Postnatal development of excitatory innervations in longitudinal smooth muscle of the chicken anterior mesenteric artery

AimsThe anterior mesenteric artery of chickens contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer. Cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle. The aim of this study was to clarify postnatal development of excitatory neurotransmission of the longitudinal smooth muscle.Main methodsMembrane potentials of smooth muscle were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS).Key findingsHistological examination showed that longitudinal smooth muscles exist in the artery at birth. EFS failed to evoke membrane response in 1-day-old chickens, though it caused depolarization (excitatory junction potential; EJP) in 12-week-old chickens. However, exogenous application of acetylcholine (ACh) or ATP produced depolarization in longitudinal smooth muscle of 1-day-old chickens, suggesting that responsiveness of smooth muscle to excitatory neurotransmitters is already established at birth. In preparations isolated from 10-day-old chickens, EFS caused EJP, which was totally blocked by atropine but not by a non-specific purinoceptor antagonist, suramin. Several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in smooth muscle of adult chickens, were expressed in the anterior mesenteric artery of 10-day-old chickens.SignificanceExcitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but develops during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in smooth muscle.     

Interaction of contractile responses in canine tracheal smooth muscle

Concentration-response curves for norepinephrine, acetylcholine, and 5-hydroxytryptamine were obtained in vitro alone and after precontraction with histamine, 5-hydroxytryptamine, or acetylcholine. Responses obtained to each agonist after precontraction were greater than responses to the agonist alone after subtraction of the force due to the precontracting stimulus. Augmentation of responses after precontraction was the greatest for norepinephrine, less for 5-hydroxytryptamine, and least for acetylcholine. Verapamil had no significant effect on the augmentation of responses to either 5-hydroxytryptamine or acetylcholine caused by precontraction. When the efficacy of acetylcholine was decreased by receptor alkylation with phenoxybenzamine, the augmentation of responses to acetylcholine caused by precontraction with histamine was significantly enhanced. Differences in the magnitude of the effect of precontraction on responses to different agonists may reflect differences in their efficiency of stimulus-response coupling in canine tracheal smooth muscle, or they may result from an increased expression of distinct receptors or receptor-mediated effects uncovered by the facilitory stimuli.     

Adrenergic regulation of contractile activity in the smooth muscle of umbilical vessels

A study was made of contractile activity produced in isolated muscle strips from human umbilical vessels by adrenomimetics and adrenoblockers. Activation of -as well as -adrenoceptors was found to cause contraction in the smooth muscle of the umbilical arteries and veins — a different effect from that occurring in other vessels. Selective shut-down of - or -receptors under the action of phentolamine and obsidane would indicate that activation of - and -adrenoceptors are responsible for mainly phasic and tonic components (respectively) of smooth muscle contraction in the umbilical vein. Obsidane was also found to inhibit the tonic component of contraction induced by oxytocin. In the smooth muscle cells of the umbilical artery, - and -receptors produce nonselective inhibition of noradrenaline-induced contraction, which obviously indicates limited differentiation in the adrenoceptors of this vessel. In view of the experimental findings obtained, application of obsidane either separately or in combination with oxytocin might be recommended for obstetrical use.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 547–551, July–August, 1989.     

Quantitative study of intimal longitudinal smooth muscle in human small mesenteric arteries

The present paper examines the results of quantitative assessment of intimal longitudinal smooth muscle (ILSM) in small arteries from normal mesentery in man. 212 vessels from 24 patients who underwent colectomy for colorectal carcinoma were studied. The mean amount of ILSM in these vessels was found to be 1.42% (range 0.00-8.90%) of external vessel diameter. A statistically significant (p = 0.018) positive correlation was demonstrated between the mean amount of ILSM in vessels from any individual patient and the level of diastolic blood pressure. It is concluded that increased intravascular tension is one factor which influences the development of ILSM in human small mesenteric arteries.     

