Prostacyclin production by the bovine aortic smooth muscle

It is well known that cultured aortic smooth muscle cells, the phenotype of which has modulated from contractile to synthetic, are able to release prostacyclin (PGI2). We have studied the release of PGI2 from cultured explants of bovine aortic media, which represent an homogeneous population of smooth muscle cells with a contractile phenotype. These explants released spontaneously huge amounts of PGI2, which was the major eicosanoid produced. PGI2 release was stimulated by serum and by serotonin. This experimental model seems useful to evaluate the contribution of smooth muscle to the biosynthesis of PGI2 by the arterial wall.     

Effect of vasoactive peptides on prostacyclin formation in cerebromicrovascular cellular elements and glia: a comparative study

The aim of the present study was to determine basal and stimulated release of prostacyclin from the separately cultured endothelial and smooth muscle cells derived from rat brain microvessels and from glial cells.The basal release of PGI₂ (measured as a 6-keto-PGF_1α formation by radioimmunoassay method) was significantly greater in cultured endothelial cells than in cultured smooth muscle or glial cells (254 ± 32, 140.7 ± 17 and 76.8 ± 5.8 pg/mg protein, respectively). Prostacyclin formation stimulated by angiotensin I, angiotensin II and bradykinin was significantly increased in the smooth muscle cells. A significant enhancement of PGI₂ formation was also observed in the glial cells exposed to angiotensin II or bradykinin. Vasoactive peptides did not affect prostacyclin production in the endothelial cells.Presented results indicate that the smooth muscle cells represent the most sensitive site of prostacyclinpeptide interaction. These data also suggest that the endothelial and the glial cells may protect the cerebromicrovascular smooth muscle by inactivating vasoactive peptides derived from either the blood or the brain.     

Role of protein kinase C in the control of vascular prostacyclin: Study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca⁺⁺ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

PGI₂ synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI₂ produced were shown as 6-keto-PGF_1α per μg of aortic tissue DNA instead of per mg wet weight. We also investigated PGI₂ synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima.Basal PGI₂ production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI₂ production by atherosclerotic aorta was also significantly higher than that of controls. PGI₂ producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI₂ production than the medial layer.Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI₂ than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI₂ production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Role of protein kinase C in the control of vascular prostacyclin: study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca++ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Prostacyclin-stimulating drugs: New prospects

SKF 525-A (proadifen), a well-known inhibitor of drug metabolism and cytochrome P-450 activity, stimulated the release of prostacyclin (PGI₂) from the rabbit aorta in vitro. The PGI₂-stimulating activity of SKF 525-A was characterized by specific structural requirements : activity was abolished by the deletion of the terminal propyl chain and increased by its elongation into an isobutyl chain; chlorination of the phenyl rings increased the potency. SKF 525-A increased the production of PGI₂ by cultured endothelial cells from bovine aorta and human umbilical vein, but had no effect on cultured smoooth muscle from the bovine aortic media. In human platelets, SKF 525-A inhibited prostaglandin and thromboxane production induced by A23187, thrombin and ADP. Simultaneous stimulation of endothelial PGI₂ and inhibition of platelet TxA₂ represents an original pharmacological profile : SKF 525-A might thus constitute the prototype of a new class of antiplatelet drugs.     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD₄, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE_2α and PGI₂ (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD₄, methacholine and histamine. LTD₄ (3–100 nM), methacoline (0.1–10 μM) or histamine (0.3–30 μM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE₂ and PGF_2α but not PGI₂, was decreased in tissues lacking epithelium. Indomethacin (1 μM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI₂ and is unlikely to be PGF_2α or PGE₂. However, the possibility remains that the basal release of PGE₂ and/or PGF_2α derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE₂ and PGF_2α which may be involved in the maintenance of baseline tone.     

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A₂ and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B₄ and 6-keto-prostaglandin F_1α, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.     

Expression of connexin43 gap functions between cultured vascular smooth muscle cells is dependent upon phenotype

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.     

Contractile responses to prostacyclin (PGI2) of isolated human saphenous and rat venous tissue

Prostacyclin (PGI₂), in a wide concentration range, produced neither contraction nor relaxation of isolated human saphenous vein. Isolated portal veins and vena cava from normal and spontaneously hypertensive rats (SHR) responded only with an increase in contractile tension when exposed to PGI₂. This constrictor effect was absent in a calcium-free buffer. PGI₂ failed to relax KCI contracted vena cava. The constrictor effect of PGI₂ on portal vein was attenuated in a glucose-free, oxygen deficient buffer. No tachyphylaxis or tolerance to the constrictor effect of PGI₂ was noted. Results emphasize that PGI₂ may produce differing effects on vascular smooth muscle tension depending on species and type of blood vessel studied.     

Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts.

