Submucosal Arterioles Research Articles

Heparin-binding EGF-like growth factor is a potent dilator of terminal mesenteric arterioles

Objective We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) protects the intestines from multiple forms of injury via direct cytoprotective effects on the intestinal mucosa. In this study, we examined the effects of HB-EGF on the hemodynamics of intestinal arterioles, the major resistance vessels that regulate blood flow to the intestines, as an additional mechanism of HB-EGF-mediated intestinal protection. Methods The hemodynamic effects of HB-EGF in rodent terminal mesenteric arterioles and human submucosal arterioles were examined ex vivo using a video dimension analyzer. Cultured human intestinal microvascular endothelial cells (HIMEC) were used to elucidate the mechanisms of HB-EGF-induced vasodilation. Results HB-EGF significantly increased vessel diameter under conditions of increasing intraluminal pressure and increased flow rate. These HB-EGF-mediated vasodilatory effects were observed in terminal mesenteric arterioles from adult rats and 3 day old rat pups. These effects were confirmed in submucosal arterioles from human intestine. Furthermore, HB-EGF significantly reduced endothelin-1-induced mesenteric arteriolar vasoconstriction. The vasodilatory effects of HB-EGF were blocked by ET B receptor antagonism in adult rat arterioles, and also by nitric oxide synthase inhibition in rat pup and human infant arterioles. In HIMEC, HB-EGF significantly increased endothelin B (ET B) receptor protein expression and provoked intracellular calcium mobilization. Conclusions HB-EGF is a potent vasodilator of the intestinal microvasculature, further supporting its use in diseases manifested by decreased intestinal blood flow, including necrotizing enterocolitis.

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found.

Loss of purinergic vascular regulation in the colon during colitis is associated with upregulation of CD39

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 microM) but did constrict in the presence of its nonhydrolyzable analog alpha,beta-methylene ATP (1 microM). alpha,beta-Methylene ADP (100 microM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.

Physiological role of inward rectifier K+ channels in vascular smooth muscle cells

K(+) channels play indispensable roles in establishing the membrane potential and in regulating the contractile tone of arterial smooth muscle cells. There are four types of K(+) channels in arterial smooth muscle: voltage-dependent K(+) (K(V)), Ca(2+)-dependent K(+) (BK(Ca)), ATP-dependent K(+) (K(ATP)), and inward rectifier K(+) (Kir2) channels. Comparatively few physiological studies have focused on Kir2 channels because they are present only in certain small-diameter cerebral and submucosal arterioles and in coronary arterial smooth muscle. Here, we review the characteristics and regulation of Kir2 channels in vascular arterial smooth muscle. Current knowledge of the predominant Kir2 channel subtype is Kir2.1, not Kir2.2 and 2.3. Electrophysiological measurements to determine the current-voltage relationship in arterial smooth muscle revealed inward rectification with a single-channel conductance of 21 pS. Kir2 channels were found to influence the resting tone of cerebral and coronary arteries based on the fact that barium (Ba(2+)) induces the constriction of these arteries at resting tone. Kir2 channels are also highly responsive to vasoconstrictors and vasodilators. For example, the vasoconstrictors endothelin-1 and angiotensin II inhibit Kir2 channel function by activating protein kinase C (PKC), and the vasodilator adenosine stimulates Kir2 channel function by increasing the level of cAMP, which subsequently activates protein kinase A (PKA). Certain pathological conditions such as left ventricular hypertrophy are associated with a decrease in Kir2 channel expression. Although our understanding of the physiological role and regulation of Kir2 channels is incomplete, it is believed that Kir2 channels contribute to the control of vascular tone in small-diameter vessels via various intracellular signalling pathways that regulate cell membrane potential.

