Mechanical and electrophysiological effects of isoprostanes on bronchial arterial smooth muscle

We investigated the mechanical and electrophysiological responses of human and porcine bronchial arterial smooth muscle to isoprostanes (metabolites of membrane lipid peroxidation). These evoked a constrictor response which was sensitive to blockade of thromboxane receptors, as well as to a non-specific tyrosine kinase inhibitor and an inhibitor of Rho-kinase. The patch clamp technique was used to characterize the K+ and Ca2+ currents in these tissues, and to show that isoprostanes caused a brief enhancement of K+ currents followed by prolonged and marked suppression of the same.     

The role of arterial smooth muscle in vasorelaxation

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.     

Relaxation of canine airway smooth muscle

Relaxation of airway smooth muscle is an inadequately understood yet critical process that, if impaired, may have significant implications for asthma. Here we explore why relaxation is an important process to consider, how it may determine airway hyperresponsiveness, and some of the factors that influence relaxation of the airway smooth muscle. These include mechanical and biochemical factors such as deep inspirations or large amplitude oscillation of the muscle, plastic properties of the muscle, the load the muscle experiences, calcium, phosphorylation of the myosin light chain, cytoskeletal proteins, and sensitization.     

Differential effects of furosemide on porcine bronchial arterial and airway smooth muscle.

Furosemide attenuates airway obstruction in asthmatic subjects when administered as an aerosol pretreatment. This protective effect of furosemide could be related to relaxation of bronchial smooth muscle or to increased bronchial blood flow. To determine whether furosemide dilates bronchial smooth muscle, isometric contractile responses in distal bronchi from young pigs were studied. In bronchial smooth muscle rings that were precontracted with 10(-5) M acetylcholine, significant relaxation occurred with 10(-8) to 3 x 10(-6) M isoproterenol but not with 10(-8) to 10(-3) M furosemide. In contrast, bronchial arteries that were precontracted with either 10(-4) M norepinephrine or 10(-8) M vasopressin significantly relaxed in response to 10(-4) to 3 x 10(-3) M and 10(-3) to 3 x 10(-3) M furosemide, respectively. We conclude that furosemide, under the described experimental conditions, relaxes airway vascular smooth muscle but not bronchial smooth muscle. These results are consistent with previous suggestions that inhaled furosemide increases blood flow to airway tissues (Gilbert IA, Lenner KA, Nelson JA, Wolin AD, and Fouke JM. J Appl Physiol 76: 409-415, 1994).     

Plasticity in canine airway smooth muscle

The large volume changes of some hollow viscera require a greater length range for the smooth muscle of their walls than can be accommodated by a fixed array of sliding filaments. A possible explanation is that smooth muscles adapt to length changes by forming variable numbers of contractile units in series. To test for such plasticity we examined the muscle length dependence of shortening velocity and compliance, both of which will vary directly with the number of thick filaments in series. Dog tracheal smooth muscle was studied because its cells are arrayed in long, straight, parallel bundles that span the length of the preparation. In experiments where muscle length was changed, both compliance and velocity showed a strong dependence on muscle length, varying by 1.7-fold and 2.2-fold, respectively, over a threefold range of length. The variation in isometric force was substantially less, ranging from a 1.2- to 1.3-fold in two series of experiments where length was varied by twofold to an insignificant 4% variation in a third series where a threefold length range was studied. Tetanic force was below its steady level after both stretches and releases, and increased to a steady level with 5-6 tetani at 5 min intervals. These results suggest strongly that the number of contractile units in series varies directly with the adapted muscle length. Temporary force depression after a length change would occur if the change transiently moved the filaments from their optimum overlap. The relative length independence of the adapted force is explained by the reforming of the filament lattice to produce optimum force development, with commensurate changes of velocity and compliance.     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Parasympathetic innervation of canine tracheal smooth muscle

