Effect of flush-perfusion on vascular endothelial and smooth muscle function

BACKGROUND: The aim of this study was to investigate how much perfusion pressure an artery can tolerate without significant loss of endothelium-dependent relaxation (EDR) and vascular contractility. METHODS: The abdominal aortas of 396 Sprague-Dawley rats were used. One hundred twenty aortas were flush-perfused for 1 or 5 minutes with cold St. Thomas' Hospital cardioplegic (STHC) solution or with the same solution but modified by the addition of 3.5% dextran 40. Three perfusion pressures were tested: 50, 100, and 150 mm Hg. Two hundred eighty vessels were subjected to pressures of 50, 150, or 300 mm Hg using saline or STHC solution at 22 degrees C or STHC solution at 4 degrees C, for 10 or 60 seconds. The vessels were investigated in organ baths. Contractility was tested with the thromboxane analogue U-46619, acetylcholine was used to investigate EDR, and papaverine to elicit endothelium-independent relaxation. RESULTS: Flush-perfusion with cold STHC solution for 5 minutes at a perfusion pressure of 50 or 100 mm Hg affected neither contractility nor EDR. Vessels exposed to a flush-perfusion pressure of 150 mm Hg for 1 or 5 minutes lost 39% (p < 0.001) and 53% (p < 0.001) of their contractility, respectively. Flush-perfusion at 150 mm Hg for 1 minute did not affect EDR, whereas 5 minutes' perfusion caused a reduction of 7% (p < 0.05). A repetition of these experiments using STHC solution with 3.5% dextran 40 added gave no significantly different results. The impairment in contractility and EDR seen after perfusion at 150 mm Hg for 5 minutes disappeared after transplantation and reperfusion for 7 days. The vessels could be distended with saline or STHC solution at a pressure of 150 mm Hg without affecting contractility at 22 degrees C. At 4 degrees C, however, this pressure was harmful to contractility. Distention at a pressure of 300 mm Hg almost abolished contractility and 7 days after transplantation there had not yet been any recovery of contractility, but 30 days after transplantation the grafts had regained their normal contractility. CONCLUSIONS: Cold STHC solution, with or without dextran 40, can be used with a perfusion pressure of 100 but not 150 mm Hg without impairing EDR or vascular smooth muscle function.     

Effect of temperature in long-term preservation of vascular endothelial and smooth muscle function

BACKGROUND: In clinical transplantation the donor organ is perfused with a cold preservation solution to obtain quick core cooling and a suitable environment for the tissue cells. Without good preservation of the vasculature, progressive deterioration of the blood flow during reperfusion may ultimately lead to the no-reflow phenomenon, even though the function of the other cells in the organ may be adequately preserved. The aim of this study was to find the optimal storage temperature for preservation of the vasculature. METHODS: The infrarenal aorta of 126 Sprague-Dawley rats were studied in organ baths: as fresh controls, after 36 hours of storage at 0.5 degrees C, 4 degrees C, 8.5 degrees C, and 22 degrees C in University of Wisconsin solution, and after 36-hour storage followed by transplantation and a lapse of 2 hours, 24 hours, and 7 days. The thromboxane analogue U-46619 was used to test contractility. Acetylcholine was used to elicit endothelium-dependent relaxation (EDR), and papaverine to elicit endothelium-independent relaxation. RESULTS: Storing the vessels at 0.5 degree C proved best regarding preservation of contractility, with a nonsignificant decrease, whereas storage at 4 degrees C and 8.5 degrees C resulted in a significant decrease after 36 hours. The contractility did not recover within 24 hours of in vivo reperfusion, but full recovery was seen after 7 days. Regardless of the preservation temperature used, a significant impairment in EDR was seen after 36 hours of storage. Two hours after transplantation, vessels stored at 4 degrees C and 8.5 degrees C showed no significant impairment in EDR, whereas those stored at 0.5 degrees C demonstrated a significant loss of EDR. After 24 hours and after 7 days, EDR was normal in all groups. CONCLUSIONS: Endothelium-dependent relaxing factor function is best preserved at 4 degrees C and 8.5 degrees C, whereas preservation of vascular smooth muscle function is best preserved at 0.5 degrees C.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K+ channel have been described in vascular smooth muscle: Voltage-activated K+ channels (Kv) are encoded by the Kv gene family, Ca(2+)-activated K+ channels (BKCa) are encoded by the slo gene, inward rectifiers (KIR) by Kir2.0, and ATP-sensitive K+ channels (KATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4-1.6, Kv2.1, Kv9.3, Kv beta 1-beta 4, slo alpha and beta, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K+ channels are modulated by physiological vasodilators, which increase K+ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate KATP channels. These include adenosine, calcitonin gene-related peptide, and beta-adrenoceptor agonists. beta-adrenoceptors can also activate BKCa and Kv channels. Several vasoconstrictors that activate protein kinase C inhibit KATP channels, and inhibition of BKCa and Kv channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BKCa channels, and possibly KATP channels. Hypoxia leads to activation of KATP channels, and activation of BKCa channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of Kv channels. Vasodilation to increased external K+ involves KIR channels. Endothelium-derived hyperpolarizing factor activates K+ channels that are not yet clearly defined. Such K+ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are eicosanoids synthesized from arachidonic acid by the cytochrome P450 eposygenase pathway. The present studies demonstrate that 8,9-, 11,12-, and 14,15-EET are rapidly taken up by porcine aortic smooth muscle cells. About half of the uptake is incorporated into phospholipids, and saponification indicates that most of this remains in the form of EET. The EETs also are converted to the corresponding dihydroxyeicosatrienoic acids (DHETs) and during prolonged incubations, additional metabolites that do not retain the EET carboxyl group are formed. Most of these products are released into the medium. However, some DHET and metabolites less polar than EET are incorporated into the phospholipids, and a small amount of unesterified EET is also present in the cells. The incorporation of 14,15-EET and its conversion to DHET did not approach saturation until the concentration exceeded 10-20 microM, indicating that vascular smooth muscle has a large capacity to utilize this EET. These findings suggest that certain vasoactive effects of EETs may be due to their incorporation by smooth muscle cells. Furthermore, through conversion to DHET and other oxidized metabolites, smooth muscle apparently has the capacity to inactivate EETs that are either formed in or penetrate into the vascular wall.     