Direct regulation of smooth muscle contractile elements by second messengers

The effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and phorbol 12,13 dibutyrate (PDBu) on the Ca2+ sensitivity of the contractile elements in the rat mesenteric artery were investigated, using a method of permeabilizing smooth muscle with Staphylococcal alpha-toxin. Both cAMP and cGMP relaxed the permeabilized rat mesenteric artery at the intracellular Ca2+ concentrations [( Ca2+]i) held constant with Ca2+ EGTA buffer and Ca2+ ionophore, ionomycin. In addition, forskolin and sodium nitroprusside which activate adenylate and guanylate cyclases, respectively, also induced relaxation at a fixed [Ca2+]i. In contrast PDBu which stimulates protein kinase C caused an increase in force at a constant [Ca2+]i which could be partially reversed by cAMP or cGMP. These results indicate that second messengers exert direct control over smooth muscle Ca2+ sensitivity of the contractile elements, which is of physiologic and pharmacologic importance.     

The pro-oxidant role of methylglyoxal in mesenteric artery smooth muscle cells

Methylglyoxal (MG), a highly reactive metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation endproducts (AGEs). The present study investigated whether methylglyoxal induced oxidative stress and activated nuclear factor kappa B (NF-kappaB) in freshly isolated and cultured smooth muscle cells (SMCs) from rat mesenteric artery. The treatment of cells with MG (50 or 100 micromol/L) induced a significant increase in AGE formation and oxidation of DCF. MG-enhanced generation of AGEs and the oxidation of DCF was markedly inhibited by antioxidant n-acetylcysteine (NAC, 600 micromol/L). MG at a concentration of 100 micromol/L increased the heme-oxygenase-1 expression in these cells. Moreover, MG activated NF-kappaB p65, indicated by an increased immuno cytochemistry stain for NF-kappaB p65 located in the nucleus after the treatment of mesenteric artery SMCs with MG. MG-induced activation of NF-kappaB p65 was inhibited by NAC. In summary, MG significantly increases oxidative stress and activates NF-kappaB p65 in mesenteric artery SMCs. The pro-oxidant role of methylglyoxal may contribute to various pathological changes of SMCs from resistance arteries.     

Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

Cell calcium and its regulation in smooth muscle

Two novel methods used to study smooth muscles-electron probe X-ray microanalysis and Ca2+-sensitive indicators (which are used for resolving, respectively, the spatial distribution and temporal distribution of calcium)-are briefly reviewed and the major findings obtained are summarized. In smooth muscle the sarcoplasmic reticulum is the major intracellular source of Ca2+; mitochondria do not play a significant role in the physiological regulation of [Ca2+]i. Under pathological conditions mitochondria can reversibly accumulate large amounts of calcium. Resting [Ca2+]i generally ranges from 80 to 200 nM, and is lower in phasic than in tonic smooth muscles. Removal of extracellular Ca2+ and Ca2+ entry blockers can reduce [Ca2+]i, but the effects of beta-adrenergic agents are variable. Increases in [Ca2+]i are triggered by electrical stimulation, depolarization with high K+, and excitatory agonists. Stretch, after a delay of several seconds, can cause an increase in [Ca2+]i in some smooth muscles. There is also a delay of approximately 200-400 ms between the initiation of the rise of Ca2+ and contraction that follows spontaneous action potentials or electrical stimulation. Agonist-induced Ca2+ release, a major mechanism of pharmacomechanical coupling, has been demonstrated in smooth muscles depolarized with high K; evidence suggests that it is mediated by G proteins that couple receptors to phospholipase C. Ca2+ release can be triggered directly in permeabilized smooth muscle with inositol 1,4,5-trisphosphate. Even though Ca2+ is the major physiological regulator of contraction, Ca2+ sensitivity of the regulatory-contractile apparatus differs in different (phasic and tonic) smooth muscles, and can be modulated in a given smooth muscle. The force [Ca2+]i ratio is higher during agonist-stimulated than during high K+-induced contractions, owing to agonist-induced increases in Ca2+ sensitivity mediated by G proteins. In some phasic smooth muscles (guinea pig ileum), the time course of the initial myosin light chain phosphorylation is extremely rapid and returns to basal levels while force remains elevated. In these smooth muscles there is also a marked decrease in the Ca2+ sensitivity of the regulatory-contractile apparatus during maintained depolarization in Ca2+-free or low Ca2+ solutions. It has been suggested that regulation of myosin light chain phosphatase plays a major role in the modulation of the Ca2+ sensitivity manifested as either potentiation or desensitization to [Ca2+]i.     

