Endothelin-induced changes in intracellular pH and Ca2+ in coronary smooth muscle: role of Na(+)-H+ exchange.

The relationship between endothelin-1 (ET-1)-induced stimulation of Na(+)-H+ exchange and intracellular free Ca2+ ([Ca2+]i) was examined in primary cultures of porcine coronary artery smooth muscle cells. Intracellular pH (pHi) and [Ca2+]i were measured using 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein and the acetoxymethyl ester of fura-2 respectively. In HCO3(-)-free buffer (pH = 7.4), ET-1 (0.1-50 nM) induced a sustained, dose-dependent increase in pHi. ET-1 (10 nM) increased pHi from 6.83 +/- 0.01 to 6.93 +/- 0.02 (P < 0.01). The alkalinization was blocked by the Na(+)-H+ exchange inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 3 microM) or by substitution of Na+ with N-methylglucamine or choline chloride (P < 0.05). Recovery of pHi in response to acidification, induced by washout of a 20 mM NH4Cl prepulse, was > 90% inhibited by EIPA (3 microM), confirming the presence of an ET-1-responsive Na(+)-H+ exchanger. Coronary smooth muscle cells responded to ET-1 with a dose-dependent, biphasic increase in [Ca2+]i which was not inhibited by manipulations (EIPA pretreatment or Na(+)-free media) shown to block the Na(+)-H+ exchanger. The ET-1-mediated alkalinization was not inhibited by removal of extracellular Ca2+ ([Ca2+]o). However, complete blockade of the ET-1-mediated [Ca2+]i response using the intracellular Ca(2+)-chelator, [bis-(2-amino-5-methylphenoxy)ethane-NNN'N'-tetraacetic acid tetraacetoxymethyl ester] (MAPTAM), in [Ca2+]o-free media, demonstrated that an increment in [Ca2+]i is required for activation of the Na(+)-H+ exchanger by ET-1. The ET-1-induced rise in [Ca2+]i was not associated with a rise in pHi in the presence of CO2/HCO3-. We conclude that: (1) activation of Na(+)-H+ exchange by ET-1 requires an increment in [Ca2+]i; and (2) ET-1 stimulates EIPA-sensitive Na(+)-H+ exchange, but this stimulation does not modulate ET-1-induced changes in [Ca2+]i.

Endothelin receptors and calcium signaling.

Endothelin (ET) is a potent vasoactive peptide produced by endothelial cells that elicits prolonged constriction in most smooth muscle preparations and dilation in others. Of three isopeptides, ET-1 is the only form constitutively released and may modulate vascular tone via binding to one of several receptor subtypes in smooth muscle. Activation of the ETA receptor is associated with pronounced vasoconstriction whereas ETB receptor occupation is linked to vasodilation. In addition, other subtypes of the ETB receptor exist, one mediating vasodilation (ETB1) and the other eliciting constriction (ETB2). An additional receptor subtype, ETC, has been identified although its physiological significance is uncertain. Distribution of these receptors varies between species and among tissue types, although it has been generally observed that ETA receptors predominate in arterial vessels whereas ETB receptors predominate on the low pressure side of the circulation. In vascular smooth muscle, an increase in intracellular Ca2+ is a common feature occurring after activation of all receptor subtypes. Upon binding of ET-1 to ETA, phospholipase C is activated and inositol triphosphate is generated. Ca2+ is then released from intracellular stores accompanied by the influx of extracellular Ca2+ and activation of the contractile machinery. The precise mechanism by which ET-1 affects intracellular Ca2+ regulation is not fully understood, but most likely involves multiple ion channels, protein kinases, and other intracellular mediators. The events coupled to non-ETA receptor signaling are poorly understood.

Alterations in human endothelial cell morphology, proliferation and function by a macrophage-derived factor.