The interaction of human platelets with one another and with the blood vessel wall is thought to be regulated in part by a balance between two arachidonic acid metabolites: thromboxane A₂, synthesized by platelets, and prostacyclin (PGI₂), synthesized by the vessel wall. We have studied the ability of cultured human vascular cells to synthesize PGI₂ from arachidonic acid. Four strains of human arterial smooth muscle cells synthesized a mean of 1.36 ng PGI₂ per 10⁵ cells, with a range of 0.2–5.3 ng PGI₂ per 10⁵ cells among the different strains. Human umbilical vein endothelial cells synthesized a mean of 7.16 ng PGI₂ per 10⁵ cells with a range of 2.3–14.0 ng per 10⁵ cells. In contrast, cultured human diploid skin fibroblasts synthesized only 0.27 ng PGI₂ per 10⁵ cells with a range of 0.05–0.6 ng per 10⁵ cells. When cultured cells were mixed with platelets, PGI₂ synthesis from added arachidonate was reduced rather than stimulated. Thus the major precursor cyclic endoperoxides utilized for PGI₂ synthesis are formed within the cells and not from endoperoxides synthesized by platelet cyclooxygenase. Aspirin has been proposed as an anti-thrombotic agent. Aspirin could be ineffective, however, if it inhibited not only platelet cyclooxygenase but that of vessel wall cells as well. Measurement of the rate constant or potency for aspirin inhibition of PGI₂ synthesis in cultured cells indicates that the cyclooxygenase in both cell types of the blood vessel wall is 14–44 fold less sensitive to aspirin inactivation than that in platelets, and appropriate levels of aspirin can selectively block human platelet thromboxane A₂ synthesis without compromising the capacity of the vasculature to produce PGI₂.     

Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells

Histamine stimulates the production of prostacyclin (PGI₂) in cultured human endothelial cells. We have examined the cell specificity of histamine-mediated PGI₂ synthesis in primary and subcultured human cells. Venous and arterial smooth muscle cells and skin fibroblasts synthesized PGI₂ from exogenous arachidonic acid, but they did not synthesize a significant amount of PGI₂ when treated with histamine. Endothelial cells, however, produced similar amounts of PGI₂ in response to histamine and arachidonic acid. Thrombin also stimulates PGI₂ production in endothelial cells. Histamine and thrombin yielded an additive production of PGI₂ when added simultaneously to endothelial cells. When histamine and thrombin were added sequentially, the amount of PGI₂ produced was not additive but equaled the amount characteristic of the first agonist alone. Following an initial treatment with histamine, endothelial cells were unable to respond to histamine for 3 hr, after which the PGI₂ biosynthetic response rapidly returned to normal by $\text{4}\text{1}\text{2}$ hr. When the initial histamine treatment was carried out under mildly alkaline conditions, the complete return of activity was delayed to 8 hr after treatment. The synthesis of PGI₂ from exogenous arachidonic acid was unaffected by prior treatment with histamine. Recovery of histamine-mediated PGI₂ production was not dependent on protein synthesis but required a component of fetal calf serum that is nondialyzable and moderately heat stable. Thus endothelial cell PGI₂ synthesis in response to a physiologic agonist is subject to several levels of regulation, reflecting not only intracellular events but also the extracellular environment.     

Cultured human skin fibroblasts and arterial cells produce a labile platelet-inhibitory prostaglandin

Human skin fibroblasts and cells cultured from human arterial smooth muscle produce a platelet-inhibitory prostaglandin in response to mechanical trauma. This prostaglandin is synthesized from an endogenous precursor rather than exogenous cyclic endoperoxides; it differs from PGE₁ and PGD₂ and resembles PGI₂ (prostacyclin) in its stability properties, being stable at pH ≥ 8.5 and labile at pH 7.4 and below. The prostaglandin synthesis pathway in these cultured cells is less sensitive to inhibition by aspirin than that in human platelets.     

The effects of thyroxine and methimazole treatment on the synthesis of prostacyclin (PGI2) in the rat

The effects of thyroxine (T4) and methimazole administration on plasma prostacyclin (PGI₂) levels $\text{in vivo}$ and on PGI₂ release by aortic rings incubated $\text{in vitro}$ were investigated in rats. Male rats were given single injection of T4 (200 μg/100 g body wt) ip every 24 h for either 3, 7 or 14 days for hyperthyroid rats. For hypothyroid rats, a group of rats were given methimazole (0.01 % in drinking water) for 14 days. PGI₂ concentrations were determined in plasma and also in the medium in which aortic rings were incubated. PGI₂ was measured as 6-keto-PGF1α by RIA. Plasma PGI₂ levels in T₄-treated groups were found to be significantly higher than those of control animals. Aortic rings obtained from rats given single injection of T₄ for 7 and 14 days showed significant increases in release of PGI₂ into the incubation medium. In contrast, rats given methimazole for 14 days showed a significant decrease in the production of PGI₂ by aortic rings without any significant changes in plasma levels. Direct addition of T₄ into the incubation medium did not cause any significant changes in PGI₂ release by aortic rings obtained from control rats.These results suggest the regulatory role of thyroid hormone in PGI₂ synthesis $\text{in vivo}$.     

