Nebivolol induces NO-mediated relaxations of rat small mesenteric but not of large elastic arteries

Nebivolol is a newer beta1-selective adrenergic receptor antagonist, which unlike classic beta-blockers, lowers systemic vascular resistance by direct vasodilator effects possibly involving NO. This study was designed to determine the effects of nebivolol on small arteries, which contribute to the most part of systemic vascular resistance. Mesenteric arteries, isolated from 9-week-old Wistar-Kyoto (WKY) rats, were studied under perfused and pressurized conditions using a video dimension analyzer. Aortic rings from the same animals were suspended in organ chambers, and isometric tension was measured. Experiments were performed during contraction to prostaglandin F2alpha. In small arteries, nebivolol (10(-9) to 3 x 10(-5) M) induced concentration-dependent relaxations (maximum, 55 +/- 8%). The relaxations were less pronounced as compared with those to acetylcholine (maximum, 99 +/- 2%; p < 0.05), but were significantly greater than those to atenolol (maximum, 2 +/- 0%; p < 0.05). Nebivolol-induced responses were markedly reduced by the NO-synthase inhibitor N(omega)-nitro-L-arginine methylester (L-NAME; 10(-4) M; maximum, 11 +/- 2%; p < 0.05). This inhibition could be entirely reversed by pretreatment with L-arginine (10(-3) M; maximum, 46 +/- 7%), a precursor of NO. In contrast to mesenteric arteries, nebivolol did not affect vascular tension of precontracted aortas. These findings indicate that nebivolol induces NO-mediated relaxations in small arteries but not large elastic vessels and therefore, independent of its antihypertensive action, might be effective in protecting the microcirculation in various cardiovascular disease states.     

Evidence against potassium as an endothelium-derived hyperpolarizing factor in rat mesenteric small arteries

1. Endothelium-derived hyperpolarizing factor (EDHF) has recently been identified as potassium released from endothelial cells into the myo-endothelial space. The present study was designed to test this hypothesis. 2. In rat small mesenteric arteries, mounted in a wire myograph, relaxation to acetylcholine or potassium was not significantly changed following incubation with oxadiazolo-quinoxalin-1-one (ODQ, 4 microM) and indomethacin (10 microM, n = 9). 3. Maximal relaxations to acetylcholine occurred in all arteries, were maintained and were significantly greater (P < 0.01, n = 9) than the transient relaxations to potassium, which only occurred in 30-40% of vessels. 4. Removal of the vascular endothelium abolished relaxant responses both to potassium and acetylcholine (P < 0.005, n = 9). 5. Compared with responses in 5.5 mM potassium PSS, relaxation responses to added potassium in arteries maintained in 1.5 mM potassium PSS were more marked and were not dependent on the presence of an intact endothelium (n = 8). 6. Incubation with BaCl2 (50 microM) significantly inhibited the maximal relaxant response to potassium in the presence of an intact endothelium in 5.5 mM potassium PSS (P < 0.05, n = 4), but had no effect on relaxation of de-endothelialized preparations in 1.5 mM potassium PSS (n = 5). 7. Treatment with ouabain (0.1 mM) abolished the relaxant response to potassium in 1.5 mM potassium PSS (P < 0.001, n = 9), but only partly inhibited the maximal relaxant response to acetylcholine in 5.5 mM potassium PSS (P < 0.01, n = 5). 8. These data show that at physiological concentrations of potassium an intact endothelium is necessary for potassium-induced relaxation in rat mesenteric arteries. Furthermore, the response to potassium is clearly different to that from acetylcholine, indicating that potassium does not mimic EDHF released by acetylcholine in these arteries.     

Functional evidence of a role for two-pore domain potassium channels in rat mesenteric and pulmonary arteries

