largely unaffected by indomathacin (10 μM) plus L-NAME (30 μM), but abolished by the combination of indomethacin, L-NAME and TBA (1 mM, n=5).
8. These data suggest that in the rabbit carotid artery: (a) ACh can induce the release of both NO and EDHF, whereas {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 only evokes the release of NO from the endothelium, (b) the diffusible EDHF released by ACh may be a cytochrome P450-derived arachidonic acid metabolite, and (c) EDHF-induced relaxation involves the opening of at least two types of K_Ca channels, whereas NO mediates vasorelaxation via a guanosine 3′: 5′-cyclic monophosphate (cyclic GMP)-mediated pathway, in which a cytochrome P450 pathway and K_Ca channels do not seem to be involved.

     

Possible mechanisms underlying the midazolam-induced relaxation of the noradrenaline-contraction in rabbit mesenteric resistance artery

1. The mechanisms underlying the midazolam-induced relaxation of the noradrenaline (NA)-contraction were studied by measuring membrane potential, isometric force and intracellular concentration of Ca²⁺([Ca²⁺]_i) in endothelium-denuded muscle strips from the rabbit mesenteric resistance artery. The actions of midazolam were compared with those of nicardipine, an L-type Ca²⁺-channel blocker.
2. Midazolam (30 and 100 μM) did not modify either the resting membrane potential or the membrane depolarization induced by 10 μM NA.
3. NA (10 μM) produced a phasic, followed by a tonic increase in both [Ca²⁺]_i and force. Midazolam (10–100 μM) did not modify the resting [Ca²⁺]_i, but attenuated the NA-induced phasic and tonic increases in [Ca²⁺]_i and force, in a concentration-dependent manner. In contrast, nicardipine (0.3 μM) attenuated the NA-induced tonic, but not phasic, increases in [Ca²⁺]_i and force.
4. In Ca²⁺-free solution containing 2 mM EGTA, NA (10 μM) transiently increased [Ca²⁺]_i and force. Midazolam (10–100 μM), but not nicardipine (0.3 μM), attenuated this NA-induced increase in [Ca²⁺]_i and force, in a concentration-dependent manner. However, midazolam (10 and 30 μM), had no effect on the increases in [Ca²⁺]_i and force induced by 10 mM caffeine.
5. In ryanodine-treated strips, which have functionally lost the NA-sensitive Ca²⁺- storage sites, NA slowly increased [Ca²⁺]_i and force. Nicardipine (0.3 μM) did not modify the resting [Ca²⁺]_i but partly attenuated the NA-induced increases in [Ca²⁺]_i and force. In the presence of nicardipine, midazolam (100 μM) lowered the resting [Ca²⁺]_i and further attenuated the remaining NA-induced increases in [Ca²⁺]_i and force.
6. The [Ca²⁺]_i-force relationship was obtained in ryanodine-treated strips by the application of ascending concentrations of Ca²⁺ (0.16–2.6 mM) in Ca²⁺-free solution containing 100 mM K⁺. NA (10 μM) shifted the [Ca²⁺]_i-force relationship to the left and enhanced the maximum Ca²⁺-induced force. Under these conditions, whether in the presence or absence of 10 μM NA, midazolam (10 and 30 μM) attenuated the increases in [Ca²⁺]_i and force induced by Ca²⁺ without changing the [Ca²⁺]_i-force relationship.
7. It was concluded that, in smooth muscle of the rabbit mesenteric resistance artery, midazolam inhibits the NA-induced contraction through its inhibitory action on NA-induced Ca²⁺ mobilization. Midazolam attenuates NA-induced Ca²⁺ influx via its inhibition of both nicardipine-sensitive and -insensitive pathways. Furthermore, midazolam attenuates the NA-induced release of Ca²⁺ from the storage sites. This effect contributes to the midazolam-induced inhibition of the NA-induced phasic contraction.

     

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 (I_K) 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 I_K, 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 μM 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 I_K or I_K,Ca at a concentration of 10 μM.
5. Leukotriene C₄ (100 nM) had no effect on either I_K or I_K,Ca in RPAMs. MK886 produced its usual increase in I_K,Ca and also blocked I_K, in the presence of leukotriene C₄. Similarly, leukotriene E₄ (100 nM) did not alter the amplitude of I_K. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 μM) did not affect I_K in RPAMs, and did not affect the response to MK886.
6. Arachidonic acid (10 μM) enhanced I_K,Ca in both RPAMs and RMAMs.
7. The results show that MK886 markedly affects both I_K 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.