Effect of manganese and verapamil on electrical and contractile responses of pulmonary artery smooth muscle induced by noradrenaline and histamine

Action of noradrenaline and histamine on the resting potential, membrane resistance and contractility of rabbit pulmonary artery muscle cells was investigated in normal and Ca-blockers (manganese and verapamil)-containing Ringer-Lock solutions. It was shown that catecholamine and histamine induced depolarization by different mechanisms. Thus, noradrenaline action is accounted for by the decreased membrane permeability to potassium ions, while the histamine-induced depolarization is a consequence of sodium and, probably, chlorine permeability. The contraction induced by the transmitters is activated primarily by the extracellular calcium ions entering the cells by two ways: via chemosensitive Ca-channels activated by adrenergic and histaminergic receptors or via potential-dependent slow Ca-channels activated by the transmitter-induced membrane depolarization. It is not excluded that during activation of muscle cells by the transmitters part of calcium is release from both intramembrane and intracellular stores.     

Src modulates contractile vascular smooth muscle function via regulation of focal adhesions

Src is a known regulator of focal adhesion turnover in migrating cells; but, in contrast, Src is generally assumed to play little role in differentiated, contractile vascular smooth muscle (dVSM). The goal of the present study was to determine if Src-family kinases regulate focal adhesion proteins and how this might affect contractility of non-proliferative vascular smooth muscle. We demonstrate here, through the use of phosphotyrosine screening, deconvolution microscopy imaging, and differential centrifugation, that the activity of Src family kinases in aorta is regulated by the alpha agonist and vasoconstrictor phenylephrine, and leads to focal adhesion protein phosphorylation and remodeling in dVSM. Furthermore, Src inhibition via morpholino knockdown of Src or by the small molecule inhibitor PP2 prevents phenylephrine-induced adhesion protein phosphorylation, markedly slows the tissue's ability to contract, and decreases steady state contractile force amplitude. Significant vasoconstrictor-induced and Src-dependent phosphorylation of Cas pY-165, FAK pY-925, paxillin pY-118, and Erk1/2 were observed. However, increases in FAK 397 phosphorylation were not seen, demonstrating differences between cells in tissue versus migrating, proliferating cells. We show here that Src, in a cause and effect manner, regulates focal adhesion protein function and, consequently, modulates contractility during the action of a vasoconstrictor. These data point to the possibility that vascular focal adhesion proteins may be useful drug discovery targets for novel therapeutic approaches to cardiovascular disease.     

Ghrelin suppression of potassium currents in smooth muscle cells of human mesenteric artery

Ghrelin is a 28-amino acid peptide hormone which modulates many physiological functions including cardiovascular homeostasis. Here we report some novel findings about the action of ghrelin on smooth muscle cells (SMC) freshly isolated from human mesenteric arteries. Ghrelin (10(-7) mol/l) significantly suppressed the iberiotoxin-blockable component of potassium currents (I(K)) and depolarized the cell membrane, while having no effect on Ca(2+) currents. Inhibition of inositol-trisphosphate (IP(3))-activated Ca(2+) release channels, depletion of sarcoplasmic reticulum (SR) Ca(2+) stores, blockade of phospholipase D (PLD) or protein kinase C (PKC) each abolished the effect of ghrelin on I(K), while the inhibition of phospholipase C (PLC) did not. These data imply that in human mesenteric artery SMC ghrelin suppresses I(K) via PLD, PKC and SR Ca(2+)-dependent signaling pathway.     