Changes in endothelial cell (EC) morphology occur at sites of physiological lymphocyte traffic and in areas of chronic inflammation. Previous studies have shown that EC shape changes also occur in vitro following exposure of EC monolayers to peripheral blood mononuclear cell (PBMC)-derived conditioned media (CM). In the present study, quantitative image analysis is used to define the cell of origin of the elongating factor(s), to examine changes in EC proliferation and function accompanying PBMC-induced human EC elongation and to identify the active PBMC-derived products responsible for this elongation. By separating mononuclear cells into subpopulations (macrophages, B cells and T cells) and adding conditioned media derived from these subpopulations to cultured ECs, the macrophage (M phi) is shown to be the primary cell of origin of the elongating factor(s). Furthermore, EC elongation is accompanied by both a dose-dependent decrease in cellular proliferation and an increase in prostacyclin production. These findings suggest that PBMC-induced changes in EC morphology may be associated with a shift from a proliferative state to a more secretory phase of the EC cycle. Finally, using recombinant factors it is shown that TNF alpha acting in combination with IL-1 may be the active PBMC-derived products which contribute to EC elongation.

Postoperative venous thromboembolism and brain tumors: Part III. Biochemical profile.

The plasminogen activator activity of 31 human brain tumor samples was assessed and the results correlated with clinical parameters and the postoperative occurrence of venous thrombosis. A significant negative correlation was found between plasminogen activator activity and venous thrombosis (P = 0.02), while positive correlations were observed between plasminogen activator activity and peritumoral brain edema (P = 0.03) and alpha 2 antiplasmin measured in the systemic circulation (P = 0.01). Tissue plasminogen activator was found in higher concentrations in meningioma tissue but did not correlate significantly with the occurrence of venous thrombosis. The results of this study suggest a local enzymatic degradation effect of plasminogen activator on thrombogenic substances present in the brain tumor and/or its environment.

Nickel inhibits endothelin-induced contractions of vascular smooth muscle.

Endothelin (ET)-induced contractions of vascular smooth muscle (VSM) are dependent on extracellular Ca2+ yet display only partial sensitivity to L-type Ca2+ antagonists. The purpose of this study was to evaluate the effect of nickel (Ni2+), a Ca2+ channel antagonist with clearly documented differential potency toward L- vs. T-type Ca2+ currents on ET-mediated contractions in VSM. Treatment of rings of left anterior descending porcine coronary artery (LAD) with Ni2+ produced a profound dose-dependent inhibition of isometric force development in response to porcine ET (ET-1). At a concentration of 360 microM, Ni2+ exerted a significant inhibitory effect on contracture in response to doses of ET-1 ranging from 3 to 100 nM. In contrast, the same concentration of Ni2+ failed to significantly affect peak force development in response to KCl depolarization (5-77 mM) or to phenylephrine (0.3-30 mM). In addition, 360 microM Ni2+ significantly inhibited the contractile response of rat aorta to 10 nM ET-1. We conclude that ET-1 activates a Ni2(+)-sensitive process in VSM which may signal an additional Ca2+ influx pathway that appears to be functionally distinct from the L-type Ca2+ channel.

Effects of EDCF and endothelin on phosphatidylinositol hydrolysis and contraction in rat aorta.

Endothelium-derived constricting factor (EDCF) and endothelin are peptidergic substances produced and released from endothelial cells that induce contraction of vascular smooth muscle. The purpose of the present study was to investigate possible mechanisms by which EDCF and endothelin elicit contraction. Exposure of rat aorta to EDCF or synthetic endothelin resulted in time- and concentration-dependent increases in tension and levels of inositol monophosphate, a breakdown product of the phosphatidylinositides. A 10-s exposure to endothelin elevated levels of inositol 1,4,5-trisphosphate. Trypsinization or heating of EDCF prevented the contraction and inositol monophosphate formation. To assess whether EDCF and endothelin may act as endogenous agonists of the dihydropyridine-sensitive Ca2+ channel, we evaluated the ability of the dihydropyridine Ca2+ channel agonist (+)-S202-791 to increase the formation of the inositol phosphates. (+)-S202-791 increased inositol monophosphate formation. However, in contrast to that elicited by EDCF and endothelin, the increase in inositol monophosphate because of (+)-S202-791 was abolished by pretreatment with the cyclooxygenase inhibitor indomethacin (10 microM). These results suggest that contractions induced by EDCFs may be mediated through activation of phospholipase C and subsequent production of second messengers.

Endothelial cell-derived vasoconstrictors: mechanisms of action in vascular smooth muscle.