     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

The expression and function of Ca(2+)-sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes

BACKGROUND AND PURPOSE: The extracellular calcium-sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate-conductance, calcium-sensitive K(+) channels (IK(Ca)) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes. EXPERIMENTAL APPROACH: Pressure myography, western blotting, sharp microelectrode and K(+)-selective electrode recordings were used to investigate the functional expression of the CaR and IK(Ca) in rat mesenteric arteries. KEY RESULTS: Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619-induced vessel contraction elevated the extracellular [K(+)] around the myocytes, and inhibition of this 'K(+) cloud' by iberiotoxin was needed to reveal calindol-induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK(Ca) expression was also diminished, but IK(Ca)-generated hyperpolarizations mediated by 1-EBIO were unaffected. CONCLUSIONS AND IMPLICATIONS: The reduced CaR-mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK(Ca) channels. Detection of CaR-mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long-term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.     

Magnesium lithospermate B dilates mesenteric arteries by activating BKca currents and contracts arteries by inhibiting Kv currents

Aim:

To examine the involvement of K⁺ channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix.

Methods:

Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K⁺ currents in single isolated mesenteric artery myocytes.

Results:

MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 μmol/L) with an EC₅₀ of 111.3 μmol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC₅₀ of 224.4 μmol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK_Ca currents (EC₅₀ 156.3 μmol/L) but inhibited K_V currents (IC₅₀ 26.1 μmol/L) in a voltage- and concentration-dependent manner.

Conclusion:

MLB dilated arteries by activating BK_Ca channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.     

Effects of propafenone and 5-hydroxy-propafenone on hKv1.5 channels

1. The goal of this study was to analyse the effects of propafenone and its major metabolite, 5-hydroxy-propafenone, on a human cardiac K⁺ channel (hKv1.5) stably expressed in Ltk⁻ cells and using the whole-cell configuration of the patch-clamp technique.
2. Propafenone and 5-hydroxy-propafenone inhibited in a concentration-dependent manner the hKv1.5 current with K_D values of 4.4±0.3 μM and 9.2±1.6 μM, respectively.
3. Block induced by both drugs was voltage-dependent consistent with a value of electrical distance (referenced to the cytoplasmic side) of 0.17±0.55 (n=10) and 0.16±0.81 (n=16).
4. The apparent association (k) and dissociation (l) rate constants for propafenone were (8.9±0.9)×10⁶ M⁻¹ s⁻¹ and 39.5±4.2 s⁻¹, respectively. For 5-hydroxy-propafenone these values averaged (2.3±0.3)×10⁶ M⁻¹ s⁻¹ and 21.4±3.1 s⁻¹, respectively.
5. Both drugs reduced the tail current amplitude recorded at −40 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a `crossover'' phenomenon when the tail currents recorded under control conditions and in the presence of each drug were superimposed.
6. Both compounds induced a small but statistically significant use-dependent block when trains of depolarizations at frequencies between 0.5 and 3 Hz were applied.
7. These results indicate that propafenone and its metabolite block hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutical range.

     

Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery

1. The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves.
2. Vasopressin (10⁻¹⁰–10⁻⁷ M) caused concentration-dependent contractions (pD₂=8.36±0.09). The V₁-receptor antagonist d(CH₂)₅Tyr(Me)AVP (10⁻⁹–10⁻⁸ M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA₂ value of 9.83 with a slope of 1.10±0.14.
3. Vasopressin (3×10 ⁻¹⁰ and 10⁻⁹ M) caused concentration-dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration-response curve for noradrenaline. The V₁-receptor antagonist induced concentration-dependent inhibitions of potentiation induced by vasopressin. The selective V₁-receptor agonist [Phe^*, Orn⁸]-vasotocin (3×10 ⁻¹⁰ and 10⁻⁹ M) induced potentiation of electrical stimulation-evoked responses which was also inhibited in the presence of the V₁ antagonist (10⁻⁸ M). In contrast, the V₂-receptor agonist deamino-8-D-arginine vasopressin (desmopressin 10⁻⁸–10⁻⁷ M) did not modify the electrical stimulation-induced responses and the V₂-receptor antagonist [d(CH₂)₅, D-Ile^*, Ile⁴, Arg⁸]-vasopressin (10⁻⁸–10⁻⁷ M) did not affect the potentiation evoked by vasopressin.
4. In artery rings contracted by 10⁻⁶ M noradrenaline in the presence of 10⁻⁶ M guanethidine and 10⁻⁶ M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency-dependent relaxations which were unaffected by 10⁻⁹ M vasopressin but abolished by 10⁻⁶ M tetrodotoxin.
5. Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl₂ to KCl depolarized vessels. The augmenting effects were inhibited by the V₁ antagonist.
6. In the presence of the calcium antagonist nifedipine (10⁻⁶ M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl.
7. The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V₁ receptor stimulation which brings about an increase in calcium entry through dihydropyridine-sensitive calcium channels.

     

Role of Ca2+-activated K+ channels in acetylcholine-induced dilatation of the basilar artery in vivo

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine.
2. Topical application of acetylcholine (10⁻⁶ and 10⁻⁵ M) increased diameter of the basilar artery from 238±7 μm to 268±7 and 288±7 μm, respectively (P<0.05 vs. baseline diameter). Iberiotoxin (10⁻⁸ M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10⁻⁸ M iberiotoxin, 10⁻⁶ and 10⁻⁵ M acetylcholine increased diameter of the basilar artery from 239±7 μm to 246±7 and 261±7 μm, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05).
3. Sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 242±9 μm to 310±12 and 374±13 μm, respectively (P<0.05 vs. baseline diameter). In the presence of iberiotoxin (10⁻⁸ M), sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 243±6 μm to 259±9 and 311±12 μm, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P<0.05).
4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener.
5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

     

Endothelium-dependent relaxation by substance P in human isolated omental arteries and veins: relative contribution of prostanoids,nitric oxide and hyperpolarization

1. The objective of the present study was to investigate human omental arteries and veins with respect to: (i) the contractile effect of the thromboxane A₂ analogue U46619, (ii) endothelium-dependency and mediators of the relaxing effect of substance P (SP) and acetylcholine (ACh).
2. Changes in isometric tension in response to administration of U46619, SP and ACh were measured in human isolated omental arteries and veins with and without endothelium. To investigate the mechanism of action of SP, the SP-induced relaxation was measured in the presence of indomethacin (cyclo-oxygenase inhibitor), N^G-monomethyl-L-arginine (L-NMMA, nitric oxide-synthase inhibitor), KCl (inhibitor of endothelium-dependent hyperpolarization), tetraethylammonium (TEA; non-selective inhibitor of K⁺-channels, with some preference for the high conductance Ca²⁺-activated K⁺-channel, BK_Ca), glibenclamide (inhibitor of the ATP-sensitive K⁺-channel) and/or clotrimazole (inhibitor of the cytochrome P450-system and the intermediate conductance Ca²⁺-activated K⁺-channel, IK_Ca).
3. U46619 contracted both the artery and the vein segments. Endothelium removal did not alter the contraction.
4. ACh caused neither contraction nor relaxation in artery and vein segments precontracted with U46619.
5. In both artery and vein segments precontracted with U46619, SP produced endothelium-dependent relaxation. The relaxation was unaffected by indomethacin, but was incompletely reduced by L-NMMA and KCl respectively. The L-NMMA-resistent relaxation was abolished in the presence of KCl.
6. TEA inhibited the SP-induced relaxation in artery and vein segments both in the presence and absence of L-NMMA and indomethacin, while glibenclamide and clotrimazole had no effect.
7. In conclusion, the SP-induced relaxation in human omental arteries and veins seems to be mediated via NO and endothelium-dependent hyperpolarization. K_ATP and I_KCa are probably not involved in the hyperpolarization, but activation of BK_Ca may contribute to the hyperpolarization. Prostanoid synthesis and the cytochrome P450-system are probably not involved in the SP-induced relaxation in this area.

     

Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteries

Background and purpose:

Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries.

