Characterizations of the α1-adrenoceptor subtypes mediating contractions of the human internal anal sphincter.

Human internal anal sphincter (IAS) is contracted by α1-adrenoceptor stimulation and thus α1-adrenoceptor agonists may be useful in treating fecal incontinence. This study characterizes the contribution of α1-adrenoceptor subtypes in contraction of human IAS and to investigate the age-related risk of patients with fecal incontinence. IAS and inferior mesenteric artery (IMA), as a predictor of systemic arterial pressure, were obtained from 11 patients. Both muscle strips were assessed by isometric-contraction experiments using phenylephrine, further in IAS, in the presence of various subtype selective α1-adrenoceptor antagonists. Immunohistochemistry and gene expression studies were performed in the same samples. The mean pEC50 values with SEM of phenylephrine in IAS (6.30 ± 0.13) were higher than those of IMA (5.60 ± 0.10). Furthermore, the age-related pEC50 change of IAS was observed between age <70 and ≥70 (6.58 ± 0.13 and 6.07 ± 0.16, respectively (P < 0.05)). In IAS, rightward shift of the concentration-response curves of phenylephrine was observed with three α1-adrenoceptor antagonists. Each pKB value of silodosin, BMY-7378 and prazosin was 9.36 ± 0.53, 7.28 ± 0.20 and 8.89 ± 0.12, respectively. These pKB values and gene expression studies indicated that α1A-adrenoceptor subtypes predominantly contributed to human IAS contraction.

Two distinct dysfunctions in diabetic mouse mesenteric artery contraction are caused by changes in the Rho A-Rho kinase signaling pathway.

Diabetic complications are associated with small artery dysfunctions. The objective of this study was to identify differences in endothelial cell-denuded mesenteric artery second branch (mesenteric artery-2) contraction, as a typical small artery, between diabetic and non-diabetic mice. Contractile responses in mesenteric artery-2 were assessed in male type 2 diabetic ob/ob mice aged 16-22 weeks and in age-matched control (Lean) mice. Phenylephrine induced dose-dependent contractions in Lean mice (1126.8 ± 28.6 mN/mm tissue at 10 µM phenylephrine; n=5), which were significantly reduced in ob/ob mice (716.8 ± 40.8 mN/mm at 10 µM phenylephrine; n=5). Exposure to high glucose (HG; twice the normal glucose [NG] concentration) enhanced phenylephrine-induced contraction in Lean (1341.4 ± 15.5 mN/mm; n=5) but not in ob/ob mice. These dysfunctions did not involve α(1)-receptor sensitization or protein kinase activity, although the calcium sensitivity of contraction was decreased in ob/ob mice. The Rho kinase inhibitor Y27632 suppressed the difference between Lean and ob/ob mice under NG conditions, which was accompanied by Rho A inactivation. Under HG conditions, glucose-dependent Rho A activation persisted in ob/ob mice whereas Rho kinase expression was reduced. These data suggest that inactivation of Rho A reduced contractibility under NG conditions, and the lack of glucose dependency is associated with reduced Rho kinase expression.

Pharmacological modulation of gut mucosal and large vessel blood flow.

BACKGROUND: Constipation, diminished gut blood flow, ischaemic colitis and drug therapy may be associated. AIM: To study the effect of constipating medication on, and the regulation of, gut blood flow. METHODS: 24 healthy females (mean age 30) received, in a double-blind, three-way crossover study: (i) placebo, (ii) ipratropium 40 microg by inhalation (positive control known to reduce rectal mucosal blood flow) and (iii) oral loperamide 4 mg. Mucosal blood flow was measured at the splenic flexure and rectum using laser Doppler flowmetry. Blood flow in the superior and inferior mesenteric arteries was measured by trans-abdominal Doppler ultrasound. RESULTS: Ipratropium decreased rectal mucosal blood flow by 16% (P=0.009) and splenic flexure mucosal blood flow by 8% (P=0.075). Loperamide caused no change in rectal (P=0.40) or splenic flexure mucosal blood flow (P=0.73). Neither treatment changed superior or inferior mesenteric artery blood flow. Splenic flexure mucosal blood flow showed a positive correlation with rectal mucosal blood flow (r=0.69; P<0.0001). CONCLUSIONS: Vasoactive agents may reduce gut mucosal blood flow in the absence of reduced large vessel flow. Constipating drugs do not necessarily reduce gut blood flow. Rectal mucosal blood flow correlates with splenic flexure mucosal flow, and potentially may be used as a more convenient surrogate for studying splenic flexure blood flow.