1. Experiments were performed to elucidate the mechanism by which alterations of extracellular pH (pH(o)) change membrane potential (E(M)) in rat mesenteric and pulmonary arteries. 2. Changing pH(o) from 7.4 to 6.4 or 8.4 produced a depolarisation or hyperpolarisation, respectively, in mesenteric and pulmonary arteries. Anandamide (10 microm) or bupivacaine (100 microm) reversed the hyperpolarisation associated with alkaline pH(o), shifting the E(M) of both vessels to levels comparable to that at pH 6.4. In pulmonary arteries, clofilium (100 microm) caused a significant reversal of hyperpolarisation seen at pH 8.4 but was without effect at pH 7.4. 3. K(+) channel blockade by 4-aminopyridine (4-AP) (5 mm), tetraethylammonium (TEA) (10 mm), Ba(2+) (30 microm) and glibenclamide (10 microm) depolarised the pulmonary artery. However, shifts in E(M) with changes in pH(o) remained and were sensitive to anandamide (10 microm), bupivacaine (100 microm) or Zn(2+) (200 microm). 4. Anandamide (0.3-60 microm) or bupivacaine (0.3-300 microm) caused a concentration-dependent increase in basal tone in pulmonary arteries. 5. RT-PCR demonstrated the expression of TASK-1, TASK-2, THIK-1, TRAAK, TREK-1, TWIK-1 and TWIK-2 in mesenteric arteries and TASK-1, TASK-2, THIK-1, TREK-2 and TWIK-2 in pulmonary arteries. TASK-1, TASK-2, TREK-1 and TWIK-2 protein was demonstrated in both arteries by immunostaining. 6. These experiments provide evidence for the presence of two-pore domain K(+) channels in rat mesenteric and pulmonary arteries. Collectively, they strongly suggest that modulation of TASK-1 channels is most likely to have mediated the pH-induced changes in membrane potential observed in these vessels, and that blockade of these channels by anandamide or bupivacaine generates a small increase in pulmonary artery tone.     

The effect of morphine in rat small mesenteric arteries

We investigated the effect of morphine in phenylephrine (PE)- or KCl-precontracted rat small mesenteric arteries. Morphine (10(-6)-10(-4) M) administration caused concentration-dependent relaxation responses in small mesenteric arteries precontracted by PE or KCl. Removal of endothelium did not significantly alter the relaxation responses to morphine. The relaxant responses to morphine were partially inhibited by pre-treatment of tissues with naloxone (NAL, 10(-5) M) for 20 min. The inhibitory effect of NAL on relaxant responses to morphine in PE- or KCl-precontracted arteries did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of endothelium-intact or endothelium-denuded arterial segments with NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) or cyclooxygenase (COX) inhibitor indomethacin (10(-5) M) or histamine H(1)-receptor blocker diphenhydramine (10(-6) M), for 20 min did not inhibit the relaxation responses to morphine. Small mesenteric arterial segment contractions induced by stepwise addition of calcium to high KCl solution with no calcium were almost completely inhibited by morphine. These findings suggested that morphine-induced relaxation responses in isolated rat small mesenteric arteries were neither dependent on endothelium nor blocked by NOS or COX inhibition but they rather seem to depend on an interaction of morphine with calcium influx pathways.     

Contribution of K+ channels and ouabain-sensitive mechanisms to the endothelium-dependent relaxations of horse penile small arteries