Experimental approach:

The non-hydrolyzable selective P2Y1 agonist ADPβS (3 µM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca²⁺]_i were obtained in the presence and absence of inhibitors of protein kinase C (PKC).

Key results:

ADPβS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 µM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 µM) or BIS-VIII (1 µM) tended to augment concentration-dependent dilatation to ADPβS (0.1–3 µM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 µM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca²⁺]_i in pressurized arteries confirmed the P2Y1 receptor but not M₃ muscarinic receptor desensitization.

Conclusions and implications:

These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.     

Modulation of relaxation to levcromakalim by S-nitroso-N-acetylpenicillamine (SNAP) and 8-bromo cyclic GMP in the rat isolated mesenteric artery

1. Levcromakalim caused concentration-dependent relaxations of methoxamine-induced tone in both endothelium-denuded and intact vessels. Its potency was reduced by the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP; 0.1 μM or 1 μM) in both denuded and intact vessels. The maximal relaxation (R_max) was reduced only in denuded vessels.
2. SNAP was more potent in endothelium-denuded than intact vessels but there were no differences in R_max. Glibenclamide (10 μM) did not affect relaxation to SNAP in endothelium-denuded or intact vessels.
3. The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) increased the potency and R_max of levcromakalim in endothelium-intact vessels. ODQ had no effect in denuded vessels.
4. ODQ (10 μM) reduced the vasorelaxant potency of SNAP in both intact and endothelium-denuded vessels by 190-fold and 620-fold, respectively.
5. 8-bromo cyclic GMP (10 or 30 μM) reduced both the potency and R_max of levcromakalim in de-endothelialized vessels, but had no effect in intact vessels although it reduced both the potency and R_max of levcromakalim in intact vessels incubated with ODQ (10 μM).
6. In the presence of ODQ (10 μM), SNAP (0.1 μM or 1 μM) reduced the potency of levcromakalim in intact vessels, without altering R_max, but had no effect in denuded vessels. SNAP (50 μM) reduced both the potency and R_max of levcromakalim in intact and endothelium-denuded vessels.
7. Therefore, although SNAP causes relaxation principally through generation of cyclic GMP, it can modulate the actions of levcromakalim through mechanisms both dependent on, and independent of, cyclic GMP; the former predominate in endothelium-denuded vessels and the latter in intact vessels.

     

Actions of 4-chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth muscle and endothelial cells

1. Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca²⁺, apparently through ryanodine-sensitive Ca²⁺ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca²⁺, by use of Ca²⁺ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells.
2. After phenylephrine-(PE, 10 μM) sensitive Ca²⁺ stores were depleted by maximal PE stimulation in Ca²⁺-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca²⁺ was restored for a 30 min refilling period. At the end of this period, Ca²⁺ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca²⁺ store.
3. In a concentration-dependent manner (30, 100 and 300 μM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca²⁺-free media. This suggests that Ca²⁺ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca²⁺-containing or Ca²⁺-free Krebs solution.
4. Restoring Ca²⁺ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca²⁺. The contraction of tissues pretreated with 300 μM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 μM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca²⁺-free and Ca²⁺-containing medium suggesting a rapid reversal of CEP effects.
5. Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K⁺ in the absence of and after 30 min pre-incubation with 30, 100 and 300 μM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K⁺ (77.6, 41.1 and 10.8% of control).
6. Some arterial rings were pre-incubated with ryanodine (30 μM) for 30 min before the construction of PE concentration-response curves. In Ca²⁺-free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9±1.2% of 100 mM K⁺ contraction, n=11) in the presence of external Ca²⁺. EC₅₀ values for PE in ryanodine-treated tissues (1.7±0.25 μM, n=16) were not significantly different from controls (2.5±0.41 μM, n=22). Maximum contractions to PE (118.5±4.4% of 100 mM K⁺ contraction, n=16) were also unaffected by ryanodine when compared to controls (129±4.2%, n=23).
7. When fura-2 loaded smooth muscle cells (n=13) and endothelial cells (n=27) were imaged for Ca²⁺ distribution, it was observed that 100 and 300 μM CEP in Ca²⁺-free medium caused Ca²⁺ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 mM) reversed the effect of CEP on intracellular Ca²⁺ to control values.
8. These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca²⁺ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5-HT and K⁺. The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca²⁺ elevation.