Intercellular signaling between the endothelial cell (EC) and vascular smooth muscle (VSM) is an important determinant of vasomotor tone. We evaluated mechanisms of action of EC-derived constrictors on VSM using conditioned medium from bovine aortic ECs in culture (EC-CM) or endothelin-1 (ET-1), and isolated coronary arteries or cultured VSM cells. EC-CM enhanced Ca2+ uptake into monolayers of rat aortic VSM and elicited sustained contractions in isolated coronary vessels in a time- and dose-dependent manner. The enhanced Ca2+ uptake and contractions were markedly attenuated by the Ca2+ channel antagonists bepridil, verapamil, and nitrendipine. EC-CM and ET-1 resulted in VSM membrane depolarization and increased excitability to electrical stimulation that was blocked by verapamil. ET-1 and EC-CM induced a dose-dependent increase in steady-state [Ca2+]i in Fura-2-loaded rat VSM cells. Most VSM responded with a rapid and transient increase in [Ca2+]i while others lacked only the transient phase. The elevated poststimulus [Ca2+]i level appeared to precede the influx of extracellular Ca2+ and contraction. EC-CM and ET-1 also resulted in time- and concentration-dependent increases in inositol monophosphate (IP) formation in rat aorta that paralleled the development of isometric force. We propose a biphasic mechanism in which the stable constrictors present in EC-CM elicit a rapid, phospholipase C-mediated mobilization of intracellular Ca2+ accompanied by or coupled to a sustained influx of extracellular Ca2+ through voltage-dependent channels.

Effect of calcium channel modulators on isolated endothelial cells.

Indirect evidence, using organic calcium channel modulators suggests that calcium channels exist in endothelial cells. Using freshly prepared and cultured bovine aortic endothelial cells, we have studied the effect of calcium channel modulators on Fura-2 fluorescence and have examined the binding of the dihydropyridine, (+)[3H]PN200-110. In both isolated primary and cultured cells, external calcium (0.5-2 mM) and bradykinin (10(-8) M) increased the intracellular calcium concentration. In cultured cells, the increase in calcium was not significantly attenuated by preincubation with nitrendipine (10(-8) M) or d-cis-diltiazem (10(-6) M). The calcium agonists (-)Bay k8644 and (+)202-791 had no effect on intracellular calcium concentration, but other agonists including ATP (10(-4) M) and thrombin (1.5 micrograms/ml) significantly increased the calcium concentration. Competition binding studies with (+)[3H]PN200-110 indicated specific binding of this ligand with a KD of 57 nM and a Bmax of 2.1 pmol/10(6) cells. While these data do not provide convincing evidence for the existence of calcium channels in cultured or fresh bovine aortic endothelial cells, explanations may yet reconcile our observations with the presence of calcium channels in these cells.

Plasma proteolytic activity following burns.

Because a number of metabolic events which are triggered by proteolysis in the bloodstream are activated following trauma, net proteolytic activity (P.A.) in the plasma of 37 pediatric burned patients was measured. The P.A. assay involved incubating plasma with a radioiodinated protein substrate and counting the isotopic activity of the hydrolyzed fragments in the acid-soluble fraction. In patients, plasma P.A. increased in direct proportion to the extent of burn injury. To examine additional trends in plasma P.A. suggested by the patient P.A. data, we measured plasma P.A. in a burned rat model: circulating P.A. was significantly elevated at 6 hours and until at least 2 weeks postburn; infection with Pseudomonas and use of the proteolytic debriding agent, Travase, each further elevated this activity; the plasma P.A. was not directly derived from the burn site. We postulate that this elevated circulating P.A. triggers some of the pathologic as well as some physiologic sequelae which follow burn trauma.

Quantitative studies of human monokine-induced endothelial cell elongation.