1. Penile small arteries (effective internal lumen diameter of 300–600 μm) were isolated from the horse corpus cavernosum and mounted in microvascular myographs in order to investigate the mechanisms underlying the endothelium-dependent relaxations to acetylcholine (ACh) and bradykinin (BK).
2. In arteries preconstricted with the thromboxane analogue U46619 (3–30 nM), ACh and BK elicited concentration-dependent relaxations, pD₂ and maximal responses being 7.71±0.09 and 91±1% (n=23), and 8.80±0.07 and 89±2% (n=24) for ACh and BK, respectively. These relaxations were abolished by mechanical endothelial cell removal, attenuated by the nitric oxide (NO) synthase (NOS) inhibitor, N^G-nitro-L-arginine (L-NOARG, 100 μM) and unchanged by indomethacin (3 μM). However, raising extracellular K⁺ to concentrations of 20–30 mM significantly inhibited the ACh and BK relaxant responses to 63±4% (P<0.01, n=7) and to 59±4% (P<0.01, n=6), respectively. ACh- and BK-elicited relaxations were abolished in arteries preconstricted with K⁺ in the presence of 100 μM L-NOARG.
3. In contrast to the inhibitor of ATP-sensitive K⁺ channels, the blockers of Ca²⁺-activated K⁺ (K_Ca) channels, charybdotoxin (30 nM) and apamin (0.3 μM), each induced slight but significant rightward shifts of the relaxations to ACh and BK without affecting the maximal responses. Combination of charybdotoxin and apamin did not cause further inhibition of the relaxations compared to either toxin alone. In the presence of L-NOARG (100 μM), combined application of the two toxins resulted in the most effective inhibition of the relaxations to both ACh and BK. Thus, pD₂ and maximal responses for ACh and BK were 7.65±0.08 and 98±1%, and 9.17±0.09 and 100±0%, respectively, in controls, and 5.87±0.09 (P<0.05, n=6) and 38±11% (P<0.05, n=6), and 8.09±0.14 (P<0.01, n=6) and 98±1% (n=6), respectively, after combined application of charybdotoxin plus apamin and L-NOARG.
4. The selective inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 μM) did not alter the maximal responses to either ACh or BK, but slightly decreased the sensitivity to both agonists, δpD₂ being 0.25±0.07 (P<0.05, n=6) and 0.62±0.12 (P<0.01, n=6) for ACh and BK, respectively. Combined application of ODQ and charybdotoxin plus apamin produced further inhibition of the sensitivity to both ACh (δpD₂=1.39±0.09, P<0.01, n=6) and BK (1.29±0.11, P<0.01, n=6), compared to either ODQ or charybdotoxin plus apamin alone.
5. Exogenous nitric oxide (NO) present in acidified solutions of sodium nitrite (NaNO₂) and S-nitroso-cysteine (SNC) both concentration-dependently relaxed penile resistance arteries, pD₂ and maximal responses being 4.84±0.06 and 82±3% (n=12), and 6.72±0.07 and 85±4% (n=19), respectively. Charybdotoxin displaced to the right the dose-relaxation curves for both NO (δpD₂ 0.38±0.06, P<0.01, n=6) and SNC (δpD₂ 0.50±0.10, P<0.01, n=5), whereas apamin only reduced sensitivity (δpD₂=0.35±0.12, P<0.05, n=5) and maximum response (65±9%, P<0.05, n=6) to SNC. ODQ shifted to the right the dose-relaxation curves to both NO and SNC. The relaxant responses to either NO or SNC were not further inhibited by a combination of ODQ and charybdotoxin or ODQ and charybdotoxin plus apamin, respectively, compared to either blocker alone.
6. In the presence of 3 μM phentolamine, 5 μM ouabain contracted penile resistance arteries by 50±6% (n=17) of K-PSS, but did not significantly change the relaxant responses to either ACh, BK or NO. However, in the presence of L-NOARG ouabain reduced the ACh- and BK-elicited relaxation from 94±3% to 16±5% (P<0.0001, n=6), and from 98±2% to 13±3% (P<0.0001, n=5), respectively. Combined application of ODQ and ouabain inhibited the relaxations to NO from 92±2% to 26±3% (P<0.0001, n=6).
7. The present results demonstrate that the endothelium-dependent relaxations of penile small arteries involve the release of NO and a non-NO non-prostanoid factor(s) which probably hyperpolarize(s) smooth muscle by two different mechanisms: an increased charybdotoxin and apamin-sensitive K⁺ conductance and an activation of the Na⁺-K⁺ATPase. These two mechanisms appear to be independent of guanylate cyclase stimulation, although NO itself can also activate charybdotoxin-sensitive K⁺ channels and the Na⁺-K⁺ pump through both cyclic GMP-dependent and independent mechanisms, respectively.

     

Elevated pressure selectively blunts flow-evoked vasodilatation in rat mesenteric small arteries

BACKGROUND AND PURPOSE: The present study investigated mechanisms underlying impaired endothelium-dependent vasodilatation elicited by elevating the intraluminal pressure in rat mesenteric small arteries. EXPERIMENTAL APPROACH: Arterial segments (internal diameter 316+/-2 microm, n=86) were mounted in a pressure myograph. The effect of elevating pressure from 50 to 120 mmHg for 1 h before resetting it to 50 mmHg was studied on endothelium-dependent vasodilatation. KEY RESULTS: In arteries constricted with U46619 in the presence of indomethacin, shear stress generated by flow, evoked vasodilatation that was abolished by an inhibitor of nitric oxide (NO) synthase, asymmetric dimethylarginine (1 mM), whereas acetylcholine-induced vasodilatation was unchanged. After elevation of intraluminal pressure for 1 h and then resetting it to 50 mmHg, vasodilatation induced by shear stress and the NO donor, S-nitrosopenicillamine was inhibited, while vasodilatation induced by a guanylyl cyclase activator, BAY 412272, and acetylcholine was unaltered. Superoxide levels sensitive to polyethylene glycol superoxide dismutase were increased in segments exposed to elevated pressure. A superoxide scavenger, tempol (300 microM), a general endothelin receptor antagonist, SB 217242 and the selective ET(A) receptor antagonist, BQ 123 preserved shear stress-evoked vasodilatation. CONCLUSIONS AND IMPLICATIONS: The present study shows that transient exposure to an elevated intraluminal pressure selectively inhibits flow-evoked NO-mediated vasodilatation, probably through activation of endothelin receptors and increased formation of superoxide. In contrast, elevation of pressure did not affect the acetylcholine-evoked endothelium-derived hyperpolarizing factor type vasodilatation in mesenteric small arteries.     