To investigate whether efferent parasympathetic fibers to the trachealsmooth muscle course through the pararecurrent nerve rather than therecurrent or the superior laryngeal nerve, we stimulated all threenerves in anesthetized dogs. We also recorded the pararecurrentnerve activity response to bronchoconstrictor stimuli and compared itwith pressure changes inside a saline-filled cuff of an endotrachealtube. Electrical stimulation (30 s, 100 Hz, 0.1 ms, 10 mA) increasedtracheal cuff pressure by 21.0 ± 3.2 and 1.3 ± 0.7 cmH₂O for the pararecurrent and the recurrent laryngealnerve, respectively. Stimulation of the superior laryngeal nerveincreased tracheal cuff pressure before, but not after, sectioning ofthe ramus anastomoticus, which connects it to the pararecurrent nerve.Intravenous administration of sodium cyanide increased pararecurrentnerve activity by 208 ± 51% and tracheal cuff pressure by14.4 ± 3.5 cmH₂O. Elevation of end-tidalPCO₂ to 50 Torr increased pararecurrent nerveactivity by 49 ± 19% and tracheal cuff pressure by 8.4 ± 3.6 cmH₂O. Further elevation to 60 Torr increasedpararecurrent nerve activity by 101 ± 33% and tracheal cuffpressure by 11.3 ± 2.9 cmH₂O. These results lead usto the conclusion that parasympathetic efferent fibers reach the smoothmuscle of the canine trachea via the pararecurrent nerve.

     

The smooth muscle cell. III. Elastin synthesis in arterial smooth muscle cell culture

Primate arterial smooth muscle cells and skin fibroblasts were examined for their ability to synthesize elastin in culture. In the presence of the lathyrogen beta-aminopropionitrile, the smooth muscle cells incorporate [3H]lysine into a lysyl oxidase substrate that was present in the medium and associated with the cell layer. A component having a mol wt of 72,000 and an electrophoretic mobility similar to that of authentic tropoelastin was isolated from the labeled smooth muscle cells by coacervation and fractionation with organic solvents. In the absence of beta-aminopropionitrile, long-term cultures of smooth muscle cells incorporated [14C]lysine into desmosine and isodesmosine, the cross-link amino acids unique to elastin. In contrast, no desmosine formation occurred in the fibroblast cultures. These characteristics demonstrate that arterial smooth muscle cells are capable of synthesizing both soluble and cross-lined elastin in culture.     

Effect of exogenous ATP on canine jejunal smooth muscle

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 +/- 0.6 mV) and EFS evoked a NANC IJP (17 +/- 0.4 mV). Omega-conotoxin GVIA (100 nM) and a low-Ca(2+), high-Mg(2+) solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 microM) and N(G)-nitro-L-arginine (100 microM). Oxyhemoglobin (5 microM) partially (38.8 +/- 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-(N, O-bis-[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine , pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, alpha, beta-methylene ATP, 2-methylthioadenosine 5'-triphosphate tetrasodium salt, and adenosine 5'-O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.     

Effects of extracellular calcium on canine tracheal smooth muscle

Strips of canine tracheal smooth muscle were studied in vitro to determine the effects of changes in the extracellular calcium (Cao) concentration on tonic contractions induced by acetylcholine and 5-hydroxytryptamine. Strips were contracted with graded concentrations of the above agents in 2.4 mM Ca, after which CaCl2 was administered to achieve final concentrations of 5.0, 10.0, and 20.0 mM. Increases in Cao to 5 mM or above caused significant relaxation of muscles contracted with 5-hydroxytryptamine but did not significantly relax muscles contracted with acetylcholine. Increases in Cao also caused significant relaxation of muscles contracted with low concentrations of K+ (20 or 30 mM). However, in 60 or 120 mM K+, increases in Cao resulted predominantly in muscle contraction. Inhibition of the Na+-K+-ATPase by ouabain (10(-5) M) or K+ depletion reversed the effects of Cao from relaxation to contraction in tissues contracted with 5-hydroxytryptamine. Increases in Cao also caused contraction rather than relaxation in the presence of verapamil (10(-6) M). We conclude that calcium has both excitatory and inhibitory effects on the contractile responses of canine tracheal smooth muscle. The inhibitory effects of Ca2+ appear to be linked to the activity of the membrane Na+-K+-ATPase.     