Progesterone inhibits arterial smooth muscle cell proliferation

Mortality from atherosclerotic cardiovascular disease is lower in premenopausal women than in age-matched men. It is also lower in postmenopausal women who take estrogens and progestins together rather than estrogens alone. Progesterone receptors were detected in human and rat aortic smooth muscle cells in vivo and in vitro (in subculture). We examined the effect of progesterone on proliferation of smooth muscle cells, important constituents of atherosclerotic plaques. Progesterone at physiologic levels inhibited DNA synthesis and proliferation in these cells in a dose-dependent manner, and pretreatment with the progesterone receptor antagonist RU486 blocked inhibition. Cyclin A and E messenger RNA levels decreased after progesterone treatment but those of cyclin B and D1 did not change. This cell cycle-dependent inhibition of arterial smooth muscle cell proliferation by progesterone may represent a mechanism for the hormone's protective effect against atherosclerosis.     

Urokinase receptor-dependent upregulation of smooth muscle cell adhesion to vitronectin by urokinase

The plasminogen activator system has been implicated in the modulation of the response to vascular injury. Although urokinase-type plasminogen activator (uPA) and its receptor (uPAR) may enhance matrix degradation as well as migration and invasion by smooth muscle cells (SMCs), their roles in cell adhesion are uncertain. Therefore, we examined the ability of uPA and uPAR to modulate adhesion of cultured human vascular SMCs to various matrices. We demonstrated a dose-dependent stimulation of adhesion by single-chain uPA (scuPA) to vitronectin (maximum 1.55-fold [+/-0. 04-fold] increase, 10 nmol/L, P<0.002) but not to laminin, collagen I, or collagen IV. Baseline adhesion to vitronectin was completely inhibited by both EDTA and RGD peptide but was restored to >40% of control in the presence of scuPA (P=0.001 and 0.046, respectively). Adhesion to vitronectin was also significantly enhanced by the amino-terminal fragment of uPA (P=0.007) and two-chain, high-molecular-weight uPA (P<0.01) but not by the low-molecular-weight fragment of uPA, which lacks the receptor-binding domain. Aprotinin, a plasmin inhibitor, had no effect on baseline or scuPA-stimulated adhesion, suggesting a plasmin-independent process. Preincubation of scuPA with soluble uPAR inhibited scuPA stimulation of adhesion by 88+/-14% (P=0.01), as did pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes glycophosphatidylinositol-anchored proteins, including uPAR. Antibodies to both alphavbeta3 and alphavbeta5 integrin inhibited baseline adhesion but not scuPA stimulation. Finally, coating plates with scuPA alone enabled cell adhesion, which could be inhibited by both soluble uPAR and anti-uPAR antibodies. These data suggest that uPA stimulates adhesion of SMCs specifically to vitronectin and that it is mediated by an interaction with uPAR. Upregulation of both proteins after vascular injury may facilitate migration through stimulation of both matrix degradation and cell adhesion.     