Leucocyte-endothelial cell interactions are important in the inflammatory response. In this study, the effect of peripheral blood mononuclear cell (PBMC) products on endothelial cell (EC) shape was examined and quantified. PBMC were obtained from normal donors by Ficoll-Hypaque separation of heparinized whole blood and cultured for 72 hr in media containing 10% fetal calf serum with and without concanavalin A (Con A). Media conditioned by PBMC or control, nonconditioned media were then added to preconfluent, first passage EC cultures derived from human umbilical veins. Conditioned media from Con A-stimulated PBMC resulted in a dose-dependent, marked elongation and whorling of cultured EC. The minimum effective concentration found to elicit a response was 1.25%, with a maximum response occurring at 10%. Quantitative morphometric analyses of treated EC indicated that the elongation was highly significant (p less than 0.001) when compared to EC incubated with control, nonconditioned media. In addition, EC elongation was accompanied by a highly significant (p less than 0.001) increase in cell area. Although less dramatic, conditioned media from unstimulated PBMC also elicited a similar, significant dose-dependent change in EC shape. Significant changes in EC shape were evident within 6 hr and continued over the time course of the experiment (40 h). Cell shape changes were partially reversible at 18 h after removal of the PBMC-conditioned media and replacement with control, nonconditioned media. The change in EC morphology induced by a PBMC-derived factor(s) suggests a mechanism by which activated leucocytes may modulate cellular traffic at the blood-vessel wall interface.

The effects of gonadotropin-releasing hormone and estradiol on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells.

This study investigated the effects of physiological concentrations of GnRH and estradiol (E2) on LH biosynthesis and release using cultured anterior pituitary cells. Pituitaries from female rats were enzymatically dispersed and cultured for 48 h in steroid-free alpha-Modified Eagle's Medium, followed by a 24-h culture in medium with or without E2. The cells were then incubated for a 4-h (Exp 1 and 2) or 8-h (Exp 3) period in medium containing radiolabeled precursors with or without GnRH. Radioactive precursor incorporation into LH was determined by immunoprecipitation, while immunoreactive LH (iLH) content was quantified by RIA. In the first experiment, all concentrations of E2 (10(-11)-10(-8) M) enhanced iLH release in response to 1 nM GnRH, confirming previous reports. GnRH increased [3H]glucosamine (3H-Gln) incorporation into LH, but had no effect on [35S]methionine (35S-Met) incorporation. The higher concentrations of E2 enhanced GnRH-stimulated 3H-Gln LH production. In the second experiment, the effects of GnRH (10(-9) M) and E2 (5 X 10(-10) M) on the incorporation of [3H]galactose, [3H]mannose, [3H]fucose, or [35S]sulfate into LH were investigated. Although all precursors were incorporated into LH, no specific effect of GnRH and/or E2 on incorporation of any of the precursors into LH was noted. In Exp 3, pituitary cells were cultured with or without 0.5 nM E2 followed by an 8-h incubation with varying physiological concentrations of GnRH (10(-11)-10(-9) M) and radiolabeled precursors (3H-Gln and 35S-Met). GnRH stimulated iLH release in a dose-dependent manner, and this response was enhanced by E2. GnRH also increased the incorporation of both 3H-Gln and 35S-Met into LH, but the dose of GnRH required for this response was dependent upon the estrogen environment. In the absence of E2, only 10(-9) M GnRH increased 3H-Gln LH and 35S-Met LH production, whereas in cells exposed to E2, all concentrations of GnRH (10(-11)-10(-9) M) increased 3H-Gln LH and 35S-Met LH production. In all experiments, the specific activity of radiolabeled LH released under basal conditions was greatly reduced by stimulation with GnRH. These results suggest that GnRH regulates both LH glycosylation and LH polypeptide synthesis and that E2 lowers the physiological concentration of GnRH necessary to stimulate this biosynthetic response. Moreover, estrogen's enhancement of GnRH-stimulated LH release appears to be due to its action on mechanisms regulating the release of previously synthesized stored hormone as well as the release of newly synthesized LH.

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells.

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.

An in vitro study of LH release, synthesis and heterogeneity in pituitaries from proestrous and short-term ovariectomized rats.

