Disruption of smooth muscle gap junctions attenuates myogenic vasoconstriction of mesenteric resistance arteries

Communication between vascular smooth muscle (VSM) cells via low-resistance gap junctions may facilitate vascular function by synchronizing the contractile state of individual cells within the vessel wall. We hypothesized that inhibition of gap junctional communication would impair constrictor responses of mesenteric resistance arteries. Immunohistochemical experiments revealed positive staining for connexin 37 (Cx37) in both endothelium and smooth muscle of rat mesenteric arterioles, whereas connexin 43 (Cx43) immunoreactivity was not detected in the mesenteric vasculature. Administration of the gap junction inhibitory peptide Gap27, which targets Cx37 and Cx43, significantly diminished myogenic vasoconstriction (8.6 +/- 3.8% of passive diameter at 100 Torr) and changes in vessel wall intracellular [Ca2+] of mesenteric resistance arteries compared with vessels treated with either vehicle (physiological saline solution) (33.5 +/- 6.1%) or a control peptide (32.1 +/- 6.5%). Administration of 18alpha-glycyrrhetinic acid, structurally distinct from Gap27, also significantly attenuated myogenic constriction compared with its vehicle control (DMSO) (9.6 +/- 3.2% vs. 23.8 +/- 4.6%). In contrast, phenylephrine-induced vasoconstriction was not altered by gap junction blockers. Attenuated myogenic vasoconstriction resulting from inhibition of gap junctions persisted after disruption of the endothelium. In additional experiments, VSM cell membrane potential was recorded in mesenteric resistance arteries pressurized to 20 or 100 Torr. VSM membrane potential was depolarized at 100 Torr compared with 20 Torr. However, VSM cells in arteries treated with Gap27 were significantly hyperpolarized (-48.6 +/- 1.4 mV) at the higher pressure compared with vehicle (-41.4 +/- 1.5 mV) and Gap20-treated (-38.4 +/- 0.7 mV) vessels. Our findings suggest that inhibition of smooth muscle gap junctions attenuates pressure-induced VSM cell depolarization and myogenic vasoconstriction.     

Disruption of the rat mesenteric arterial bed endothelial function by air perfusion

The impact of air perfusion on the endothelial function of the rat mesenteric arterial bed (MAB; perfused with Krebs' bicarbonate plus indomethacin) was compared to that of the NO synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME). Air shifted the dose-response curve for the alpha-adrenoceptor agonist, norepinephrine (NE) to the left (ED_50%: 2.9 ± 0.7 to 0.9 ± 0.7 μg, P < 0.05); maximal vasoconstriction did not change. L-NAME produced a similar increase in midrange sensitivity (ED_50% 1.4 ± 0.7 μg, P < 0.05) and a 20% increase in maximum (152 ± 6 to 183 ± 7 mmHg, P < 0.05). Electromechanical stimulation with potassium chloride (KCL) was not modified by reserpine. Neither air nor L-NAME modified midrange sensitivity to KCL. L-NAME produced a 17% increase in maximum (91 ± 4 to 107 ± 5 mmHg, P < 0.05); reserpine abolished the latter effect. Air and L-NAME diminished endothelium-dependent vasodilation elicited by carbachol. Air did not modify endothelium-dependent vasodilation elicited by sodium nitroprusside; this response was potentiated by L-NAME. In summary, air and L-NAME produced similar effects on receptor-dependent activation of the endothelial L-arginine nitric oxide (NO) pathway. Potentiation by L-NAME of the maximal electromechanical response suggests the existence of a tone-dependent NO system. Abolition of the latter response by reserpine suggests that this system is of sympathetic origin.     

Effect of vagotomy on dynamics of mesenteric lymphatic vessels in the rat

The functional modulation of lymphatic vessels may be closely associated with intact structures of the vagus nerve. In the present study, the vagotomy was done in Wistar rat to investigate the effect of vagus nerves on dynamic changes of mesenteric lymphatic vessels. After denervation, the mesenteric lymphatics showed significant decreases in contraction rate, diameter in the static state and overall contractile activity under a microscopic observation. The lymphatic contraction rhythm and valve movement became irregular and inconsistent. These findings indicated that the lymphatic innervation might be an important factor for active lymph formation and transportation.     

Increases in NPY in non-sympathetic nerve fibres supplying rat mesenteric vessels after immunosympathectomy.

The effect of nerve growth factor (NGF) deprivation on developing peripheral peptide-containing nerves has been examined in Wistar rats. Animals were treated from birth for 7 days with antibodies to NGF (10 microliters/g body weight) and killed at 4 or 8 weeks of age. The nerves of the mesenteric and femoral blood vessels, vas deferns and bladder were viewed with histochemical and immunohistochemical techniques. The effectiveness of anti-NGF treatment was monitored by viewing catecholamine (CA)-containing nerves, which were virtually absent from the blood vessels, but were little affected in the vas deferens and bladder in both age groups. Immunoreactivity for substance P and calcitonin gene-related peptide was slightly reduced in the blood vessels. Immunoreactivity for neuropeptide Y (NPY) was reduced in the femoral blood vessels by 88% at both ages, but reductions in NPY immunoreactivity (NPY-IR) in the mesenteric vessels varied with age. In the mesenteric artery at 4 weeks, NPY-IR was reduced by 96% from control values, but at 8 weeks it was reduced by only 37%. Acute sympathectomy with 6-OHDA treatment reduced NPY-IR in the mesenteric artery by 98% at 4 weeks and 93% at 8 weeks. It is proposed that the increase in NPY-IR but not CA-containing nerves in the mesenteric artery between 4 and 8 weeks after immunosympathectomy is due to compensatory innervation from a non-sympathetic source (probably enteric neurons) that is available to mesenteric, but not to femoral blood vessels.     