Collateral ventilation and pulmonary arterial smooth muscle in the coati

Collateral ventilation can participate in ventilation-perfusion regulation by shifting normoxic gas into hypoxic lung regions. In species lacking collateral pathways, such as cattle and swine, ventilation-perfusion balance must rely heavily on hypoxic vasoconstriction, which may explain why their muscular pulmonary arteries are much thicker than those of other animal species. The presence of these unusually muscular vessels in turn may account for the vigorous pressor response to acute hypoxia in these species. The only other species known to lack collateral ventilation is the coati. To determine whether coatis fit the pulmonary circulatory pattern of cattle and swine, we measured pulmonary arterial wall dimensions and pulmonary vascular responsiveness to acute airway hypoxia in 11 adult coatis. Hypoxia caused impressive pulmonary arterial hypertension [normoxia = 17 +/- 1 (SE) Torr, hypoxia = 40 +/- 2 Torr, cardiac output unchanged]. The medial thickness of muscular pulmonary arteries (50-300 microns) was 17.1 +/- 1.8% (SD) of external diameter, a thickness unprecedented in normotensive adult mammals. We conclude that coatis fit the pattern of other species lacking collateral ventilation, since they have thick-walled pulmonary arteries and a vigorous pressor response to hypoxia.     

MAP kinases and vascular smooth muscle function

Integrins are a subclass of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. Integrins influence transendothelial migration of lymphocytes and monocytes and are suitable targets for experimental immunotherapy. They are critically involved in the pathogenesis of autoimmune neuritis and abnormally expressed in human neuropathies. Also, the role of integrins in myelination, neurite outgrowth, and nerve regeneration suggests that they could be involved in the recovery phase of immune-mediated neuropathies. We investigated by immunohistochemistry the expression of a number of integrin subunits during the course of experimental autoimmune neuritis (EAN). Results were compared with the human immune neuropathy Guillain-Barre syndrome (GBS) and extended in vitro. Inflammation and demyelination in both EAN and GBS induced the down-regulation of beta4 integrin in Schwann cells (SCs), whereas loss of alpha2 was noted only in EAN. When axonal loss was present, SCs displayed alpha5 integrin, in both EAN and GBS. In vitro, basal lamina and inflammatory cytokines modulated the expression of beta4 in SCs, but they did not influence alpha2 and alpha5 expression. Finally, integrins were differentially expressed in blood vessels during EAN. In conclusion, the spatiotemporal changes in integrin expression may be used to characterize, stage, and better understand the pathogenesis and evolution of inflammation during GBS and EAN. This may help to establish useful, novel therapy for immune-mediated neuropathies.     

Interaction between growth factors and kinins in arterial smooth muscle cells

Bradykinin receptors are present on vascular smooth muscle cells; however, the regulation and biological function of these receptors is unclear. To address these questions the interaction between growth factors and kinins in cultured arterial smooth muscle cells has been examined. Based upon the data a hypothesis is presented that platelet-derived growth factor (PDGF) upregulates cell surface bradykinin B2 receptors on arterial smooth muscle cells. The biological effect of the increase in B2 receptors is currently unclear but under certain conditions they may enhance mitogenesis. These mitogenic effects however, are strongly opposed by the effects of bradykinin acting via a B1-type of receptor which mediates potent inhibition of growth factor-induced mitogenesis.     

Urokinase expression in transduced endothelial cells

Increased thromboresistance through the release of lytic agents by endothelial cells may improve the patency of endothelial lined prosthetic grafts. We have evaluated the expression of urokinase from cells transduced with a retrovirus containing the gene for a human preprourokinase. Endothelial cells were enzymatically harvested from canine external jugular vein in nine animals and grown to confluence in culture. One-third of these cells served as controls, and the remaining two-thirds were transduced via incubation with an LXSN-type retroviral vector carrying the urokinase gene and a neomycin resistance gene. Successfully transduced cells were selected by incubation with 400 micrograms/mL G418 and pure cultures grown to confluence. Supernatants from confluent control and experimental cell cultures after 48 hours in defined, serum-free medium were assayed for human urokinase concentration and overall enzyme activity. ELISA quantitation of concentration using mouse antihuman urokinase antibody showed 0.15 +/- 0.11 ng/mL/hr/10(6) cells in the transduced cell supernatant; no measurable concentration was found in the control cells. (P < 0.01) Overall (human plus canine) enzyme activity of urokinase was determined using an indirect spectrophotometric assay based on plasminogen activation (ploug U/mL). Transduced cells showed activities of 0.12 at 10 days and 0.45 at confluence; control cell activity was 0.0 and 0.15, respectively. (P < 0.05) These data show that endothelial cells can be transduced with a urokinase expressing gene that increases the release of this thrombolytic agent. Lining small diameter prosthetic grafts with these cells may improve their thromboresistance and long-term patency.     