Administration of the thromboxane receptor antagonist vapiprost in a mouse model of DSS‐induced colitis

Thromboxane has been implicated in the constriction of intestinal submucosal arterioles in mice rendered colitic with dextran sodium sulfate (DSS). In the present study, C57BL/6 mice given 40 kD DSS were administered one of three doses of the thromboxane receptor antagonist vapiprost: 0.3, 0.5, and 1.0 mg/kg/day, or no vapiprost as a control. DSS and vapiprost were given in drinking water (5% w/v for DSS) for a period of 6 days, concluding with an assessment of a disease activity index comprising stool consistency, rectal bleeding, and weight loss. The presence of loose stools decreased by 30–40% with all doses of vapiprost. Rectal bleeding decreased by ~25% with the lower dose of vapiprost, and by ~40% with the two higher doses. However, vapiprost was unable to prevent DSS‐induced weight loss. The overall disease activity index (0–4 scale) decreased from 2.9 ± 0.1 (no vapiprost) to 2.3 ± 0.3 (p<0.05) with the higher vapiprost doses of 0.5 and 1.0 mg/kg/day. In summary, vapiprost attenuates the disease activity index of DSS‐induced colitis in mice, primarily by decreasing the presence of loose stools and rectal bleeding. Supported by CCFA and NIH (P01DK043785).

Sympathetic vasoconstrictor regulation of mouse colonic submucosal arterioles is altered in experimental colitis

Recent studies suggest that altered neural regulation of the gastrointestinal microvasculature contributes to the pathogenesis of inflammatory bowel disease. Therefore, we employed video microscopy techniques to monitor nerve-evoked vasoconstrictor responses in mouse colonic submucosal arterioles in vitro and examined the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis. Nerve stimulation (2-20 Hz) caused frequency-dependent vasoconstrictor responses that were abolished by tetrodotoxin (300 nm) and guanethidine (10 microm). The P2 receptor antagonist suramin (100 microm) or the alpha(1)-adrenoceptor antagonist prazosin (100 nm) reduced the vasoconstriction and the combination of suramin and prazosin completely abolished responses. Nerve-evoked constrictions of submucosal arterioles from mice with TNBS colitis were inhibited by prazosin but not suramin. Superfusion of ATP (10 microm) resulted in large vasoconstrictions in control mice but had no effect in mice with colitis whereas constrictions to phenylephrine (3 microm) were unaffected. P2X(1) receptor immunohistochemistry did not suggest any alteration in receptor expression following colitis. However, Western blotting revealed that submucosal P2X(1) receptor expression was increased during colitis. In contrast to ATP, alphabeta-methylene-ATP (1 microm), which is resistant to catabolism by nucleotidases, constricted control and TNBS arterioles. This indicates that reduced purinergic transmission to submucosal arterioles may be due to increased degradation of ATP during colitis. These data comprise the first description of the neural regulation of mouse submucosal arterioles and identify a defect in sympathetic regulation of the GI vasculature during colitis due to reduced purinergic neurotransmission.

The presence of EGF- and IGF-1-receptors in the small intestine of fetal, neonatal and weaned piglets

Abstract During development, gut morphology and functions change. Some of these are mediated by milk-borne factors, e.g. insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF), of which the effects are in part dictated by the distribution of their receptors (R). EGFR- and IGF-1R-immunoreactivity (IR) was evaluated in samples of the fetal, neonatal and weaned porcine small intestine. EGFR-IR first appeared in the neonatal duodenum and was located basolaterally in villar and to a lesser extent in cryptal enterocytes. In weaned pigs, EGFR appeared apically in enterocytes with a region-dependent distribution along the villus–crypt axis. IGF-1R-IR was observed in smooth muscle cells in each age group. From the last trimester of gestation onward IGF-1R-IR was seen at the apical and basolateral side of villar enterocytes and in submucosal arterioles. Thus, the age- and region-dependent presence of EGFR and IGF-1R has to be taken into account when evaluating effects of EGF and IGF-1 on growth or repair.