Action of ryanodine on neurogenic responses in rat isolated mesenteric small arteries

1. Rat mesenteric arteries (∼250 μm) were set up in a single-channel isometric myograph designed to allow fluorescence measurements concurrent with field stimulation of intramural nerves. Vessels were loaded with 6 μM fura-2AM for 2 h and simultaneous recordings of neurogenic contraction (force) and intracellular calcium [Ca²⁺]_i were obtained. In other experiments, arteries were loaded with 1 μCi ml⁻¹ [³H]-noradrenaline (NA) for 30 min in order to measure release of [³H]-NA in response to field stimulation to examine whether ryanodine directly inhibited neuronal release of NA.
2. Arteries were activated by single intermittent field stimulation or continuously to excite intrinsic sympathetic nerves, or by cumulative addition of noradrenaline (1 nM–10 μM) to the bathing solution.
3. Pre-incubation with ryanodine markedly inhibited the contraction and [Ca²⁺]_i release in response to single-pulse nerve stimulation. Ryanodine also inhibited an early phasic component of the response to continuous field stimulation and reduced the rate of rise in force in response to continuous field stimulation. However, stable maximal contraction and [Ca²⁺]_i in response to continuous field stimulation as well as maximal responses to exogenous NA were unaffected. Release of [³H]-NA in response to single intermittent field stimulation was not affected by ryanodine when compared to vehicle.
4. Our results suggest that brief intermittent activation of intramural sympathetic nerves increases [Ca²⁺]_i and contracts small arteries primarily by releasing Ca²⁺ from a ryanodine-sensitive intracellular store. In contrast, the stable rise in tone and [Ca²⁺]_i resulting from continuous nerve stimulation may largely depend on sources of Ca²⁺ other than the ryanodine-sensitive intracellular store.

     

Notoginsenoside Ft1 activates both glucocorticoid and estrogen receptors to induce endothelium-dependent,nitric oxide-mediated relaxations in rat mesenteric arteries

Panax notoginseng (Burk.) F.H. Chen has been used traditionally for the treatment of cardiovascular diseases. Notoginsenoside Ft1 (Ft1) is a bioactive saponin from the leaves of P. notoginseng. Experiments were designed to determine whether or not Ft1 is an endothelium-dependent vasodilator. Rat mesenteric arteries were suspended in organ chambers for the measurement of isometric tension during phenylephrine-induced contractions. The cyclic guanosine monophosphate (cGMP) level was assessed using enzyme immunoassay. The phosphorylation and protein expressions of endothelial nitric oxide synthase (eNOS), glucocorticoid receptors (GR), estrogen receptors beta (ERß), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) were determined by Western blotting. The localization of GR and ERß were determined by immunofluorescence staining. Ft1 caused endothelium-dependent relaxations, which were abolished by l-NAME (inhibitor of nitric oxide synthases) and ODQ (inhibitor of soluble guanylyl cyclase). Ft1 increased the cGMP level in rat mesenteric arteries. GR and ERß were present in the endothelial layer and their antagonism by RU486 and PHTPP, respectively, inhibited Ft1-induced endothelium-dependent relaxations and phosphorylations of eNOS, Akt and ERK1/2. Inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin and ERK1/2 by U0126 reduced Ft1-evoked relaxations and eNOS phosphorylation. Taken in conjunction, the present findings suggest that Ft1 stimulates endothelial GRs and ERßs with subsequent activation of the PI3K/Akt and ERK1/2 pathways in rat mesenteric arteries. This results in phosphorylation of eNOS and the release of NO, which activates soluble guanylyl cyclase in the vascular smooth muscle cells leading to relaxations.     

Potassium channel activation and relaxation by nicorandil in rat small mesenteric arteries