Voltage-dependent Ca2+ channels in arterial smooth muscle cells

The past years have seen some significant advances in our understanding of the functional and molecular properties of voltage-dependent Ca2+ channels in arterial smooth muscle. Molecular cloning and expression studies together with experiments on native voltage-dependent Ca2+ channels revealed that these channels are built upon a molecular structure with properties appropriate to function as the main source for Ca2+ entry into arterial smooth muscle cells. This Ca2+ entry regulates intracellular free Ca2+, and thereby arterial tone. We summarize several avenues of recent research that should provide significant insights into the functioning of voltage-dependent Ca2+ channels under conditions that occur in arterial smooth muscle. These experiments have identified important features of voltage-dependent Ca2+ channels, including the steep steady-state voltage-dependence of the channel open probability at steady physiological membrane potentials between -60 and -30 mV, and a relatively high permeation rate at physiological Ca2+ concentrations, being about one million Ca2+ ions/s at -50 mV. This calcium permeation rate seems to be a feature of the pore-forming Ca2+ channel alpha1 subunit, since it was identical for native channels and the expressed alpha1 subunit alone. The channel activity is regulated by dihydropyridines, vasoactive hormones and intracellular signaling pathways. While the membrane potential of smooth muscle cells primarily regulates arterial muscle tone through alterations in Ca2+ influx through dihydropyridine-sensitive voltage-dependent ('L-type') Ca2+ channels, the role of these channels in the differentiation and proliferation of vascular smooth muscle cells is less clear. We discuss recent findings suggesting that other Ca2+ permeable ion channels might be important for the control of Ca2+ influx in dedifferentiated vascular smooth muscle cells.     

Bilirubin levels in subarachnoid clot and effects on canine arterial smooth muscle cells

BACKGROUND AND PURPOSE: Previous studies have suggested that bilirubin is a potential contributor to cerebral vasospasm. The purpose of this investigation was to determine whether bilirubin accrues in subarachnoid clot, whether its vasoconstrictive effect could involve a direct action on arterial smooth muscle cells, and, if so, whether bilirubin affects their Ca2+ uptake. METHODS: Subarachnoid clots were analyzed for bilirubin using high-performance liquid chromatography. The length and 45Ca2+ uptake of vascular smooth muscle cells enzymatically dissociated from canine carotid arteries were measured before and after exposure to bilirubin solution. Additional experiments were conducted on cultured smooth muscle cells from canine basilar artery and on ATP-depleted cardiac myocytes. RESULTS: Mean +/- SE bilirubin concentration in experimental clot was 263 +/- 35.7 mumol/L. Vascular smooth muscle cells exposed to bilirubin showed progressive shortening (P < .01) and an increased uptake of 45Ca2+ (P < .001). Contraction was prevented by Ca(2+)-free media but not by verapamil. Experiments with heart myocytes showed that bilirubin caused an increased uptake of 45Ca2+ but not of [14C]sucrose. CONCLUSIONS: The results indicate that bilirubin accrues in subarachnoid clot, that it exerts a direct constrictive effect on arterial smooth muscle cells, and that this effect is associated with an increased uptake of Ca2+. Studies on heart myocytes suggest that the Ca2+ uptake induced by bilirubin could be due to a selective increase in membrane permeability to Ca2+.     

Electrical coupling between smooth muscle cells and endothelial cells in pig coronary arteries

The control of smooth muscle cells by endothelial cells has been well established by the identification of vasoactive factors released by the endothelial cells. In contrast, the possibility that smooth muscle cells influence the endothelial cells has been considered rarely. Some results suggest possible electrical communication between the smooth muscle and the endothelial cells but proof is lacking. We therefore tested for electrotonic conduction of signals from smooth muscle cells to endothelial cells. The endothelium was removed from half of a strip of porcine coronary artery. In a partitioned chamber, rectangular hyperpolarization or depolarization was applied to the de-endothelialized region by field stimulation. The resulting membrane potential changes in the smooth muscle cells spread electrotonically along the media into the area with intact endothelium. We recorded from endothelial cells to determine whether this electrical signal spreads into endothelial cells. Hyperpolarization or depolarization initiated in smooth muscle cells was recorded consistently in endothelial cells. This demonstrates a functional electrotonic propagation from smooth muscle to endothelial cells.     