Accelerated communication the direct contractile effect of gastrin releasing peptide on isolated gastric smooth muscle cells of the guinea pig

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether gastrin releasing peptide (GRP) can cause contraction by exerting a direct action on muscle cells. In addition, the inhibitory effect of 8-( N,N-diethylamino )-octyl-3,4,5-trimethoxybenzoate hydrochloride ( TMB-8 ), an inhibitor of intracellular Ca²⁺ release, and verapamil, a Ca²⁺ channel blocker, on the GRP-induced contraction of gastric smooth muscle cells were examined. GRP elicited a contractile response of gastric muscle cells in a dose-dependent manner. The ED₅₀ was 13 pM. TMB-8 significantly inhibited the contractile effect of GRP in gastric muscle cells. These results demonstrate the direct action of GRP on the gastric smooth muscle cells of the guinea pig, and the importance of Ca²⁺-release from intracellular calcium stores in the contractile response to GRP.     

Inhibitory effects of interleukin-6 on release of PGI2 by cultured human pulmonary artery smooth muscle cells

We evaluated the effect of interleukin-6 (IL-6) on the production of prostacyclin (PGI₂) by cultured human pulmonary artery smooth muscle cells (HPASMC). Incubation of these cells for up to 48 h with IL-6 led to a dose- and time-dependent decrease in the concentration of PGI₂ in the culture medium. The incubation of HPASMC with 10 μg/ml of lipopolysaccharide (LPS), 200 U/ml of IL-1β or 500 U/ml of TNFα for 24 hr significantly increased the concentration of PGI₂ in the medium. However, the addition of IL-6 to a medium containing LPS, IL-1β, or TNFα significantly inhibited the stimulatory effect of those substances on PGI₂ production. Such inhibition was closely related to the concentration of IL-6. IL-6 may counteract the roles of LPS and of other cytokines on the regulation of pulmonary vascular tension in endotoxin- and cytokine-mediated disorders such as sepsis and the acute respiratory distress syndrome (ARDS).     

Smooth muscle cells in aortic cultures of untreated and cholesterol-fed rabbits

Summary Smooth muscle cells were identified in aortic cultures from which the explants had not been removed. They appeared in cultures from intact aorta within 10 days and in cultures from aorta with plaques in 15 days. Myometrial smooth muscle cells grew within 2–4 days.Differences in growth rate of smooth muscle cells from various sources were explained as were differences in growth of aortic and subcutaneous fibroblasts. Morphologic characteristics of smooth muscle cells and fibroblasts were described in details. Peculiarities of migration of smooth muscle cells in vitro were discussed.This study was supported by grant HE-02534 from the National Heart Institute.     

Expression and distribution of InsP(3) receptor subtypes in proliferating vascular smooth muscle cells

The expression and distribution of types 1, 2, and 3 inositol 1,4, 5-trisphosphate receptor (InsP(3)R) in proliferating, primary cultures of rat aortic smooth muscle were compared to fully developed and differentiated rat aortic smooth muscle. Subtype-specific InsP(3)R antibodies revealed that the expression of type 1 InsP(3)R was similar in cultured aortic cells and aorta homogenate but expression of type 2 and 3 InsP(3)R subtypes was increased 3-fold in cultured aortic cells. The distribution of the type 1 InsP(3)R was located throughout the cytoplasm; type 2 InsP(3)R was found closely associated with the nucleus and at the plasma membrane; type 3 InsP(3)R was distributed predominantly around the nucleus. Alterations in InsP(3)R subtype expression and localization may have important functions in regulating intracellular calcium release around the nucleus when vascular smooth muscle cells switch to a more proliferating phenotype.     

The effect of radiation on prostacycliln (PG2) productnio by cultured endothelial cells

The effect of ionizing irradiation on the synthesis of prostacyclin (PGI₂) by cult8erd bovine aortic endothelial cells was detemined. PGI₂ was measuured in the culture medium by a radioimmunoassay for 6-Kto PGF_1α. Two phenomena were observed following irradiation: a) Cells which suffered an immediate radiation damage (1000–5000 rads) released high quantities of PGI₂ to the culture medium. This was due to a de novo synthesis of PGI₂ stimulated by radiation induced cellular damage, since pretreatment with aspirin of the endothelial cell monolayers resulted in a marked inhibition of PGI₂ release following irradiation. b) Metabolically active cells which remained confluent and firmly attaached to the culture dish following single, low and intermediate doses (200–1200 rads) radiation, exhibited a marked decreased in their capacity to synthesize PGI₂ upon exposure to various stimuli of the archidonic acid cascade (arachidonic acid, melittin, ionophore A23187 and PGH₂. Similar results were observed with cells treated with fractionated radiation.The quantities of PGI₂ produced by the endothelial cells decreased as a function of the dose of radiation and time interval between irradiation and subsequent stimulation. Radiation had a minimal effect on the nonthrombogenic properties of the endothelial cells, as evidenced by the small increase in the platelet adherence to the endothelial cells. The effect of radiation on PGI₂ pruction by the vascular endothelium may be relevant to the development of radiation induced capillary occlusions, and the enhancement of atherosclerotic lesions in large vesses.