1. We used whole-cell patch clamp to investigate the currents activated by nicorandil in smooth muscle cells isolated from rat small mesenteric arteries, and studied the relaxant effect of nicorandil using myography.
2. Nicorandil (300 μM) activated currents with near-linear current-voltage relationships and reversal potentials near to the equilibrium potential for K⁺.
3. The nicorandil-activated current was blocked by glibenclamide (10 μM), but unaffected by iberiotoxin (100 nM) and the guanylyl cyclase inhibitor LY 83583 (1 μM). During current activation by nicorandil, openings of channels with a unitary conductance of 31 pS were detected.
4. One hundred μM nicorandil had no effect on currents through Ca²⁺ channels recorded in response to depolarizing voltage steps using 10 mM Ba²⁺ as a charge carrier. A small reduction in current amplitude was seen in 300 μM nicorandil, though this was not statistically significant.
5. In arterial rings contracted with 20 mM K⁺ Krebs solution containing 200 nM BAYK 8644, nicorandil produced a concentration-dependent relaxation with mean pD₂=4.77±0.06. Glibenclamide (10 μM) shifted the curve to the right (pD₂=4.32±0.05), as did 60 mM K⁺. LY 83583 caused a dose-dependent inhibition of the relaxant effect of nicorandil, while LY 83583 and glibenclamide together produced greater inhibition than either alone.
6. Metabolic inhibition with carbonyl cyanide m-chlorophenyl hydrazone (30 nM), or by reduction of extracellular glucose to 0.5 mM, increased the potency of nicorandil.
7. We conclude that nicorandil activates K_ATP channels in these vessels and also acts through guanylyl cyclase to cause vasorelaxation, and that the potency of nicorandil is increased during metabolic inhibition.

     

Involvement of T-type calcium channels in excitatory junction potentials in rat resistance mesenteric arteries

1. We investigated the role of voltage-operated calcium channels in sympathetic transmission and depolarization-induced contractions in the rat mesenteric artery. In particular, we investigated the role of the T-type voltage-operated calcium channels (T-channels) in mediating excitatory junction potentials (EJPs). 2. EJPs were evoked by electrical field stimulation (trains of five stimuli at 0.9 Hz) in small mesenteric arteries. The average resting membrane potential was -59.8+/-0.5 mV (n=65). Trains of stimuli evoked individual EJPs with the peak EJP of 6+/-0.2 mV (n=34) occurring with the second stimulus. Trains of EJPs were inhibited 90% by tetrodotoxin (0.1 micro M) or by omega-conotoxin GVIA (GVIA, 10 nM) indicating their neural origin. 3. The EJPs were not inhibited by the L-type calcium channel blocker nicardipine at 0.1 micro M, a concentration sufficient to abolish the contraction to potassium depolarization. However, mibefradil (3 micro M), considered a relatively selective T-channel antagonist, inhibited the EJPs by about 50%. This concentration of mibefradil did not inhibit GVIA-sensitive electrically-evoked twitches of the rat vas deferens. Thus the action of mibefradil in reducing EJPs is unlikely to be due to either inhibition of L- or N-type channels but is probably due to inhibition of T-channels. 4. The finding that Ni(2+) (300 micro M), an inhibitor of T-type calcium channels, also reduced EJP amplitude by about 80% but did not block electrically-evoked twitches in the rat vas deferens, further supports an important role of T-channels in mediating small depolarizations associated with the EJPs evoked by sympathetic nerve stimulation.     

Blockade of swelling-induced chloride channels by phenol derivatives.

1. In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine Kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2. RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3. RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4. The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5. The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein.     

Comparison of noradrenaline and lysosphingolipid-induced vasoconstriction in mouse and rat small mesenteric arteries

1 We have compared vasoconstriction responses in isolated mesenteric small arteries from mice and rats as elicited by KCl, noradrenaline and the lysosphingolipids sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). 2 Contractile responses to KCl and noradrenaline, but not those of S1P or SPC, were significantly related to vessel diameter in both species. 3 When comparing vessels of similar diameter, contractile responses for KCl and the three agonists were much smaller in mice than in rats, e.g. 8.3 +/- 0.4 vs. 14.7 +/- 0.7 mn for noradrenaline. 4 Based upon the antagonist rank order of potency of prazosin (pKB 8.80) > B8805-033 (pKB 7.89) > yohimbine (pKB 6.18) approximately BMY 7378 (pA2 6.03), noradrenaline responses in mice were mediated solely via alpha1A-adrenoceptors, similar to what repeatedly has been shown in rats. 5 The S1P3 receptor antagonist suramin (100 microM) significantly inhibited responses to S1P and SPC in rats but not in mice, and did not affect noradrenaline responses in either species. 6 We conclude that for any given diameter, mouse mesenteric arteries develop less contraction in response to various stimuli. Noradrenaline acts via alpha1A-adrenoceptors in both species. Responses to S1P and SPC differ between both species with regard to suramin-sensitivity indicating involvement of different receptor subtypes for lysosphingolipids in both species.     