Isolated rat inferior mesenteric artery response to adenosine: possible participation of Na+/K+-ATPase and potassium channels.

Adenosine (10(-7)-3 x 10(-4) M) produced concentration-dependent and endothelium-independent relaxation of isolated rat inferior mesenteric artery. Application of indomethacin (10(-5) M) or N(G)-nitro-L-arginine (10(-5) M) did did not alter adenosine-elicited relaxation. Conversely, in the presence of high concentration of K+ (100 mM), ouabain (10(-4)) or combination of tetraethylammonium (5 x 10(-4) M) and glibenclamide (10(-6) M), adenosine-evoked relaxant effect was significantly reduced. In K+-free solution, 1-3 mM potassium induced relaxation, which was partially reversed by ouabain (10(-4) M). 1,3-Dipropyl-8-cyclopentylxanthine (10(-9) M), an A1-receptor antagonist, did not affect adenosine-evoked relaxation. Oppositely, 8-(3-chlorostyryl)-caffeine (3 x 10(-7)-10(-6) M), a selective A2A receptor antagonist, significantly inhibited adenosine-induced relaxation in a concentration-dependent manner (pA2 = 6.74). These results indicate that in the isolated rat inferior mesenteric artery, adenosine produces endothelium-independent relaxation, which is partly induced by activation of smooth muscle adenosine A2A receptors, and further mediated by the activation of smooth muscle Na+/K+-ATPase and opening of mixed population of K+ channels.

Effect of peptide Vilon on the content of transforming growth factor-beta and permeability of microvessels during experimental chronic renal failure.

We studied the effect of Vilon in rats 2, 4, and 6 months after the onset of chronic renal failure. Subcutaneous injection of Vilon significantly decreased serum concentration of transforming growth factor-beta(1) and permeability of mesenteric microvessels in rats 2 months after the onset of chronic renal failure. Our results indicate that the preparation produces a potent homeostatic effect in the early period of chronic renal failure.

Argan (Argania spinosa) oil lowers blood pressure and improves endothelial dysfunction in spontaneously hypertensive rats.

Traditionally hand-pressed argan oil, obtained from Argania spinosa seeds, is eaten raw in south-west Morocco; its rich composition of tocopherols, MUFA and PUFA make a study of its actions on risk factors for CVD, such as hypertension, interesting. The effects of 7 weeks of treatment with argan oil (10 ml/kg) on the blood pressure and endothelial function of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats were investigated. Systolic blood pressure and heart rate were measured every week by the tail-cuff method and endothelial function was assessed by carbachol (10(-8) to 10(-4) M)-induced relaxations of aortic rings and small mesenteric arteries pre-contracted with phenylephrine. Argan-oil administration reduced the mean blood pressure of SHR after the fifth week of treatment (P<0.05) and increased (P<0.01) the endothelial responses of arteries from SHR. The NO synthase inhibitor, L-N-omega-nitroarginine (3 x 10(-5) M) revealed a greater participation of NO in the relaxant effect after the treatment. When cyclooxygenase (COX) was blocked with indomethacin (10(-5) M), an involvement of COX products in the endothelium-dependent response was characterized. Enzyme immunoassay of thromboxane B2 showed a significant decrease (P<0.05) in the release of thromboxane A2 in both aorta and small mesenteric artery after argan-oil treatment of SHR. Experiments in the presence of the thromboxane A2-prostaglandin H2 receptor antagonist ICI 192,605 (10(-5) M) confirmed this result. Results after incubation with the antioxidants superoxide dismutase and catalase suggested that a decreased oxidative stress might contribute to explain the beneficial effects of argan-oil treatment.