Stimulation of splanchnic afferents reflexly relaxes tracheal smooth muscle in dogs

Although chemical stimulation of abdominal visceral afferents has been shown to reflexly increase cardiovascular and ventilatory function, the effect of stimulating these afferents on airway smooth muscle is unknown. Therefore, we recorded transverse smooth muscle tension from an innervated segment of trachea in chloralose-anesthetized dogs while we topically applied capsaicin (200 micrograms/ml) and bradykinin (0.01-10 micrograms/ml) to the serosal surfaces of the stomach, small intestine, and gallbladder. Application of these irritant substances to the stomach and small intestine decreased tracheal tension and increased mean arterial pressure. However, application of capsaicin and bradykinin to the gallbladder had only small effects on both of these variables. Cutting the splanchnic nerves abolished or greatly attenuated the decreases in tension and increases in mean arterial pressure, whereas cutting the vagi had no effect on them. We conclude that stimulation of splanchnic afferent endings in the stomach and small intestine reflexly relaxes tracheal smooth muscle in dogs. This effect may be one component of the constellation of autonomic responses reflexly evoked by abdominal visceral pain and inflammation.     

The proteome and secretome of human arterial smooth muscle cells

Smooth muscle cells (SMCs) play a crucial role in cardiovascular disorders. A differential proteomic approach should help to elucidate SMC dysfunctions involved in these diseases. With this goal in mind, we plotted the first 2-dimensional (2-D) maps of the proteome and secretome of human arterial smooth muscle cell (ASMC). Intracellular and secreted proteins were extracted from a primary culture of SMCs obtained from patients undergoing coronary artery bypass surgery (n = 11) and separated by 2-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two protein patterns specific to the ASMC proteome and secretome, respectively. A total of 121 and 40 distinct intracellular and secreted polypeptide spots, corresponding to 83 and 18 different proteins, respectively, were identified by matrix-assisted laser desorption/ionization mass spectrometry. The 2-D reference maps and database resulting from this study confirm that SMCs are involved in a wide range of biological functions. They could constitute a useful tool for a wide range of investigators involved in vascular biology, allowing them to investigate SMC protein changes associated with cardiovascular disorders or environmental stimuli.     

Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells

Plasma fibronectin promotes modulation of rat arterial smooth muscle cells from a contractile to a synthetic phenotype during the first few days in primary culture. This process includes cell adhesion and spreading, loss of myofilaments, and formation of a widespread rough endoplasmic reticulum and a prominent Golgi complex. The structural reorganization is accompanied by activation of overall RNA and protein synthesis. Moreover, the cells gain the ability to replicate their DNA and divide in response to platelet-derived growth factor. Here, it is demonstrated that the power of fibronectin to bring about this change in the differentiated properties of the smooth muscle cells resides in a 105-kD cell-binding fragment, whereas a 70-kD collagen-binding fragment and a 31-kD heparin-binding fragment are inactive in this respect. Laminin, another adhesive glycoprotein and a component of the basement membrane that normally surrounds arterial smooth muscle, was contrarily found to maintain the cells in a contractile phenotype. However, with increasing time more and more cells went through the modulation into a synthetic phenotype. This "catch-up" was counteracted by a peptide that contained the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser). Hence, it is possible that the delayed modulation on laminin was due to production of fibronectin by the cells themselves. In support of this notion, fibronectin isolated from smooth muscle cultures was found to be as effective as plasma fibronectin in stimulating the phenotypic modulation. Moreover, using a combination of chemical, immunochemical, and immunocytochemical methods, it was demonstrated that the cells secreted fibronectin as well as laminin at an increasing rate during the first 4 d in primary culture and, notably, cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. Newly synthesized fibronectin was incorporated into a network of pericellular and intercellular fibrils, whereas laminin formed a more diffuse layer covering the cells in a basement membrane-like manner. Taken together, the findings suggest diverse roles for fibronectin and laminin in the control of the differentiated properties of arterial smooth muscle cells. They further indicate that the ability of arterial smooth muscle cells to produce fibronectin and laminin early in primary culture is not directly related to the phenotypic state as determined morphologically and by measurement of overall rates of RNA and protein synthesis. This may be due to the cells being able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of caldesmon in arterial smooth muscle