NO and KATP channels underlie endotoxin-induced smooth muscle hyperpolarization in rat mesenteric resistance arteries

1 Smooth muscle membrane potential and tension measurements were made in isolated mesenteric resistance arteries from rats exposed to bacterial endotoxin (lipopolysaccharide, LPS; 10 mg kg(-1), i.p.) for 3 h to mimic septic shock syndrome. 2 Over this period, rats developed an endotoxaemic response, assessed in vivo as a 41+/-4 mmHg drop in mean blood pressure, vascular hyporeactivity to noradrenaline (1 microg kg(-1), i.v.) and a significant increase in core body temperature. 3 In mesenteric small resistance arteries from these rats (o.d. 180 - 240 microm), phenylephrine (0.01-3 microm)-evoked contraction was not altered when compared with arteries from sham-operated animals, but the concentration-relaxation curve to acetylcholine (ACh; 0.01 - 3 microm) displayed a small, but significant, shift to the right. 4 The smooth muscle resting membrane potential (-70.3+/-1.6 mV) in arteries from LPS-treated rats was significantly greater than in control arteries (-55.4+/-1.2 mV), but in both cases the smooth muscle was depolarized to a similar potential by the application of N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mm; -54.1+/-2.3 vs -52.4+/-2.5 mV) or glibenclamide (10 microm; -55.0+/-2.1 vs -50.4+/-2.0 mV). 5 ACh (1 microm) elicited a maximal hyperpolarization, which ranged from -14.7+/-3.2 mV (in arteries from LPS-treated rats) to -20.6+/-2.4 mV (in arteries from sham-operated rats), and was not altered by the presence of L-NAME. Levcromakalim (1 microm) increased the smooth muscle membrane potential by around -24 mV in arteries from both sets of experimental animals. 6 These results indicate that at the level of the resistance vasculature, endotoxaemia is associated with pronounced smooth muscle hyperpolarization reflecting the action of NO on KATP channels. These changes were not associated with vascular hyporeactivity or depressed endothelial cell function in vitro, suggesting that mesenteric resistance arteries may not contribute to equivalent changes in vivo.     

Differential effects of glucose on agonist-induced relaxations in human mesenteric and subcutaneous arteries

BACKGROUND AND PURPOSE: Acute periods of hyperglycaemia are strongly associated with vascular disorder, yet the specific effects of high glucose on human blood vessel function are not fully understood. In this study we (1) characterized the endothelial-dependent relaxation of two similarly sized but anatomically distinct human arteries to two different agonists and (2) determined how these responses are modified by acute exposure to high glucose. EXPERIMENTAL APPROACH: Ring segments of human mesenteric and subcutaneous arteries were mounted in a wire myograph. Relaxations to acetylcholine and bradykinin were determined in a control (5 mM) and high glucose (20 mM) environment over a 2 and 6 h incubation period. KEY RESULTS: Bradykinin-induced relaxation in both sets of vessels was mediated entirely by EDHF whilst that generated by acetylcholine, though principally generated by EDHF, also had contribution from prostacyclin and possibly nitric oxide in mesenteric and subcutaneous vessels, respectively. A 2-h incubation of high glucose impaired bradykinin-induced relaxation of subcutaneous vessels whilst, in contrast, the relaxation generated by bradykinin in mesenteric vessels was enhanced at the same time point. High glucose significantly augmented the relaxation generated by acetylcholine in mesenteric and subcutaneous vessels at a 2 and 6 h incubation point, respectively. CONCLUSIONS AND IMPLICATIONS: Short periods of high glucose exert a variable influence on endothelial function in human isolated blood vessels that is dependent on factors of time, agonist-used and vessel studied. This has implications for how we view the effects of acute hyperglycaemia found in patients with diabetes mellitus as well as other conditions.     

L-tryptophan ethyl ester dilates small mesenteric arteries by inhibition of voltage-operated calcium channels in smooth muscle

BACKGROUND AND PURPOSE

L-tryptophan (L-W) is a precursor of the vasoconstrictor, 5-HT. However, acute administration of L-W ethyl ester (L-Wee) lowered blood pressure. The mechanism of action is unknown. This study compares the vascular effects of L-W and L-Wee in intact animals, isolated aortic rings, small mesenteric arteries (MA) and explores possible mechanisms by studies in vascular smooth muscle cells (VSMC) of MA.

EXPERIMENTAL APPROACH

Effects of L-W or L-Wee (5–50 mg kg⁻¹, i.v.) on mean arterial pressure (MAP) and heart rate (HR) were determined in male Sprague-Dawley rats. The effects of L-W and L-Wee on basal tone and of phenylephrine- or KCl-induced contractions of aortic and MA rings were assessed. Effects of L-Wee and L-W on voltage-operated calcium channels (VOCC) of VSMC of MA were also examined in patch-clamp studies.