TTX-sensitive voltage-gated Na+ channels are expressed in mesenteric artery smooth muscle cells

The presence and properties of voltage-gated Na+ channels in mesenteric artery smooth muscle cells (SMCs) were studied using whole cell patch-clamp recording. SMCs from mouse and rat mesenteric arteries were enzymatically dissociated using two dissociation protocols with different enzyme combinations. Na+ and Ca2+ channel currents were present in myocytes isolated with collagenase and elastase. In contrast, Na+ currents were not detected, but Ca2+ currents were present in cells isolated with papain and collagenase. Ca2+ currents were blocked by nifedipine. The Na+ current was insensitive to nifedipine, sensitive to changes in the extracellular Na+ concentration, and blocked by tetrodotoxin with an IC50 at 4.3 nM. The Na+ conductance was half maximally activated at -16 mV, and steady-state inactivation was half-maximal at -53 mV. These values are similar to those reported in various SMC types. In the presence of 1 microM batrachotoxin, the Na+ conductance-voltage relationship was shifted by 27 mV in the hyperpolarizing direction, inactivation was almost completely eliminated, and the deactivation rate was decreased. The present study indicates that TTX-sensitive, voltage-gated Na+ channels are present in SMCs from the rat and mouse mesenteric artery. The presence of these channels in freshly isolated SMC depends critically on the enzymatic dissociation conditions. This could resolve controversy about the presence of Na+ channels in arterial smooth muscle.     

Properties of smooth muscle hyperpolarization and relaxation to K+ in the rat isolated mesenteric artery

Smooth muscle membrane potential and tension in rat isolated small mesenteric arteries (inner diameter 100-200 microm) were measured simultaneously to investigate whether the intensity of smooth muscle stimulation and the endothelium influence responses to exogenous K+. Variable smooth muscle depolarization and contraction were stimulated by titration with 0.1-10 microM phenylephrine. Raising external K+ to 10.8 mM evoked correlated, sustained hyperpolarization and relaxation, both of which were inhibited as the smooth muscle depolarized and contracted to around -38 mV and 10 mN, respectively. At these higher levels of stimulation, raising the K+ concentration to 13.8 mM still hyperpolarized and relaxed the smooth muscle. Relaxation to endothelium-derived hyperpolarizing factor, released by ACh, was not altered by the level of stimulation. In endothelium-denuded arteries, the concentration-relaxation curve to K+ was shifted to the right but was not depressed. In denuded arteries, relaxation to K+ was unaffected by the extent of prior stimulation and was blocked with 0.1 mM ouabain but not with 30 microM Ba2+. The ability of K+ to stimulate simultaneous hyperpolarization and relaxation in the mesenteric artery is consistent with a role as an endothelium-derived hyperpolarizing factor activating inwardly rectifying K+ channels on the endothelium and Na+-K+-ATPase on the smooth muscle cells.     

Prenatal hypoxia inhibited propionate-evoked BK channels of mesenteric artery smooth muscle cells in offspring

As a common complication of pregnancy, gestational hypoxia has been shown to predispose offspring to vascular dysfunction. Propionate, one of short-chain fatty acids, exerts cardioprotective effects via reducing blood pressure. This study examined whether prenatal hypoxia impaired propionate-stimulated large-conductance Ca²⁺-activated K⁺ (BK) channel activities in vascular smooth muscle cells (VSMCs) of offspring. Pregnant rats were exposed to hypoxia (10.5% oxygen) and normoxia (21% oxygen) from gestational day 7-21. At 6 weeks of age, VSMCs in mesenteric arteries of offspring were analysed for BK channel functions and gene expressions. It was shown firstly that propionate could open significantly BK single channel in VSMCs in a concentration-dependent manner. Antagonists of G protein βγ subunits and inositol trisphosphate receptor could completely suppress the activation of BK by propionate, respectively. Gα_i/o and ryanodine receptor were found to participate in the stimulation on BK. Compared to the control, vasodilation and increments of BK NPo (the open probability) evoked by propionate were weakened in the offspring by prenatal hypoxia with down-regulated Gβγ and PLCβ. It was indicated that prenatal hypoxia inhibited propionate-stimulated BK activities in mesenteric VSMCs of offspring via reducing expressions of Gβγ and PLCβ, in which endoplasmic reticulum calcium release might be involved.