Vasorelaxation and hyperpolarisation of rat small mesenteric artery by the quaternary anion tetraphenylboron.

This study characterises the vasorelaxation and hyperpolarisation effects of the negatively charged quaternary compound tetraphenylboron (TPB) in the rat small mesenteric artery. Segments of rat small mesenteric artery were mounted in a myograph and vessel tone and membrane potential were measured simultaneously. In vessels pre-contracted with vasopressin (0.3-0.6 nM), U46619 (30-90 nM) or methoxamine (0.3-3 microM), TPB (0.1-100 microM) produced a marked endothelium-independent relaxation. However, vasorelaxation responses to TPB were abolished in tissues pre-contracted with K(+) (50 mM), and significantly inhibited by glibenclamide (glib, 10 microM). In the absence of tone, TPB (1-30 microM) caused a concentration-dependent membrane hyperpolarisation of rat mesenteric artery smooth muscle cells, which was not dependent on the endothelium, but sensitive to glibenclamide (10 microM). In methoxamine (0.3-3 microM) pre-contracted vessels, the relaxation response was associated with a marked hyperpolarisation, which was also sensitive to glibenclamide (10 microM), further inhibited by a combination of K(+) channel blockers (glib [10 microM], charybdotoxin [100 nM], apamin [100 nM], 4-aminopyridine [1 mM] and Ba(2+) [30 microM]) and abolished by 50 mM K(+). The results of this study show that TPB causes a vasorelaxation and hyperpolarisation response in the rat small mesenteric artery through a direct action on the vascular smooth muscle. TPB exerts its effects partially via the activation of K(ATP) channels, but also by another mechanism involving K(+)-dependent hyperpolarisation.

Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice.

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg x kg-1 x day-1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

Multiple P2Y receptors mediate contraction in guinea pig mesenteric vein.

Vasoconstrictor responses to exogenous adenine and pyrimidine nucleotides were measured in endothelium-denuded segments of guinea pig mesenteric vein and compared with responses in mesenteric artery. The rank order of potency for nucleotides in veins was: 2-MeSADP = 2-MeSATP > UTP > ATPgammaS = alpha,betaMeATP > UDP = ATP > ADP >> beta,gamma-D-MeATP = beta,gamma-L-MeATP. In contrast 2-MeSADP, UTP, and UDP were inactive in arteries, and the rank order of potency of other nucleotides differed; that is, alpha,betaMeATP > beta, gamma-D-MeATP > beta,gamma-L-MeATP = ATPgammaS = 2-MeSATP > ATP > ADP. In veins, UTP, ATP, and 2-MeSATP were more efficacious contractile agents than alpha,beta MeATP. In addition, the ability to desensitize responses to these nucleotides and inhibit them with various blockers differed. The response to alpha,betaMeATP in veins exhibited rapid desensitization and was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS) and suramin. The response to 2-MeSATP in veins did not desensitize; nor was it inhibited by prior alpha,betaMeATP desensitization, but it was inhibited by PPADS, suramin, and the selective P2Y(1) receptor antagonist adenosine 3',5'-bisphosphate (ABP, 10-100 microM). Responses to ATP and UTP in veins did not desensitize and were not inhibited by PPADS, suramin, ABP, or alpha, betaMeATP desensitization. In conclusion, our results suggest that venous contraction to a variety of nucleotides is mediated in large part by P2Y receptors including P2Y(1) receptors and an UTP-preferring P2Y receptor. A small component of contraction also appears to be mediated by P2X(1) receptors. This receptor profile differs markedly from that of mesenteric arteries in which P2X(1) receptors predominate.

Pharmacological characterisation of cannabinoid CB(1) receptors in the rat and mouse.