SQ 14,225 attenuates the vascular response to norepinephrine in the rat mesenteric arteries.

SQ 14, 225, a new angiotensin-converting enzyme inhibitor, attenuated the vascular contraction induced by norepinephrine in the perfused rat mesenteric vascular bed, while SQ 20, 881, an another converting enzyme inhibitor, did not have any effect on the vascular reactivity. Furthermore, this effect of SQ 14, 225 was not altered in the presence of bradykinin or angiotensin II in the perfusate. These results suggest that SQ 14, 225 may have a direct antihypertensive effect which is not mediated by the inhibition of angiotensin-converting enzyme.     

Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.     

Mathematical modeling of thrombus growth in mesenteric vessels

Richardson’s phenomenological mathematical model of the thrombi growth in microvessels is extended to describe the realistic features of the phenomenon. The main directions of the generalization of Richardson’s model are: (1) the dependence of platelet activation time on the distance from the injured vessel wall; (2) the non-homogeneity of the platelet distribution in blood flow in the vicinity of the vessel wall; (3) the adequate choice of the phenomenological function describing the dependence of blood velocity on the thrombus size. The generalization of the model corresponds to the main experimental results and theoretical considerations concerning thrombus formation obtained in recent years. The extended model permits to achieve qualitative agreement between model and experimental data.     

Kit-like immunopositive cells in sheep mesenteric lymphatic vessels

Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.     

Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.     

Adrenergic innervation of human mesenteric blood vessels.

A fluorescent histochemical technique has been applied to study the adrenergic innervation of human superior mesenteric arteries obtained at autopsy. Specific catecholamine fluorescence was demonstrated in the smaller branches of this artery taken from three infants and one child. No specific fluorescence was seen in arteries from three adult subjects.     

Detection of 15-hydroxyprostaglandin dehydrogenase in bovine mesenteric blood vessels

Two types of 15-hydroxyprostaglandin dehydrogenase (NAD⁺ and NADP⁺ dependent) were demonstrated in bovine mesentric arteries and veins. The 15-hydroxyprostaglandin dehydrogenase activity was found in the high-speed supernatant, suggesting that these enzymes are associated with the cytoplasmic fraction of the blood vessels. The levels of activities of both NAD⁺- and NADP⁺-dependent dehydrogenases were similar in mesentric blood vessels. Prostaglandin F_2α was preferred to the prostaglandin E₂ as subtrate by both NAD⁺ and NADP⁺ dependent enzymes. The presence of 15-hydroxyprostaglandin dehydrogenase in blood vessels may play a siginificant role in the regulation of intracellular levels of prostaglandins of the E and F series in blood vessels.     

Impaired arachidonic acid-mediated dilation of small mesenteric arteries in Zucker diabetic fatty rats

Arachidonic acid (AA) is a precursor of important vasoactive metabolites, but the role of AA-mediated vasodilation in Type 2 diabetes is not known. Using Zucker diabetic fatty (ZDF) rats, we examined the effects of AA in small mesenteric arteries preconstricted with endothelin. In ZDF rat mesenteric arteries, 1 microM AA produced only one-third the amount of dilation as in vessels from lean control animals. In lean control rats, the effect of AA was significantly and predominantly inhibited by the lipoxygenase inhibitors baicalein and cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC). However, baicalein and CDC had no effect on AA-mediated dilation in ZDF rat mesenteric arteries. The major [3H]AA metabolite produced by isolated mesenteric arteries in both lean and ZDF rats was 12-hydroxyeicosatetraenoic acid (12-HETE), but the amount of [3H]12-HETE produced by ZDF rat vessels was only 36% of that of control vessels. In addition, 12-HETE produced similar amounts of dilation in lean and ZDF rat mesenteric arteries. Immunoblot analysis showed an 81% reduction in 12-lipoxygenase protein in ZDF rat mesenteric arteries. Immunofluorescence labeling showed strong nitrotyrosine signals in ZDF rat mesenteric arteries that colocalized with 12-lipoxygenase in endothelium, and 12-lipoxygenase coprecipitation with anti-nitrotyrosine antibodies was enhanced in ZDF rat vessels. We conclude that AA-mediated relaxation in ZDF rat small mesenteric arteries is impaired due to reduced 12-lipoxygenase protein and activity. Increased oxidative stress and nitration of 12-lipoxygenase may underlie the impairment of AA-mediated relaxation in small mesenteric arteries of diabetic rats.     

Mechanisms of bradykinin-mediated dilation in newborn piglet pulmonary conducting and resistance vessels

Bradykinin (BK) is a potent dilator of the perinatal pulmonary circulation. We investigated segmental differences in BK-induced dilation in newborn pig large conducting pulmonary artery and vein rings and in pressurized pulmonary resistance arteries (PRA). In conducting pulmonary arteries and veins, BK-induced relaxation is abolished by endothelial disruption and by inhibition of nitric oxide (NO) synthase with nitro-L-arginine (L-NA). In PRA, two-thirds of the dilation response is L-NA insensitive. Charybdotoxin plus apamin and depolarization with KCl abolish the L-NA-insensitive dilations, findings that implicate the release of endothelium-derived hyperpolarizing factor (EDHF). However, endothelium-disrupted PRA retain the ability to dilate to BK but not to ACh or A-23187. In endothelium-disrupted PRA, dilation was inhibited by charybdotoxin. Thus in PRA, BK elicits dilation by multiple and duplicative signaling pathways. Release of NO and EDHF contributes to the response in endothelium-intact PRA; in endothelium-disrupted PRA, dilation occurs by direct activation of vascular smooth muscle calcium-dependent potassium channels. Redundant signaling pathways mediating pulmonary dilation to BK may be required to assure a smooth transition to extrauterine life.