KEY RESULTS

Administration of L-Wee, but not L-W, evoked a rapid and transient dose-dependent decrease in MAP and HR. While both agents failed to affect basal tone, L-Wee decreased, concentration-dependently, (I_max > 98%) tension responses to phenylephrine and KCl in an endothelium-independent manner in aorta (IC₅₀ 2 mM) and MA (IC₅₀ 17 µM). L-Wee evoked concentration-dependent inhibition of VOCC currents (IC₅₀ 12 µM; I_max 90%) in VSMC of MA.

CONCLUSIONS AND IMPLICATIONS

Esterified L-W (L-Wee), but not L-W, preferentially relaxed resistance vessels rather than conduit vessels. These effects were associated with blockade of VOCC by L-Wee. Our findings suggest that the falls in MAP and HR induced by L-Wee were due to blockade of VOCC by L-Wee.     

Testosterone-induced vasorelaxation in the rat mesenteric arterial bed is mediated predominantly via potassium channels

We have investigated the involvement of nitric oxide and K(+) channels in the vasorelaxant responses to physiologically-relevant concentrations of testosterone in the rat isolated mesenteric arterial bed. Testosterone (100 pM - 10 microM) elicited concentration-dependent relaxations in the isolated mesenteric arterial bed (pEC(50)=9.47 (9.22 - 9.73, 95% CI), maximal relaxation, R(max)=62.8+/-2.0%, n=6). A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM) or removal of the endothelium significantly inhibited maximal relaxations to testosterone (L-NAME: R(max)=51.4+/-1.1%, P<0.01, n=6; endothelium-denuded: R(max)=46.9+/-2.8%, P<0.001, n=5). Raising the extracellular K(+) concentration to 30 and 60 mM, or pre-treatment with 300 microM tetrabutylammonium chloride (TBA), a calcium-activated K(+) channel inhibitor, abolished vasorelaxations induced by testosterone. A selective inhibitor of ATP-sensitive K(+) (K(ATP)) channels, glibenclamide (10 microM) and an inhibitor of voltage-sensitive K(+) (K(V)) channels, 4-aminopyridine (4-AP, 1 mM) did not affect testosterone-induced responses. Vasorelaxation to 1 microM testosterone was significantly (P<0.05) inhibited by 100 nM charybdotoxin (ChTx), an inhibitor of large conductance calcium-activated K(+) (BK(Ca)) channels (control: 63.3+/-9.9%, n=6; ChTx: 11.9+/-12.7%, n=3). Neither the testosterone receptor antagonist, flutamide (10 microM) nor an aromatase inhibitor, aminoglutethimide (10 microM) inhibited testosterone-induced responses. In conclusion, the present findings demonstrate, in the rat isolated mesenteric arterial bed, that testosterone causes acute vasorelaxations at physiologically relevant concentrations which are, in part, mediated via NO- and endothelium-dependent pathways. However, the activation of BK(Ca) channels plays a substantial role in testosterone-induced vasorelaxation.     

Vanilloid receptors on capsaicin-sensitive sensory nerves mediate relaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arteries

In the present study, the vasodilator actions of methanandamide and capsaicin in the rat isolated mesenteric arterial bed and small mesenteric arterial segments were investigated. Methanandamide elicited concentration-dependent relaxations of preconstricted mesenteric arterial beds (pEC(50)=6.0+/-0.1, E(max)=87+/-3%) and arterial segments (pEC(50)=6.4+/-0.1, E(max)=93+/-3%). In arterial beds, in vitro capsaicin pre-treatment blocked vasorelaxation to 1 and 3 microM methanandamide, and reduced to 12+/-7% vasorelaxation to 10 microM methanandamide. Methanandamide failed to relax arterial segments pre-treated in vitro with capsaicin. In arterial beds from rats treated as neonates with capsaicin to cause destruction of primary afferent nerves, methanandamide at 1 and 3 microM did not evoke vasorelaxation, and relaxation at 10 microM methanandamide was reduced to 26+/-4%. Ruthenium red (0.1 microM), an inhibitor of vanilloid responses, attenuated vasorelaxation to methanandamide in arterial beds (pEC(50)=5.6+/-0.1, E(max)=89+/-1%). Ruthenium red at 1 microM abolished the response to 1 microM methanandamide, and greatly attenuated relaxation at 3 and 10 microM methanandamide in arterial beds. In arterial segments, ruthenium red (0.15 microM) blocked vasorelaxation to methanandamide, but not to CGRP. In arterial segments, the vanilloid receptor antagonist capsazepine (1 microM) inhibited, and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8 - 37) (3 microM) abolished, methanandamide-induced relaxations. CGRP(8 - 37), but not capsazepine, attenuated significantly relaxation to exogenous CGRP. These data show that capsaicin and ruthenium red attenuate vasorelaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments. In addition, CGRP(8 - 37) and capsazepine antagonize responses to methanandamide in mesenteric arterial segments. In conclusion, vanilloid receptors on capsaicin-sensitive sensory nerves play an important role in the vasorelaxant action of methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments.     