Endothelial Nitric Oxide Synthase in Human Intestine Resected for Necrotizing Enterocolitis

To determine the expression and function of endothelial nitric oxide synthase (eNOS) in submucosal arterioles harvested from human intestine resected for necrotizing enterocolitis (NEC) or congenital bowel disease. eNOS expression was determined by using immunohistochemistry. The arteriolar diameter was measured in vitro at pressures of 10 to 40 mm Hg and also in response to the eNOS agonist acetylcholine (ACh), the exogenous nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine, and the smooth muscle relaxant papaverine. Arteriolar release of NO in response to ACh was determined with a Sievers NOAnalyzer. Hemodynamics were also determined at flow rates of 50 and 100 microL/min. eNOS was present in microvessels from both groups, but NEC arterioles failed to demonstrate physiological evidence of eNOS function: they constricted in response to pressure, failed to dilate or generate NO in response to ACh, and failed to dilate in response to flow. However, they dilated in response to exogenous NO and papaverine, indicating functional vascular smooth muscle and vasodilator reserve. eNOS-derived NO, a vasodilator in the newborn intestine, did not contribute to vasoregulation in arterioles harvested from intestine resected for NEC. These vessels were constricted; lack of eNOS-derived NO may contribute to this vasoconstriction.

Changes in vasoconstrictor and vasodilator neurotransmitters in nerves supplying arterioles in developing colorectal polyps

To examine the changes that occur in the immunohistochemistry of vasoconstrictor and vasodilator transmitters in nerves supplying early and advanced colorectal polyps. We studied the perivascular innervation of submucosal arterioles of colorectal polyps (n = 18) and the innervation of the epithelial layer of polyps compared to normal controls (n=8), using immunohistochemical markers for the neurotransmitters; noradrenaline (NA) (marker used; tyrosine hydroxylase (TH)), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), and calcitonin gene-related polypeptide (CGRP). (Advanced polyps; villous adenomas>1.5 cm, polyps with severe dysplasia or partial carcinoma). In submucosal arterioles there was a progressive decrease from controls through early polyps to advanced polyps in TH and NPY perivascular immunoreactivity (P<0.015 for both). VIP and SP immunoreactivity was higher in early polyps compared to controls, but markedly reduced in advanced polyps (P<0.05 for VIP). Sparse CGRP immunoreactivity was present in polyps only. Neural VIP and SP immunoreactivity in the lamina propria of polyp mucosa was more intense than in controls. There is a decrease in vasoconstrictor neurotransmitters NPY and NA (shown by TH) around submucosal arterioles of both early and advanced polyps, but an increase in the vasodilator neurotransmitters, particularly VIP, in early colorectal polyps. These results suggest a predominantly vasodilatory neural influence in early polyps, perhaps indicating a mechanism that maintains polyp growth.

Intraperitoneal instillation of polihexanide produces hypotension and vasodilation: in vivo and in vitro study in rats

Treatment of peritonitis may include abdominal lavage with a local disinfectant polihexanide, available as 0.04% solution, which is often accompanied by hypotension. We examined the effects of peritoneal installation of polihexanide or NaCl 0.9% (10 ml each, for 10 min; polihexanide n=5, NaCl n=5) on mean arterial pressure in healthy rats and, using intravital microscopy, measured in seven other animals the diameter of terminal ileum submucosal arterioles and venules before and after local superfusion with polihexanide. Furthermore, in an in vitro isometric preparation of rat thoracic aortal rings, with and without endothelium, we tested the effects of cumulative concentrations of polihexanide on vascular basic tension and on tension elicited by phenylephrine and KCl. It was found that polihexanide peritoneal instillation produced a decrease in mean arterial pressure, while superfusion with polihexanide caused local vasodilation of intestinal wall blood vessels. In vitro, polihexanide produced endothelium-dependent relaxation in the preparations pre-contracted with phenylephrine (EC(50), polihexanide 0.04% solution 2.53+/-0.16 vs. 1.36+/-0.16, n=4, P<0.05; polihexanide 4.02+/-0.12 vs. 3.21+/-0.10, n=12, P<0.001;+ vs. - endothelium, respectively; -log g%) which (in aortae +endothelium) could be attenuated by either N(G)-nitro-L-arginine methyl ester, a nitric oxide generation inhibitor, or 1H-(1,2,4)oxodiazolo-(4,3-a)quinoxalin-1-one, an inhibitor of guanylyl cyclase. The relaxing effect of polihexanide (aortae -endothelium) was not affected by K(+)-channel blocking agents charybdotoxin, tetraethylammoniumchloride, glibenclamide or 4-aminopyridine, while polihexanide had no effects on 40-mM KCl contractions. This implies that polihexanide may promote nitric oxide liberation, potassium channel activation and vasodilation that may result in hypotension.

