Muscular composition of the gastric groove in the golden hamster

The golden hamster possesses a forestomach and a glandular stomach. The gastric groove connects the cardia to the glandular stomach and is situated on the lesser curvature of the stomach. The constitution of the muscle fibers in the gastric groove was investigated. The gastric groove consisted of two lips and a groove floor. The muscle coat of the lips was composed of a mixture of smooth and striated muscle fibers. The smooth muscle fibers were components of the cardiac muscle loop. The striated muscle fibers were extensions from the esophageal inner circular muscle layer, and invaded about half the length of the lips. The muscle coat of the groove floor consisted of an inner circular muscle layer made up of smooth muscle fibers, and the outer longitudinal muscle layer of the striated muscle fibers extended from the esophageal outer longitudinal muscle layer. The present study revealed that the muscle coat of the gastric groove in the golden hamster was composed of smooth and striated muscle fibers, and that these striated muscle fibers were extensions of the esophageal muscle coat.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta-mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL-1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta-induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

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The effects of endothelin (ET) 1 on the release of somatostatin (SS) and thyrotropin-releasing hormone (TRH) from the rat stomach were studied in vitro. The rat stomach was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) for 20 min. The amounts of SS and TRH released into the medium were measured by individual radioimmunoassays. With the addition of ET-1, the release of SS from the rat stomach was inhibited significantly in a dose-related manner, whereas TRH released from the stomach was enhanced significantly. These effects of ET-1 on SS or TRH release were blocked by BQ-485, a blocker of ETA receptor. These findings suggest that ET-1 inhibits SS and stimulates TRH release from the rat stomach in vitro, and that these effects are mediate via ETA receptor.     

The biologic effects of growth factor-toxin conjugates in models of vascular injury depend on dose, mode of delivery, and animal species

Toxin-conjugates, complexes designed from the fusion of tissue toxins and pathology-specific ligands, offer the potential for targeted cytotoxic therapy. Some have postulated that the recurrent failure of these conjugates to exhibit benefit in animal models of vascular injury arose because the timing and frequency of conjugate delivery were insufficient to meet the demands of the arterial wall. Previous data suggest that increasingly frequent dosing would lead to superior inhibition of intimal hyperplasia. We now report on the biological effects of the controlled release of a recombinant conjugate of basic fibroblast growth factor (bFGF) and the plant toxin saporin (SAP), bFGF-SAP. Alginate/heparin-Sepharose microspheres and films were designed as drug carriers to control release the bFGF-SAP conjugate or bFGF alone in small doses. When bFGF-SAP-incorporated microspheres or films were implanted adjacent to balloon angioplastied porcine carotid arteries, the controlled release of bFGF-SAP over the four-week study stimulated rather than inhibited hyperplasia. When these same devices were used in cell culture, unexpected findings were produced. bFGF-SAP reduced in vitro bovine vascular smooth muscle cell growth at high concentrations (1-10 microgram/mL) but increased smooth muscle cell growth at lower concentrations (up to 1 microgram/mL). Microsphere controlled-released bFGF-SAP ( approximately 60 ng/mL over 4 days) stimulated the growth of smooth muscle cells more than any of the tested bolus applications of the conjugate. These data provide cause to reconsider our acceptance of controlled release technology as the answer to all forms of drug delivery problems, and to apply more rigorous means of matching the kinetics of drug delivery to the kinetics of the vascular response to injury.     

HR 720, a novel angiotensin receptor antagonist inhibits the angiotensin II-induced trophic effects, fibronectin release and fibronectin-EIIIA+ expression in rat aortic vascular smooth muscle cells in vitro