We have isolated caldesmon (Mr = 145,000), by immunoprecipitation, from [32P]orthophosphate-loaded porcine carotid arteries. In resting muscles, caldesmon was phosphorylated to 0.45 mol of PO4/mol protein, while the 20,000-dalton myosin regulatory light chain (LC20) was phosphorylated to less than 0.05 mol/mol. After stimulation by KCl (110 mM) for 75 min and phorbol 12,13-dibutyrate (PDBu, 1 microM) for 60 min, caldesmon phosphorylation levels rose to 0.96 and 1.1 mol/mol, respectively. LC20 phosphorylation increased to 0.49 mol/mol at 1 min of stimulation by KCl and decreased to 0.17 mol/mol at 60 min. With PDBu, phosphate incorporation into LC20 rose only slightly, reaching 0.09 mol/mol after 90 min. Muscles contracted with histamine (10 microM) or ouabain (1 microM) also demonstrated elevated levels of phosphate incorporation into caldesmon. In these muscles, LC20 phosphorylation levels were less than 0.05 mol/mol. Three major phosphopeptides of indistinguishable mobility were identified on maps of caldesmon from resting, KCl-stimulated, and PDBu-stimulated muscles. There was, however, little similarity between the phosphopeptide maps of caldesmon phosphorylated in intact tissue and maps of purified caldesmon phosphorylated in vitro by protein kinase C (Ca2+/phospholipid-dependent enzyme) or Ca2+/calmodulin kinase II.     

Inhibitory effects of epoxyeicosatrienoic acids on volume-activated chloride channels in rat mesenteric arterial smooth muscle

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by cytochrome P450 epoxygenases in endothelial cells. It has previously been shown that EETs activate K(+) channels, which are important for the hyperpolarization and dilation of blood vessels. However, the effects of EETs on other ion channels have been less well studied. We investigated the effects of EETs on volume-activated Cl(-) channels (VACCs) in rat mesenteric arterial smooth muscle cells. Whole-cell patch clamp recording demonstrated that hypotonic solution and guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) induced a 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)- and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive VACC current in the primary cultured rat mesenteric arterial smooth muscle cells. The VACC current was inhibited by EETs and the order of potency was 8,9-EET>5,6-EET>11,12-EET>14,15-EET. The inhibitory effects of EETs could be reversed by 14,15 epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET analog), Rp-cGMP and KT-5823 (protein kinase G inhibitors). Interestingly, the inhibitory effects of EETs on VACCs were not influenced by Rp-cAMP (a protein kinase A antagonist) but it could be abolished by NF-449 (a Gs protein inhibitor), indicating the involvement of cAMP but not protein kinase A. In conclusion, our results demonstrate that EETs inhibit VACCs in rat mesenteric arterial smooth muscle cells through a cGMP-dependent pathway, which is probably due to the cross-activation by cAMP. This mechanism may be involved in the regulation of cell volume and membrane potential.     

Inhibitory effects of epoxyeicosatrienoic acids on volume-activated chloride channels in rat mesenteric arterial smooth muscle

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by cytochrome P450 epoxygenases in endothelial cells. It has previously been shown that EETs activate K⁺ channels, which are important for the hyperpolarization and dilation of blood vessels. However, the effects of EETs on other ion channels have been less well studied. We investigated the effects of EETs on volume-activated Cl⁻ channels (VACCs) in rat mesenteric arterial smooth muscle cells. Whole-cell patch clamp recording demonstrated that hypotonic solution and guanosine 5′-[γ-thio]triphosphate (GTPγS) induced a 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)- and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive VACC current in the primary cultured rat mesenteric arterial smooth muscle cells. The VACC current was inhibited by EETs and the order of potency was 8,9-EET > 5,6-EET > 11,12-EET > 14,15-EET. The inhibitory effects of EETs could be reversed by 14,15 epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET analog), Rp-cGMP and KT-5823 (protein kinase G inhibitors). Interestingly, the inhibitory effects of EETs on VACCs were not influenced by Rp-cAMP (a protein kinase A antagonist) but it could be abolished by NF-449 (a Gs protein inhibitor), indicating the involvement of cAMP but not protein kinase A. In conclusion, our results demonstrate that EETs inhibit VACCs in rat mesenteric arterial smooth muscle cells through a cGMP-dependent pathway, which is probably due to the cross-activation by cAMP. This mechanism may be involved in the regulation of cell volume and membrane potential.