Venular Constriction of Submucosal Arterioles Induced by Dextran Sodium Sulfate

Leukocyte and platelet adherence in postcapillary venules has been speculated to induce constriction of closely paired arterioles. This mechanism was investigated in the current study, in a model of intestinal inflammation induced by dextran sodium sulfate (DSS; 5,000 molecular weight). Closely paired, parallel arterioles and venules in the submucosa of the mouse intestine were observed using fluorescent intravital microscopy. Arterioles in control mice were compared to arterioles in mice given 5% DSS in drinking water for 11 days. DSS induced an inflammatory response in which fluorescently labeled leukocytes and platelets were observed adhering to venules. Arterioles constricted and flow decreased significantly when the arterioles were paired with venules having adherent leukocytes and platelets, although the decreases in flow and diameter appeared to be more dependent on platelet than leukocyte adherence. No significant constriction was observed in arterioles paired with venules having minimal platelet adherence. Inhibition of thromboxane with 100 mg/kg ozagrel induced a significant dilation of arterioles in DSS mice that was absent in control mice. The results are consistent with a proposed mechanism in which thromboxane constricts submucosal arterioles when the arterioles are closely paired with platelet-bearing venules in DSS-induced inflammation.

Colonic blood flow responses in experimental colitis: time course and underlying mechanisms

Human inflammatory bowel diseases (IBD) are associated with significant alterations in intestinal blood flow, the direction and magnitude of which change with disease progression. The objectives of this study were to determine the time course of changes in colonic blood perfusion that occur during the development of dextran-sodium-sulfate (DSS)-induced colonic inflammation and to address the mechanisms that may underlie these changes in blood flow. Intravital microscopy was used to quantify blood flow (from measurements of vessel diameter and red blood cell velocity) in different-sized submucosal arterioles of control and inflamed colons in wild-type (WT) mice. A significant (18-30%) reduction in blood flow was noted in the smallest arterioles (<40 microm diameter) on days 4-6 of DSS colitis. The arteriolar responses to bradykinin in control and DSS-treated WT mice revealed an impaired endothelium-dependent, but not endothelium-independent, vasodilation in the inflamed colon. However, this impaired vasodilatory response to bradykinin after DSS treatment was not evident in mutant mice that overexpress Cu,Zn-superoxide dismutase. Rescue of the bradykinin-induced vasodilation during DSS colitis was also observed in mice that are genetically deficient in the NAD(P)H oxidase subunit gp91(phox). These findings indicate that the decline in blood flow during experimental colitis may result from a diminished capacity of colonic arterioles to respond to endogenous endothelium-dependent vasodilators like bradykinin and that NAD(P)H oxidase-derived superoxide plays a major role in the induction of the inflammation-induced endothelium-dependent arteriolar dysfunction.

Sensory peptide neurotransmitters mediating mucosal and distension evoked neural vasodilator reflexes in guinea pig ileum

The aim was to determine the role CGRP and/or tachykinins released from sensory neural mechanisms in enteric neural vasodilator pathways. These pathways project through the myenteric plexus to submucosal vasodilator neurons. Submucosal arterioles were exposed in the distal portion of an in vitro combined submucosal-myenteric guinea pig ileal preparation, and dilation was monitored with videomicroscopy. Vasodilator neural reflexes were activated by gently stroking the mucosa with a fine brush or by distending a balloon placed beneath the flat-sheet preparation in the proximal portion. Dilations evoked by mucosal stroking were inhibited 64% by the CGRP 8-37 and 37% by NK3 (SR 142801) antagonists. When the two antagonists were combined with hexamethonium, only a small vasodilation persisted. Balloon distension-evoked vasodilations were inhibited by NK3 antagonists (66%) but were not altered by CGRP 8-37. In preparations in which myenteric descending interneurons were directly activated by electrical stimulation, combined application of CGRP 8-37 and the NK antagonists had no effect. Stimulation of capsaicin sensitive nerves in the myenteric plexus did not activate these vasodilator reflexes. These findings suggest that mucosal-activated reflexes result from the release of CGRP and tachykinins from enteric sensory neurons. Distension-evoked responses were significantly blocked by NK3 antagonists, suggesting that stretch activation of myenteric sensory neurons release tachykinins that activate NK3 receptors on myenteric vasodilator pathways.