The role of cannabinoid CB(1) receptors in sympathetic neurotransmission was characterised in nerve-mediated responses of isolated right atria, vasa deferentia and small mesenteric resistance arteries using the cannabinoid CB(1) receptor agonists Delta(9)-tetrahydrocannabinol, CP 55,940 and anandamide and the cannabinoid CB(1)-selective antagonist SR 141716A. In the mouse vas deferens, the twitch response was completely inhibited by each of the putative cannabinoid receptor agonists with pIC(50) values of CP 55,940, 9.2+/-0.1; Delta(9)-tetrahydrocannabinol, 8.4+/-0.1; anandamide, 7.1+/-0.1. SR 141716A 10-100 nM was a competitive antagonist of all three agonists with a pK(B) value of 8.4-8.6, consistent with an interaction at the cannabinoid CB(1) receptor. In the rat vas deferens CP 55,940 (0.01-10 microM) inhibited the contractions to a significant extent (88.5+/-0.5% at 10 microM; pIC(50) of 7.1+/-0.1) while Delta(9)-tetrahydrocannabinol and anandamide (both up to 10 microM) were inactive. CP 55,940 exhibited low potency in rat compared with mouse vas deferens and the rat concentration-response curve was not competitively antagonised by SR 141716A (100 nM) or SR 144528 (10 nM-10 microM), suggesting an interaction at a receptor(s) distinct from cannabinoid CB(1) or CB(2). Sympathetic nerve-induced tachycardia in rat and mouse atria, and rat mesenteric artery smooth muscle contractile responses to perivascular nerve stimulation, were not inhibited by Delta(9)-tetrahydrocannabinol, CP 55,940 or anandamide up to 1 microM. These data indicate that cannabinoid CB(1) receptor activation inhibits sympathetic neurotransmission only in the mouse vas deferens and thus point to species and regional differences in cannabinoid CB(1) receptor involvement in pre-synaptic inhibition of sympathetic neurotransmission and CP 55,940 may have inhibitory actions in rat vas deferens unrelated to cannabinoid receptor activity.

Nitric oxide is a sensory nerve neurotransmitter in the mesenteric artery of guinea pig.

Previous studies have shown that the guinea pig inferior mesenteric artery receives spinal sensory vasodilatory innervation, which can be activated by colon distention and electrical nerve stimulation. In the present study, we investigated the hypotheses that nitric oxide synthase (NOS) is present in guinea pig primary sensory neurons in the dorsal root ganglion (DRG) and in nerve fibers surrounding the mesenteric arteries, and that nitric oxide (NO) is a sensory neurotransmitter in the inferior mesenteric artery in vitro. Double-labeling immunohistochemistry showed that neuronal NOS-IR was found in 12% of cells of guinea pig thoracic and lumbar DRGs; in 95.1% of these cells it was colocalized with substance P (SP), and SP immunoreactivity (SP-IR) was present in 23% of cells of the same DRGs. Neuronal NOS-like immunoreactivity was localized in nerve fibers surrounding guinea pig mesenteric artery and 25% of them were double stained with SP-IR. Endothelium-denuded inferior mesenteric artery preparations in vitro were incubated with guanethidine (30 microns, 30 min) and pre-contracted with methoxamine (30 microns). The NO donors, sodium nitroprusside (1 micron) and L-nitrosocysteine (300 microns), produced 91.0 +/- 5.5 and 90.4 +/- 9.6% vasodilatation of total vasodilatation in the vessel segments, respectively, which was capsaicin- or tetrodotoxin-insensitive. Repetitive electrical field stimulation of the preparations produced a frequency-dependent vasodilatation which was reduced by pretreatment with capsaicin or by tetrodotoxin (10 microns). The NOS inhibitor N omega-nitro-L-arginine (L-NNA) (100 microns, 30 min) diminished the nerve-evoked vasodilatation from 41.8 +/- 8.4 to 21.4 +/- 9.7% at 2 Hz and from 50.8 +/- 5.6 to 19.0 +/- 7.3% at 15 Hz (P < 0.05), whereas NG-nitro-L-arginine methyl ester (L-NAME, 100 microns-1 mM) did not significantly inhibit the relaxation. The stereo isomer nitro-D-arginine (D-NNA, 100 microns, 30 min) was ineffective. These findings suggest that NO is a neurotransmitter released from primary sensory nerves which mediates vasodilation in vitro.