Effect of serum on vascular smooth muscle function

We investigated the in vitro and in vivo effects of KT6352, a derivative of indolocarbazole compound, on murine megakaryocytopoiesis. When serum-free megakaryocyte (Meg) colony assay was performed with 100 U/mL of recombinant mouse interleukin-3 (rmIL-3), the addition of 1x10(-11)M to 1x10(-9)M of KT6352 increased the number of Meg colonies. An additional increase of Meg colonies by KT6352 was observed in the serum-free culture containing rmIL-3 plus recombinant mouse interleukin-6 or rmIL-3 plus recombinant mouse stem cell factor. KT6352 did not stimulate Meg colony formation without rmIL-3. When KT6352 was administered intraperitoneally to normal BALB/c male mice at a dose of 10 mg/kg daily for 5 consecutive days, a 2.1-fold increase in the platelet count was observed on day 14, and the prolonged thrombocytopoiesis was detectable from 9 to 27 days after KT6352 administration. A marked increase in the white blood cell count was also observed from 5 to 14 days after KT6352 treatment. Before the gradual increase of platelet counts, 8 days after KT6352 administration, a marked increase in the number of colony-forming units of megakaryocytes (CFU-Megs) in bone marrow and spleen was observed, and a substantial increase in the number of splenic CFU-Megs was observed 14 and 23 days after KT6352 administration. Bone marrow Meg ploidy analysis by two-color flow cytometry showed a shift in the modal ploidy class from 16 to 32 and an increase in the frequency of 64 cells in KT6352-treated mice. These results suggest a possible therapeutic benefit of KT6352 in the management of thrombocytopenia.     

Tissue factor is rapidly induced in arterial smooth muscle after balloon injury

Tissue factor (TF) is a major activator of the coagulation cascade and may play a role in initiating thrombosis after intravascular injury. To investigate whether medial vascular smooth muscle provides a source of TF following arterial injury, the induction of TF mRNA and protein was studied in balloon-injured rat aorta. After full length aortic injury, aortas were harvested at various times and the media and adventitia separated using collagenase digestion and microscopic dissection. In uninjured aortic media, TF mRNA was undetectable by RNA blot hybridization. 2 h after balloon injury TF mRNA levels increased markedly. Return to near baseline levels occurred at 24 h. In situ hybridization with a 35S-labeled antisense rat TF cRNA probe detected TF mRNA in the adventitia but not in the media or endothelium of uninjured aorta. 2 h after balloon dilatation, a marked induction of TF mRNA was observed in the adventitia and media. Using a functional clotting assay, TF procoagulant activity was detected at low levels in uninjured rat aortic media and rose by approximately 10-fold 2 h after balloon dilatation. Return to baseline occurred within 4 d. These data demonstrate that vascular injury rapidly induces active TF in arterial smooth muscle, providing a procoagulant that may result in thrombus initiation or propagation.     

Mitogen-activated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation

Smooth muscle cell proliferation and migration is important in arteriosclerosis. In this process, cytokines and growth factors are upregulated and bind to their respective receptors, which in turn stimulate mitogen-activated protein (MAP) kinases. MAP kinases then relay signals to the nucleus that activate quiescent smooth muscle cells. Phosphatases downregulate MAP kinases. We investigated the role of a dual-specificity tyrosine phosphatase, MAP kinase phosphatase-1 (MKP-1), in smooth muscle cell proliferation. MKP-1 expression was high in arterial tissue by Northern analysis, and MKP-1 message was detected mainly in the arterial smooth muscle layer by in situ hybridization. After balloon injury of the rat carotid artery, expression of MKP-1 decreased greatly, whereas that of MAP kinases, especially p44 MAP kinase, increased. The time course of the reduction in MKP-1 message correlated with increased tyrosine phosphorylation and elevated p44 MAP kinase enzymatic activity. In rat arterial smooth muscle cells overexpressing MKP-1, growth was arrested in the G1 phase and entry into the S phase was blocked. A reduction in MKP-1 expression may contribute in part to proliferation of smooth muscle cells after vascular injury, possibly through a decrease in dephosphorylation of p44 MAP kinase.     