Distribution and chemical coding of corticotropin-releasing factor-immunoreactive neurons in the guinea pig enteric nervous system

Immunofluorescence was used to study immunoreactivity (IR) for corticotropin-releasing factor (CRF) in the guinea pig enteric nervous system. CRF-IR was expressed in both the myenteric and the submucosal plexuses of all regions of the large and small intestine and the myenteric plexus of the stomach. CRF-IR nerve fibers were present in the myenteric and submucosal plexuses, in the circular muscle coat, and surrounding submucosal arterioles. Most of the CRF-IR fibers persisted in the myenteric and submucosal plexuses after 7 days in organotypic culture. CRF-IR was not coexpressed with tyrosine hydroxylase-IR or calcitonin gene-related peptide-IR fibers. The proportions of CRF-IR cell bodies in the myenteric plexus increased progressively from the stomach (0.6%) to the distal colon (2.8%). Most of the CRF-IR myenteric neurons (95%) had uniaxonal morphology; the remainder had Dogiel type II multipolar morphology. CRF-IR cell bodies in the myenteric plexus of the ileum expressed IR for choline acetyltransferase (56.9%), substance P (55.0%), and nitric oxide synthase (37.9%). CRF-IR never colocalized with IR for calbindin, calretinin, neuropeptide Y, serotonin, or somatostatin in the myenteric plexus. CRF-IR cell bodies were more abundant in the submucosal plexus (29.9-38.0%) than in the myenteric plexus. All CRF-IR neurons in submucosal ganglia expressed vasoactive intestinal peptide-IR and were likely to be secretomotor/vasodilator neurons. CRF-IR neurons did not express IR for the CRF(1) receptor. CRF(1)-IR was expressed in neuronal neighbors of those with CRF-IR. Collective evidence suggests that VIPergic secretomotor neurons might provide synaptic input to neighboring cholinergic neurons.

Effects of insulin on the acetylcholine-induced hyperpolarization in the guinea pig mesenteric arterioles

Background: Insulin induces endothelium-dependent vasodilatation, which may be casually related to the insulin resistance and hypertension. Endothelium-derived nitric oxide (NO) is the most important mechanism of insulin-induced vasodilatation, and a possible contribution of endothelium-derived hyperpolarizing factor (EDHF) is also considered. Attempts were made to observe the effects of insulin on acetylcholine (ACh)-induced hyperpolarization in the submucosal arteriole of the guinea pig ileum, the objective being to investigate possible involvement of EDHF in the actions of insulin. Methods: Conventional microelectrode techniques were applied to measure the membrane potential of smooth muscle cells in the submucosal arteriole. EDHF-induced hyperpolarization was elicited by ACh in the presence of both N ω-nitro- l-arginine ( l-NNA) (100 μM) and diclofenac (1 μM). Results: The resting membrane potential was −70.9 mV, and Ba 2+ (0.5 mM) depolarized the membrane to −33.0 mV. Insulin (10 μU/ml to 100 mU/ml) did not change the membrane potential in the absence or presence of Ba 2+. In the presence of Ba 2+, ACh (3 μM) hyperpolarized the membrane with two components, an initial large hyperpolarization followed by a slow and small one. Low concentration of insulin (100 μU/ml) did not alter the ACh-induced hyperpolarization. High concentration of insulin (100 mU/ml) shortened the time required to reach the peak amplitude and tended to increase the peak amplitude of the ACh-induced hyperpolarization. Conclusions: The data show that insulin enhances the ACh-induced hyperpolarization in the submucosal arterioles of the guinea pig ileum. The results suggested that EDHF also accounts for one of the endothelial factors involved in the insulin-induced vasodilatation.

Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several mediators of vasodilation, which include prostacyclin (PGI(2)), nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently defined the role of nitric oxide and PGI(2) in the dilation of submucosal intestinal arterioles from patients with normal bowel function. However, significant endothelium-dependent dilator capacity to ACh remained after inhibiting both these mediators. The current study was designed to examine the potential role of EDHF in human intestinal submucosal arterioles. ACh elicited endothelium-dependent relaxation in the presence of inhibitors of nitric oxide synthase and cyclooxygenase (23 +/- 10%, n = 6). This ACh-induced relaxation was inhibited and converted to constriction by catalase (-53 +/- 10%, n = 6) or KCl (-30 +/- 3%, n = 7), whereas 17-octadecynoic acid and 6-(2-propargylloxyphenyl) hexanoic acid, two inhibitors of cytochrome P450 monooxygenase, had no significant effect (3 +/- 1% and 20 +/- 8%, n = 5, respectively). Exogenous H(2)O(2) elicited dose-dependent relaxation of intact microvessels (52 +/- 10%, n = 7) but caused frank vasoconstriction in arterioles denuded of endothelium (-73 +/- 8%, n = 7). ACh markedly increased the dichlorofluorescein fluorescence in intact arterioles in the presence of nitric oxide synthase and cyclooxygenase inhibitors compared with control and compared with catalase-treated microvessels (363.6 +/- 49, 218.8 +/- 10.6, 221.9 +/- 27.9, respectively, P < 0.05 ANOVA, n = 5 arbitrary units). No changes in the dichlorofluorescein fluorescence were recorded in vessels treated with ACh alone. These results indicate that endothelial production of H(2)O(2) occurs in response to ACh in human gut mucosal arterioles but that H(2)O(2) is not an EDHF in this tissue. Rather, we speculate that it stimulates the release of a chemically distinct EDHF.

Submucosal secretomotor and vasodilator reflexes.

The presence of neuronal reflexes within the intestine that modulate mucosal ion secretion and blood flow have been recognized for many years, but the organization of these reflexes was unclear. This review highlights important findings from recent in vitro guinea-pig studies which have shown that both intrinsic primary afferent neurones (IPANs) and extrinsic primary afferent neurones (EPANs) can respond to chemical and/or mechanical stimuli to activate pathways, the afferent and efferent elements of which are confined to the walls of the intestine. Enteric neuronal pathways involve both myenteric and submucosal plexus neurones whereas capsaicin-sensitive afferent nerves evoke secretion by stimulating submucosal secretomotor neurones and vasodilation by direct actions on the submucosal arterioles. In this review, the cellular mechanisms involved in these pathways are described and the implications of these findings are discussed.

Long vasodilator reflexes projecting through the myenteric plexus in guinea-pig ileum.

This study examined enteric neural reflexes activating submucosal cholinergic vasodilator motoneurons, which innervate the final resistance vessels regulating mucosal blood flow. Videomicroscopy was employed to monitor dilatation of submucosal arterioles in in vitro preparations from guinea-pig ileum. Balloon distension of intact lumen evoked reflex vasodilatation and flat sheet preparations were employed to separate mucosal mechanical stimulation from intestinal distension. Mucosal stroking and balloon distension of the orad segment evoked vasodilatations > 1.5 cm from the stimulating site. Mucosal stimulation was blocked by combined 5-HT3/5HT4 antagonists but distension-evoked responses were unaffected. Distension-evoked responses were also unaffected by nifedipine (5 microM) or nifedipine (1 microM) and wortmannin (300 nM), suggesting stretch activation rather than stretch-activated contraction was involved. Mucosal and distension-evoked responses were completely blocked when the myenteric plexus was surgically lesioned and were significantly inhibited by hexamethonium. The muscarinic antagonist 4-DAMP, which inhibits vasodilatations evoked by submucosal cholinergic vasodilator neurons, blocked dilatations elicited by mucosal stimulation and balloon distension. Maximal dilatations evoked with either sensory modality could be further enhanced when stimulated with the second modality. Dilatations evoked by stimulation of the aborad segment were similar to those elicited in the orad segment. In conclusion, sensory mechanisms in the mucosa and muscularis propria activate vasodilator pathways in the myenteric plexus which project for significant distances in both ascending and descending directions before innervating submucosal arterioles. These reflexes could co-ordinate mucosal blood flow during multiple motor events such as peristalsis and intestinal mixing between propulsive events.