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.

Microvascular disease and anastomotic dehiscence in the colon.

Platelet-derived serotonin released in response to tissue manipulation during surgery may contribute to mesenteric arterial vasospasm leading to postoperative anastomotic leakage after colorectal resection. Organ bath experiments were used to demonstrate the efficacy of naftidrofuryl fumarate (NFT) to oppose serotonin-induced vasoconstriction of human mesenteric arteries. Cumulative dose-response curves were derived with and without NFT at 10(-9) and 10(-6) mol/l concentrations. The difference in maximal contractility between the three sets of curves (n = 8 for each) was significant (P < 0.0001). Sensitivity to serotonin in each of the three curves was measured by calculating the concentration for half-maximal response; differences were again significant (P < 0.0001). NFT reduced serotonin-induced contractility in a dose-dependent fashion in rings of human mesenteric arteries in vitro. This suggests a possible role for NFT in reducing mesenteric vasospasm in colorectal surgery.

Inferior mesenteric artery back pressure and collateral circulation to the distal colon.

The contribution of the hypogastric and superior mesenteric arteries to inferior mesenteric artery collateral (back) pressure (r-IMA) was measured in eight patients who were free from arterial disease and were operated on for small sigmoid carcinoma. Peak and mean r-IMAs were measured after clamping both common iliac arteries and the middle colic artery together with the marginal artery of Drummond. Measurements were repeated after the injection of 50 mg papaverine into the inferior mesenteric artery. For comparison the r-IMA was normalized against the radial artery pressure to create the r-IMA:radial artery pressure ratio. At peak systolic pressure the r-IMA:radial artery pressure was approximately 0.6. This fell with crossclamping of the middle colic artery and marginal artery of Drummond; a slightly greater fall was observed when the common iliac arteries were clamped. The findings were similar when mean pressures were compared. The changes in pressure ratio observed after collateral clamping were slightly amplified by injection of papaverine. These data suggest that the hypogastric arteries make at least as great or a slightly greater contribution to r-IMA than do the middle colic artery plus marginal artery of Drummond. The data also indicate the presence of other substantial collaterals. These findings stress the importance of the hypogastric collateral supplying the sigmoid colon when the inferior mesenteric artery is acutely occluded as it is during aneurysm resection.

Effect of antihypertensive treatment on response to endothelin of resistance arteries of hypertensive rats.

In other studies, we noted decreased reactivity of resistance arteries to endothelin-1 (ET-1) in hypertensive rats and humans. To determine whether these changes are reversible with antihypertensive treatment, we examined a hypertensive model exquisitely sensitive to angiotensin I-converting enzyme (ACE) inhibition, the two-kidney, one-clip (2K, 1C) Goldblatt hypertensive rat. Rats were allowed to become hypertensive for 6 weeks. At this point, either the clip was removed, or rats were treated with 5-10 mg/kg/day cilazapril, 100-150 mg/kg/day metoprolol, or 25 mg/kg/day hydralazine, or were left untreated. After 8 weeks more, mesenteric resistance arteries were examined after mounting on a wire-myograph. Blood pressure (BP) and heart/body weight ratio were normalized in unclipped and cilazapril-treated rats. Plasma renin activity (PRA) was normalized only in the unclipped group. Media width, media/lumen ratio, and media cross-sectional area were similar in control normotensive, unclipped, and cilazapril-treated rats and increased in untreated hypertensive, metoprolol, and hydralazine groups. Dose-response curves of resistance arteries to ET-1 were significantly blunted in untreated hypertensive rats (maximum active tension = 2.3 +/- 0.3 vs 3.4 +/- 0.1 N/m in control, p < 0.01), metoprolol-treated (2.2 +/- 0.4 N/m), and hydralazine-treated rats (2.1 +/- 0.5 N/m), and were normalized in cilazapril (3.3 +/- 0.1 N/m) and unclipped rats (3.2 +/- 0.1 N/m). Similar effects were noted in response to norepinephrine (NE) and arginine vasopressin (AVP).(ABSTRACT TRUNCATED AT 250 WORDS)