The aim of this study was to evaluate the direct trophic effects of angiotensin II (AII) on rat vascular smooth muscle cells obtained from a single cellular isolate. Cell volume, protein synthesis, fibronectin (FN) release and FN-EIIIA+ mRNA isoform expression were analyzed in parallel. The effects of HR 720, a novel AT1 angiotensin receptor antagonist with some AT2 receptor affinity, were compared with those of selective AT1 antagonist EXP 3174. Both HR 720 and EXP 3174 inhibited in a concentration-dependent manner the maximum increase in cell volume induced by 10(-9) M Sar1-All (IC50 = 0.49 x 10(-9) M and 0.79 x 10(-9) M, respectively). Maximum [3H]leucine incorporation was also achieved at 10(-9) M All. HR 720 blocked the increase in protein synthesis with potency similar to EXP 3174; the respective IC50 values were 1.04 x 10(-9) M and 1.36 x 10(-9) M. All dose-dependently increased FN release, which was also equally inhibited by about 50% with both compounds at 10(-6) M. Furthermore, All enhanced FN-EIIIA+ mRNA in rat vascular smooth muscle cells (VSMC), which indicated a modulation of FN isoform expression which was inhibited by angiotensin II antagonists. In conclusion, All induced parallel and concentration-dependent increases in cell volume, protein synthesis, FN release and FN-EIIIA+ mRNA expression in vascular smooth muscle cells. These effects appeared to be essentially mediated by AT1 receptor stimulation as indicated by the equal inhibitory effects of HR 720 and EXP 3174.     

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Purines of ATP, ADP, AMP and adenosine released from rat caudal artery with and without endothelium and the isolated smooth muscle and endothelial cells were examined, in order to determine the source. Treatment of intact segments of caudal arteries with noradrenaline (10 microM) for 3 min induced a large release of ATP, ADP, AMP and adenosine. However, if the artery segments had been denuded of their endothelial lining, noradrenraline induced only a slight release of purines. Endothelial cells in primary culture prepared from caudal arteries, when exposed to noradrenaline for 3 min released large amounts of purines, whereas vascular smooth muscle cells prepared similarly and passaged endothelial cells did not release purines upon exposure to noradrenaline. These results indicate that, of smooth muscle and endothelial cells of the vascular wall, only intact endothelial cells react to alpha-adrenoceptor stimulation by releasing adenine nucleotides and adenosine.     

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.     

Dietary beta-adrenoceptor agonists have a persistent effect on nitric oxide synthesis in rat cultured smooth muscle cells

Several compounds including lipopolysaccharide and sympathomimetics stimulate the expression of the inducible nitric oxide synthase in vascular smooth muscle cells. We evaluated the effect of clenbuterol on nitric oxide (NO) production by vascular smooth muscle cells of the rat aorta in culture. Wistar rats were divided into three diet groups (control, clenbuterol and washout). Aortic vascular smooth muscle cells from rats from these 3 diet groups were cultured in the presence and absence of lipopolysaccharide and/or beta-adrenoceptor agonists. NO release was measured by Griess reagent. Clenbuterol or salbutamol added to cells from control rats potentiated lipopolysaccharide-induced NO release. Cells from rats fed on clenbuterol, in a medium without beta-adrenoceptor agonists, showed a similar potentiation, even after a 10-day washout period. The addition of beta-adrenoceptor agonists to the latter cells did not increase NO production. NG-Nitro-L-arginine decreased nitrite production in lipopolysaccharide-stimulated cells. Our results demonstrate that dietary clenbuterol has a persistent 'ex vivo' effect on lipopolysaccharide-induced NO production by cultured vascular smooth muscle cells.     

Beta-adrenoceptors on smooth muscle, nerves and inflammatory cells

Although the bronchodilator action of beta 2-adrenoceptor agonists in asthma is largely due to relaxation of airway smooth muscle, these agents have other effects which may contribute to their anti-asthma action. Human airway smooth muscle contains only beta 2-receptors which, when stimulated, stimulate a rise in intracellular cAMP and activation of PKA (protein kinase A), which in turn phosphorylates several cellular proteins, resulting in relaxation. However, beta-agonists also influence membrane K+ channels and induce smooth muscle relaxation without a rise in cAMP, and this mechanism appears to be the major feature of bronchodilatation in asthma. There is also evidence that beta-agonists may modulate neurotransmission in airways via prejunctional receptors on airway nerves, both sensory and motor. Blockade of prejunctional beta 2-receptors in asthma patients may lead to marked rise in acetylcholine release, with severe bronchoconstriction. Although beta-agonists have little or no effect on the chronic inflammatory response which underlies chronic airway hyper-responsiveness, they do inhibit the release of histamine from mast cells in vitro. The presence of beta-receptors has also been detected not only on mast cells but also on eosinophils, macrophages, lymphocytes and neutrophils, but beta-agonists have little or no inhibitory action on the activities of all these cells due to rapid tachyphylaxis.     