Acquired microvascular dysfunction in inflammatory bowel disease: loss of nitric oxide-mediated vasodilation

Background & Aims: Inflammatory bowel disease (IBD; i.e., Crohn’s disease, ulcerative colitis) is characterized by refractory inflammatory ulceration and damage to the intestine. Mechanisms underlying impaired healing are not defined. Because microvascular dysfunction resulting in diminished vasodilatory capacity and tissue hypoperfusion is associated with impaired wound healing, we hypothesized that microvascular dysfunction may also occur in chronic IBD. Methods: Intact submucosal arterioles from control, involved, and uninvolved IBD specimens were assessed using in vitro videomicroscopy to assess endothelium-dependent vasodilation in response to acetylcholine (Ach) and fluorescence microscopy to detect oxyradicals. Results: Normal microvessels dilated in a dose-dependent and endothelium-dependent manner to Ach (maximum, 82% ± 2%; n = 34). Inhibition of nitric oxide synthase with N G -nitro- l-arginine methyl ester ( l-NAME) reduced maximal dilation to 54% ± 6% ( P < 0.05, n = 7), and further reduction was observed after inhibiting cyclooxygenase (indomethacin; 23% ± 10%, n = 6). Chronically inflamed IBD microvessels showed significantly reduced Ach-induced vasodilation (maximum, 15% ± 2%; n = 33), with no effect of l-NAME. Indomethacin eliminated the remaining Ach-induced vasodilation, resulting in frank vasoconstriction (−54% ± 9%, n = 6). Uninvolved IBD gut vessels and non-IBD inflammatory controls responded in a fashion similar to normal vessels. IBD-involved microvessels generated significantly higher levels of reactive oxygen species compared with control and uninvolved IBD vessels ( P < 0.01). Conclusions: Human intestinal microvessels from chronically inflamed IBD show microvascular endothelial dysfunction, characterized by loss of NO-dependent dilation that may contribute to reduced perfusion, poor wound healing, and maintenance of chronic inflammation.

EDHF is not K+ but may be due to spread of current from the endothelium in guinea pig arterioles.

Endothelium-derived hyperpolarizing factor (EDHF)-attributed hyperpolarizations and relaxations were recorded simultaneously from submucosal arterioles of guinea pigs with the use of intracellular microelectrodes and a video-based system, respectively. Membrane currents were recorded from electrically short segments of arterioles under single-electrode voltage clamp. Substance P evoked an outward current with a current-voltage relationship that was well described by the Goldman-Hodgkin-Katz equation for a K+ current, consistent with the involvement of intermediate- and small-conductance Ca2+-activated K+ channels. 1-Ethyl-2-benzimidazolinone relaxed the arterioles and evoked hyperpolarizations that were blocked by charybdotoxin, but not by iberiotoxin. Application of K+ induced depolarization under conditions in which EDHF evoked hyperpolarization. The Ba2+-sensitive component of the K+-induced current was inwardly rectifying, in contrast to the outwardly rectifying current evoked by substance P. EDHF-attributed hyperpolarizations in dye-identified smooth muscle cells were indistinguishable from those recorded from dye-identified endothelial cells in the same arterioles. These results provide evidence that EDHF is not K+ but may involve electrotonic spread of hyperpolarization from the endothelial cells to the smooth muscle cells.