Evidence for a role of collagen synthesis in arterial smooth muscle cell migration

Migration of smooth muscle cells (SMCs) and collagen synthesis by SMCs are central to the pathophysiology of vascular disease. Both processes can be induced shortly after vascular injury; however, a functional relationship between them has not been established. In this study, we determined if collagen synthesis was required for SMC migration, using ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl-4-hydroxylase, and 3,4-DL-dehydroproline (DHP), a proline analogue, which we demonstrate inhibit collagen elaboration by porcine arterial SMCs. SMCs exposed to EDHB or DHP attached normally to collagen- and vitronectin-coated substrates; however, spreading on collagen but not vitronectin was inhibited. SMC migration speed, quantified by digital time-lapse video microscopy, was significantly and reversibly reduced by EDHB and DHP. Flow cytometry revealed that expression of beta1 integrins, through which SMCs interact with collagen, was unaffected by EDHB or DHP. However, both inhibitors prevented normal clustering of beta1 integrins on the surface of SMCs, consistent with a lack of appropriate matrix ligands for integrin engagement. Moreover, there was impaired recruitment of vinculin into focal adhesion complexes of spreading SMCs and disassembly of the smooth muscle alpha-actin-containing cytoskeleton. These findings suggest that de novo collagen synthesis plays a role in SMC migration and implicates a mechanism whereby newly synthesized collagen may be necessary to maintain the transcellular traction system required for effective locomotion.     

Leukotrienes in acetylcholine-induced contraction of esophageal circular smooth muscle in experimental esophagitis

BACKGROUND & AIMS: Phospholipase A2 (PLA2) participates in acetylcholine (ACh)-induced contraction of esophageal circular smooth muscle. Because PLA2, arachidonic acid, and its metabolites are involved in inflammatory responses, their role after induction of experimental esophagitis was examined. METHODS: Experiments were performed in esophageal smooth muscle cells (ESO) isolated by enzymatic digestion from the circular layer of normal and esophagitis animals. Content of peptidoleukotrienes (leukotriene [LT] C4, LTD4, and LTE4) was measured in esophageal circular muscle tissue. RESULTS: The cytosolic PLA2 antagonist trifluoromethyl ketone analogue of arachidonic acid inhibited ACh-induced contraction of normal and esophagitis ESO. Inhibition by secreted PLA2 antagonists AM5 and MJ33 was significantly greater in esophagitis ESO. The lipoxygenase inhibitor nordihydro-guaiaretic acid and the LTD4 antagonist ICI 198,615 inhibited ACh-induced contraction of esophagitis but not of normal ESO. Secreted PLA2 and LTD4 contracted normal ESO more than esophagitis ESO. However, in esophagitis, ESO contraction was increased by threshold diacylglycerol concentration. Resting levels of LTs were greater in esophagitis than in normal circular esophageal muscle and increased in response to ACh in esophagitis but not in normal esophageal muscle. CONCLUSIONS: Esophagitis shifts the signal transduction pathway activated by ACh. Esophagitis increased the contribution of secreted PLA2 and of LTs to ACh-induced contraction.     

The effect of sodium based hypo-osmolality on arterial smooth muscle reactivity in vitro

The study tested the hypothesis that the reduced [Na+]e and hypo-osmolality of normal pregnancy are causally linked to the attenuation of vascular smooth muscle reactivity in vitro. Aortic rings from nonpregnant female rats were incubated in physiological medium containing 114 mM NaCl/l and the contractile responses to phenylephrine, KCl and CaCl2 as well as the relaxations to acetylcholine and KCl were compared with those of rings incubated in normal medium containing 119 mM NaCl/l. There was no solute substituted for the lowered [Na+]. Experiments with phenylephrine were repeated using de-endothelialized rings and intact rings pretreated with indomethacin. Contractile responses of intact rings (n = 11) in hypo-osmolar solution to phenylephrine were significantly (P < 0.001) lower than of those in normal medium (n = 11). Responses were partially restored by endothelial denudation but not in the presence of indomethacin. Relaxations to acetylcholine (n = 7 for hypo-osmolar; n = 6 for normal solution) and KCl (n = 7 for each of hypo- and normal osmolar) were significantly enhanced (P < 0.05) in rings incubated in hypo-osmolar solution. There was no significant difference between the responses of the rings to KCl, and CaCl2 in either solution. These effects are similar to some of those previously described for vascular smooth muscle in normal pregnancy suggesting that the reduced [Na+]e and hypo-osmolarity of normal pregnancy may be contributing to the diminished vascular reactivity.