Differential effects of IFN-beta1b on the proliferation of human vascular smooth muscle and endothelial cells

The effect of human interferon (IFN)-beta1b (Betaseron) on the proliferation of cultured human vascular smooth muscle and endothelial cells was tested in vitro. IFN-beta1b inhibited thymidine incorporation and growth of primary cultures of human aortic and coronary artery smooth muscle in a concentration-dependent manner. The same concentrations of IFN-beta1b did not inhibit thymidine incorporation or growth of primary cultures of human aortic or coronary artery endothelial cells. IFN-beta1b induced the expression of MxA (an antiviral protein induced by type I IFNs) in both smooth muscle and endothelial cells, suggesting that both cell types express receptors for type I IFNs. The growth-inhibitory effect of IFN-beta1b could be mimicked by commercially available human IFN-beta, but not by IFN-alpha2 or IFN-alpha8. The effect of IFN-beta1b was species specific, as it did not inhibit thymidine incorporation in aortic smooth muscle cells derived from pig, rabbit, rat, or mouse. The action of IFN-beta1b on smooth muscle cells persisted for at least 4 days following a 24 h preincubation with IFN-beta1b. Human vascular smooth muscle cells treated with IFN-beta1b did not release lactate dehydrogenase, nor did they show any morphologic change, suggesting that IFN-beta1b was not toxic to the human vascular smooth muscle cells. IFN-beta1b inhibited vascular smooth muscle growth while having no growth-inhibitory effect on endothelial cells obtained from the same blood vessel, making it a potential candidate for treating pathologic conditions where abnormal vascular smooth muscle proliferation is implicated, such as restenosis following balloon angioplasty or smooth muscle proliferation following vascular stenting.     

Effect of phospholipase C inhibitor U-73122 on antigen-induced airway smooth muscle contraction in guinea pigs

The importance of phospholipase C (PLC) in airway smooth muscle contraction was studied, using an inhibitor of PLC, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Tracheas from ovalbumin (OA)-sensitized guinea pigs contracted rapidly after exposure to low concentrations of antigen (OA). However, tracheas treated with U-73122 for 10 min prior to the addition of antigen, demonstrated a 3 log rightward shift in the OA dose-response curve with an IC50 of 7 microM. The analogue of U-73122, 1-[6[[17 beta-3-methoxyestra-1,3,5 trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73433), was approximately 5-fold less active in inhibiting smooth muscle contraction. In addition to the inhibition of antigen-induced smooth muscle contraction, U-73122 inhibited carbachol- and leukotriene D4-induced smooth muscle contraction. Furthermore, U-73122 inhibited in a dose-dependent manner antigen-induced histamine release from guinea pig tracheal tissue. The inhibition of smooth muscle contraction by U-73122 correlated well with the inhibition of polyphosphoinositide mediates smooth muscle contractile responses to muscarinic agonists and leukotrienes as well as antigenic-induced contraction.     