Blockade of glutamatergic and GABAergic receptor channels by trimethyltin chloride

1. Organotin compounds such as trimethyltin chloride (TMT) are among the most toxic of the organometallics. As their main target for toxicity is the central nervous system, the aim of the present study was to investigate the effects of TMT on receptor channels involved in various processes of synaptic transmission. 2. The Xenopus oocyte expression system was chosen for direct assessment of TMT effects on voltage-operated potassium channels and glutamatergic and GABAergic receptors, and hippocampal slices from rat brain for analyzing TMT effects on identified synaptic sites. 3. TMT was found to be ineffective, at 100 micromol l(-1), against several potassium- and sodium-operated ion channel functions as well as the metabotropic glutamate receptor. 4. The functions of the ionotropic glutamate and the GABA(A) receptor channels were inhibited by TMT in micromolar concentrations. Thus, at a maximum concentration of 100 micromol l(-1), around 20-30% of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and GABA(A) receptor-mediated ion currents and 35% of the N-methyl-D-aspartate receptor-mediated ion currents were blocked. 5. In the hippocampal slice model, the inhibitory effects of TMT were much stronger than expected from the results on the ion channels. Bath application of TMT significantly reduced the amplitudes of evoked excitatory postsynaptic field potentials in a concentration-dependent and nonreversible manner. 6. Induction of long-term potentiation, recorded from the CA1 dendritic region, was inhibited by TMT and failed completely at a concentration of 10 micromol l(-1). 7. In general, TMT affects the excitatory and inhibitory synaptic processes in a receptor specific manner and is able to disturb the activity within a neuronal network.     

Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric arteries

1. We have studied the effect of endothelin-1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist.
2. Endothelin-1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration-dependent (1–100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α-toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin-1-induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin A1.
3. In intact small arteries tyrphostin A23 inhibited the force developed to endothelin-1 at all concentrations studied; at higher concentrations (10 and 100 nM) the profile of contraction was altered from a sustained to a transient response. Tyrphostin A1 inhibited the contractile response to endothelin-1 at all concentrations except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endothelin-1 (100 nM) in the absence of extracellular calcium.
4. In rat α-toxin permeabilized mesenteric arteries endothelin-1 caused a concentration-dependent increase in force in the presence of 10 μM GTP and low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin A1 did not affect endothelin-1-induced calcium sensitization of force.
5. We conclude that increased tyrosine phosphorylation is important in the contractile response induced by endothelin-1 in intact small mesenteric arteries. Furthermore our data implicate activation of this signalling pathway in the tonic phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.

     

TRPC3 is involved in flow- and bradykinin-induced vasodilation in rat small mesenteric arteries

AIM: To test the possible involvement of TRPC3 in agonist-induced relaxation and flow-induced vasodilation in rat small mesenteric arteries. METHODS: Male Sprague-Dawley rats were used in the present study. After 72 h-treatment of antisense oligo via tail vein injection, isometric tension and isobaric diameter measurement were carried out with isolated mesenteric artery segments by using either a Pressure Myograph or a Multi Myograph system. Endothelial [Ca(2+)]i changes were measured with a MetaFluor imaging system in response to flow or to 30 nmol/L bradykinin. RESULTS: Immunohistochemical study showed that the 72 h-treatment of antisense oligo via tail vein injection markedly decreased the TRPC3 expression in mesenteric arteries, indicating the effectiveness of the antisense oligo. Isometric tension and isobaric diameter measurement showed that, although the antisense oligo treatment did not affect histamine-, ATP-, and CPA-induced relaxation, it did reduce the magnitude of flow-induced vasodilation by approximately 13% and decreased bradykinin-induced vascular relaxation with its EC50 value raised by nearly 3-fold. Endothelial [Ca(2+)]i measurement revealed that treatment of the arteries with antisense oligos significantly attenuated the magnitude of endothelial [Ca(2+)]i rise in response to flow and to 30 nmol/L bradykinin. CONCLUSION: The results suggest that TRPC3 is involved in flow- and bradykinin-induced vasodilation in rat small mesenteric arteries probably by mediating the Ca(2+) influx into endothelial cells.