It is known that acute ovariectomy (OVX) greatly attenuates the pituitary luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) in vitro. The present study evaluated possible quantitative and/or qualitative differences in the biosynthesis and secretion of LH in pituitaries from proestrous and acutely (72 h) OVX rats. Paired anterior pituitary glands were incubated for 4 h in a medium containing +/- 10 nM GnRH. Pituitary and secreted LH were measured by radioimmunoassay with differences in total LH (tissue plus medium) +/- GnRH being indicative of GnRH-stimulated LH synthesis. Qualitative changes in LH were evaluated by isoelectrofocusing (IEF). The results show that the major form of LH stored in and released from the pituitaries consisted of LH molecules with an isoelectric point (pI) in the alkaline pH range (alkaline LH), and a lesser amount (approximately 30%) of LH molecules in the acidic pH range (acidic LH). The ratio of alkaline/acidic LH observed in the pituitary and medium was similar in the proestrous and OVX groups, although the amount of alkaline and acidic LH release in response to GnRH was 2-3 times greater in the proestrous group. In both groups, the alkaline/acidic LH ratio of secreted LH was higher in the presence of GnRH than in its absence. Alkaline LH synthesis was increased by GnRH in both groups, with the response being greater in the proestrous than in the OVX group; GnRH-stimulated acidic LH synthesis was observed only in the proestrous group. In both groups, the amount of LH synthesized was about 60% of the amount released, which suggests that LH synthesis does not fully account for differences in GnRH-stimulated LH release. Treatment of pituitary extracts with neuraminidase decreased acidic LH, and proportionately increased alkaline LH. These results suggest that the quality of LH stored in and secreted from pituitaries of proestrous and OVX rats is similar, and that there is a preferential release of the major alkaline LH isoform in response to GnRH. The ovarian steroid environment, presumably estradiol, proportionately increases the amount of alkaline and acidic LH released, and differentially affects the amounts of the various isoforms synthesized in response to GnRH. The charge heterogeneity of alkaline and acidic LH may be related to the sialic acid content of the LH molecule.

Characterization of a coronary vasoconstrictor produced by cultured endothelial cells.

The vasoactive effects of media obtained from bovine aortic endothelial cells (EC) in culture were directly tested on isolated rings of the porcine left anterior descending coronary artery. Increasing concentrations of EC-conditioned culture media resulted in progressive dose-dependent increments in isometric tension in porcine, bovine, and canine coronary arteries; the response did not require an intact endothelium. Control (nonconditioned) media and that conditioned by fibroblasts or vascular smooth muscle cells in culture had negligible effects on vessel tone. The vasoconstriction required extracellular Ca2+ and was unaffected by inhibitors of cyclooxygenase and lipoxygenase or by antagonists to the alpha- or beta-adrenergic, serotonergic, histaminergic, or cholinergic receptor systems. Calibrated gel filtration of the media indicated a molecular weight of 8,500 for the vasoactive factor; treatment of the EC-conditioned media with either sodium dodecyl sulfate, trypsin, alkali, or with acid hydrolysis completely abolished the vasoconstrictive effect. These findings and others now provide evidence for the existence of an EC-derived polypeptide vasoconstrictor that may be important in the regulation of vascular smooth muscle contractility.

Streptokinase-dependent delayed activation of horse plasminogen.

Complete activation of purified horse plasminogen to plasmin was obtained with a 1:10 molar ratio of streptokinase to plasminogen after 5 min of incubation at 37 degrees C. At a 1:1 molar ratio, maximal activity did not appear until 15-30 min, while at a ratio of 6:1 complete activation was delayed for 120-180 min. Gel filtration studies of isotopically labeled streptokinase and horse plasminogen suggest that the delay was due to impaired formation of a streptokinase-plasminogen complex. The predominant streptokinase moiety within the streptokinase-plasmin complex which forms from the streptokinase-plasminogen complex had a molecular weight of about 25000. The streptokinase-horse plasmin complex activated bovine plasminogen and was relatively stable. Native streptokinase was rapidly modified by horse plasmin predominantly to a fragment with a molecular weight comparable to that of the streptokinase moiety within the horse streptokinase-plasmin complex, about 25000 daltons. Partial characterization of horse plasminogen revealed no striking differences from human plasminogen in terms of molecular weight, N-terminal analysis and amino acid composition. However, horse plasminogen did not react with antibodies to human plasminogen, and its isoenzymes were more acidic than those of the human. Further characterization of horse plasminogen will be required to ascertain whether activation by streptokinase can serve as a model for the altered kinetics which have recently been described for the activation of aberrant types of human plasminogen.