Thromboxane A2 stimulated signal transduction in vascular smooth muscle

Thromboxane A2 (TXA2) is a potent, labile vasoconstrictor which stimulates vessel contraction through vascular smooth muscle TXA2 receptors differing from those in platelets. We studied TXA2-stimulated events in cultured adult rat aortic smooth muscle cells. The stable TXA2 mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) competed for TXA2 agonist binding to vascular smooth muscle cells with an IC50 of 10 +/- 1 nM. In fura-2-loaded cells, U46619 increased free cytosolic Ca++ concentration with an EC50 of 49 +/- 14 nM. The increase in free cytosolic Ca++ was rapid, transient and independent of extracellular Ca++ or Ca++ antagonists and thus was due to release from intracellular stores. U46619-mediated Ca++ release was temporally associated with phosphorylation of myosin light chains, increased accumulation of 1,4,5-inositol trisphosphate (EC50 = 32 +/- 4 nM) and cytoplasmic acidification from pH 7.06 +/- 0.01 to 7.00 +/- 0.02 (P = .02). Ca++ release was 53% attenuated by the phospholipase C inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione. In rat aortic rings U46619 caused TXA2 receptor-mediated contractions (EC50 of 28 +/- 2 nM) which were not attenuated by removal of extracellular Ca++ from the superfusion buffer. Together, these results suggest that agonist occupation of TXA2 receptors produces vascular smooth muscle contraction through initial activation of phospholipase C with production of 1,4,5-inositol phosphate, release of intracellular calcium stores and phosphorylation of myosin light chains associated with cellular acidification, presumably via activation of Ca++ ATPase.     

Electron-microscopic radioautography of DNA-synthesizing cells of growing rat smooth muscle tissue

DNA-synthesizing cells of rat stomach muscle tissue following 50% resection of the fundal part, identical cells of the vena cava after disturbance of the blood out flow, and of the appendix on contraction of the ascending part of the large intestine were studied by electron microscopic autoradiography. DNA synthesis in the differentiated myocyte nuclei of the stomach muscle tissue, and of the "activated" myocytes of the stomach and the vein was observed. There were signs of asynchronous DNA synthesis in the nuclei of some smooth muscle cells and fibroblasts.     

Sarcoplasmic reticulum-sarcolemma interactions and vascular smooth muscle tone

A characteristic of vascular smooth muscle cell morphology is a close apposition of its peripheral sarcoplasmic reticulum (SR) with the sarcolomma; this arrangement gives rise to important functional interactions whereby the peripheral SR regulates Ca2+ influx and vascular tone. We review here the key evidence supporting the following aspects of SR-sarcolemma interactions while establishing a conceptual framework encompassing (i) the SR ultrastructure and functions, (ii) the integration of the sarcolemmal Na+-Ca2+ exchanger and the peripheral SR in the mediation of a bidirectional Ca2+ exchange between the peripheral SR and the extracellular space, (iii) the existence of a higher myoplasmic free Ca2+ concentration [Ca2+]myo in the subsarcolemmal space formed between the sarcolemma and the peripheral SR relative to the [Ca2+]myo of the inner myoplasm in the resting smooth muscle cell, (iv) the division of the subsarcolemmal space into functional microdomains, (v) the existence of spontaneous localized bursts of Ca2+ release from the peripheral SR (Ca2+ sparks) towards the sarcolemma, (vi) the physiological triggering of nonlocalized Ca2+ release from the peripheral SR by Ca2+ influx (Ca2+-induced Ca2+ release), and (vii) capacitative Ca2+ entry in vascular smooth muscle. We present an overview of the physiological and pathological implications of these interactions.     

Effect of docosahexaenoic acid on smooth muscle cell functions

There is increasing evidence that fish oil-enriched diets attenuate the progression of several types of human and experimental renal, intestinal and cardiovascular disorders, including hypertension. Docosahexaenoic acid (DHA), may be one of the active biological component. We previously reported that dietary DHA suppressed the progression of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). The purpose of this study is to clarify the in vitro effect of DHA on cultured smooth muscle cell functions such as cell growth, hypertrophy, NO release, and intracellular Ca2+ metabolism, which are involved in the regulatory mechanisms of vascular tone. Addition of DHA to the culture medium of aortic smooth muscle cells isolated from SHRSP and normotensive Wistar Kyoto rats (WKY) had no significant effects on cell growth or on cell hypertrophy induced by angiotensin II as measured by flow cytometry. DHA had no stimulatory effect on interleukin-1beta (10 ng/ml)-induced nitric oxide release from smooth muscle cells of SHRSP, but rather slightly inhibited it. However, the treatment of smooth muscle cells with DHA (30 microM) for 2 days significantly suppressed the increase in intracellular Ca2+ concentration induced by angiotensin II, but not by thapsigargin. This was due to the suppression of Ca2+ influx, as determined by Mn2+ influx experiment. These results indicate that DHA specifically suppresses Ca2+ mobilization into smooth muscle cells. This may be one of the mechanisms by which dietary DHA prevents the development of hypertension in SHRSP.     

Antigenicity of janin, a new protein from smooth muscle

The exact nature of smooth muscle autoantibodies is still unclear. The antigen(s) which they are directed against remain unknown. Janin, a so far unknown smooth muscle protein, was obtained from smooth muscle tissue extracts and purified by means of three different procedures. Its mol. w. was estimated at 60,000 daltons. It stimulated a specific anti-janin antibody in rabbits as shown by tube test, double diffusion in agar and indirect immunofluorescence. The new protein absorbed eighteen out of 104 human smooth muscle positive sera that were not absorbed with smooth muscle actin, myosin, heavy meromyosin, light meromyosin, tropomyosin and brain tubulin. This would vindicate a view that smooth muscle autoantibodies differ from patient to patient with regard to the autoantigen(s) involved.     

Decreased levels of a soluble form of the human adhesion receptor CD58 (LFA-3) in sera and synovial fluids of patients with rheumatoid arthritis

The mode of interaction between muramyl dipeptide (MDP), a compound with immunopharmacological activities, and 5-hydroxtryptamine (5-HT, serotonin) was studied in isolated nerve-smooth muscle preparations of the carp stomach. Application of exogenous 5-HT evoked direct smooth muscle contractions; electric neurogenic stimulation evoked twitches due to release of 5-HT from nerve endings. Contractions evoked by a high concentration of 5-HT (3-30 microM) were resistant to atropine and potentiated in the presence of MDP. Isamoltan (5-HTID antagonist) decreased the amplitude of contractions, whereas ketanserin (5-HT2 antagonist) and MDL 72,222 (5-HT3 antagonist) had no effect. The addition of low concentrations (0.1-1.5 microM) of 5-HT did not contract the preparation but caused a decrease in the amplitude of neurogenic twitches, which might be due to the presynaptic inhibition of serotonin release. This effect of 5-HT was not changed by isamoltan or ketanserin, but it was largely reduced in the presence of 5-HT3 antagonists tropisetron and MDL 72,222. This inhibitory effect of 5-HT on twitch amplitude was potentiated by MDP. The interaction of MDP with the serotonergic system thus involved not only potentiation of the postsynaptic effect of higher 5-HT concentrations, which might have been mediated via the 5-HT1 subsystem, but also presynaptic inhibition. MDP enhancement of 5-HT's inhibitory effect, mediated via 5-HT3 receptors, might represent a new feature in mutual 5-HT-MDP interactions.     

Airway structure and function in Eisenmenger's syndrome

The responsiveness of airways from patients with Eisenmenger's syndrome (n = 5) was compared with that in airways from organ donors (n = 10). Enhanced contractile responses to cholinergic stimulation were found in airways from patients with Eisenmenger's syndrome. The maximal responses to acetylcholine, carbachol, and parasympathetic nerve stimulation in airway tissue from these patients were 221%, 139%, and 152%, respectively, of the maximal responses obtained in donor tissue. Further, relaxation responses to isoproterenol and levocromakalim were absent (n = 2) or markedly impaired (n = 3) in airways from patients with Eisenmenger's syndrome. This attenuated relaxation response was nonspecific in that it was also absent after vasoactive intestinal peptide, sodium nitroprusside, papaverine, and electrical field application. These observations can most likely be explained by a decrease in intrinsic smooth muscle tone, as precontraction of airways revealed relaxation responses that were equivalent to those obtained in donor tissues. Morphometric analysis of tissues used for the functional studies revealed no differences in the airway dimensions (internal perimeter) or airway wall components (e.g., smooth muscle, cartilage) or total area to explain these observations. Although the mechanism for this observed decrease in intrinsic airway smooth muscle tone is not certain, it may be due to alteration in the substructure of the airway wall or, alternatively, may result from the continued release of depressant factors in the vicinity of the smooth muscle which permanently alters smooth muscle responsiveness.