Vasoconstrictive peptides induce endothelin-1 and prostanoids in human cerebromicrovascular endothelium.

Vasoconstrictive peptides and prostanoids have been implicated in the pathogenesis of hypertension and vasospasm. Recently, we have shown that human cerebromicrovascular endothelium [human brain endothelial cells (HBEC)] constitutively produces both endothelin-1 (ET-1) and prostanoids. The vasoactive peptides, arginine vasopressin (AVP) or angiotensin II (ANG II), stimulated secretion of both immunoreactive ET-1 and prostanoids from HBEC by a receptor-mediated induction of phospholipase C (PLC) and PLA2. The release of constitutive or AVP- or ANG II-induced ET-1 occurred at different rates during the 24-h incubation of HBEC in serum-free medium. The temporal profile of AVP-stimulated production of prostanoids differed from that of ANG II. AVP-induced release of prostaglandin D2 (PGD2) persisted for 24 h, whereas ANG II-stimulated PGD2 was only seen during the first 4 h of incubation. ANG II maximally stimulated PGI2 secretion during the 4- to 8-h interval, whereas AVP did not stimulate PGI2 secretion. Dexamethasone (Dxm), indomethacin (Indo), and nordihydroguaiaretic acid, the respective inhibitors of PLA2-cyclooxygenase II, cyclooxygenase, and lipoxygenase, increased both constitutive and AVP- or ANG II-stimulated secretion of ET-1. Dxm also decreased AVP- or ANG II-stimulated production of PGD2 and PGF2 alpha. These results indicate an interrelationship between HBEC production of ET-1 and prostanoids, which may play a role in regulating cerebral microcirculation.

Profile of prostaglandins induced by endothelin-1 in human brain capillary endothelium.

The vasoactive peptide, endothelin-1 (ET-1) has been implicated in the pathophysiology of various diseases. Recently, we have shown that human brain endothelial cells both secrete and express immunoreactive ET-1 high-affinity ETA receptors coupled to activation of phospholipase C (PLC). The present study demonstrates concentration-dependent stimulation of prostanoids [thromboxane B2 (TxB2), prostaglandin F2 alpha (PGF2 alpha), 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) prostaglandin E2 (PGE2), and prostaglandin D2 (PGD2)] production by ET-1 in capillary endothelial cells derived from human brain (HBCEC). The increase in the vasoconstrictive prostanoids TxA2 and PGF2 alpha temporally preceded that of the vasodilatory PGI2, PGE2 and PGD2, and was seen after 15 min of incubation with ET-1 (10 nM). Increased production of vasodilatory prostanoids was observed between 4-8 h of incubation, whereas normalization of both vasoconstrictive and vasodilatory prostaglandins occurred 24 h after addition of ET-1. Both ET-1-stimulated prostanoid and IP3 production were inhibited by BQ123, a specific antagonist of ETA receptors. ET-1-induced prostanoid secretion by HBCEC was also inhibited by dexamethasone (50 microM) and diminished by neomycin (50 microM) and verapamil (10 microM) but not by nifedipine. Phorbol myristate ester potentiated ET-1-stimulated prostanoid secretion, whereas it inhibited IP3 production. Data indicate that ET-1 activates phospholipase A2 (PLA2) and PLC in HBCEC by different intracellular mechanisms. The subsequently induced secretion of vasoactive prostanoids by HBCEC may contribute both qualitatively and temporally to the vasoactive actions of ET-1.

Adrenergic receptors coupled to adenylate cyclase in human cerebromicrovascular endothelium.

Cultured endothelium derived from three microvascular fractions of human brain was used to characterize adrenergic receptors coupled to adenylate cyclase activity. Catecholamines (norepinephrine, epinephrine) and their analogs (isoproterenol, phenylephrine, 6-fluoronorepinephrine) dose-dependently stimulated endothelial production of cAMP. Antagonists for beta 1 and beta 2 receptors (propranolol, atenolol, and butoxamine) and for alpha 1-receptors (prazosin) dose-dependently blocked cAMP formation induced by the tested adrenergic agonists. Clonidine, an alpha 2 > alpha 1-agonist, also inhibited isoproterenol-stimulated production of cAMP while yohimbine (alpha 2 > alpha 1 antagonist) augmented the norepinephrine or epinephrine-induced accumulation of cAMP. Cholera toxin-induced ADP ribosylation of the stimulatory guanine nucleotide binding protein (Gs) abolished the stimulatory effect of norepinephrine, epinephrine, phenylephrine or 6-fluoronorepinephrine on cAMP formation. ADP ribosylation of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin had no effect on either phenylephrine- or 6-fluoronorepinephrine-induced production of cAMP while it increased the norepinephrine and epinephrine-induced accumulation of cAMP. These findings represent the first documentation of beta 1-, beta 2-, alpha 1 and alpha 2-adrenergic receptors linked to adenylate cyclase in endothelium derived from human brain microvasculature. These data also indicate that activation of endothelial alpha 1 -adrenergic receptors is mediated by a signal transduction mechanism associated with Gs protein. The results strongly support the presence of various receptor-controlled adrenergic regulatory mechanisms on human cerebromicrovascular endothelium.

Secretion of immunoreactive endothelin-1 by capillary and microvascular endothelium of human brain.

Modulation of immunoreactive endothelin-1 (IR-ET-1) production by vasoactive substances was investigated in cultured endothelial cells (EC) derived from capillaries and microvessels of human brain. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester, and calcium ionophore enhanced the secretion of IR-ET-1. The known vasoconstrictive peptides, angiotensin II (Ang II) and arginine-vasopressin (AVP) dose-dependently stimulated the endothelial secretion of IR-ET-1. The angiotensin and vasopressin-inducible production of IR-ET-1 was completely inhibited by their respective receptor antagonists [Sar1, Ala8]-angiotensin II and [1-6 (beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-O-methyl-tyrosine]. The results indicate that the peptide-stimulated secretion of IR-ET-1 is receptor-mediated in EC which have specific angiotensin II and arginine-vasopressin receptors. These findings represent the first demonstration of IR-ET-1 production by capillary and microvascular endothelium of human brain.

Prostaglandin D2 in cultured capillary and microvascular endothelium of human brain.

Production of prostaglandin D2 (PGD2) was investigated in cultured endothelial cells derived from capillaries and microvessels (small and large) of human brain using radioimmunoassays. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester and calcium ionophore greatly stimulated the secretion of endothelial PGD2. Secretion of PGD2 induced by vasoconstricting peptides, angiotensin II and arginine-vasopressin, was almost completely abolished by their respective specific receptor antagonists [Sar1, Ala8]-Ang II and [1-6(beta-mercapto-beta,beta-cyclopentamethylene propionic acid) 2-O-methyltyrosine]. Thus, the augmented production of PGD2 by angiotensin II and arginine-vasopressin is a receptor-mediated event. It also indicates that the EC have specific angiotensin II and arginine-vasopressin (V1) receptors. This study represents the first demonstration of vasoactive agents modulating PGD2 production in capillary and microvascular endothelium of human brain.

Dopaminergic receptors linked to adenylate cyclase in human cerebromicrovascular endothelium.

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)

[Metabolic turnover of catecholamines in the heart of rats after nonfatal burns]. / Metabolicki obrt kateholamina u srcu pacova posle nesmrtne oparotine.

The content and metabolic turnover of noradrenaline (NA) and adrenaline (A) have been studied in the hearts of rats subjected to nonlethal scalding. The results of the study have shown that in an early phase of the general reaction of the organism to thermal trauma it has come to the decreased NA content while in the later phase the NA content was in the limits of control values. The increased contents of Na and A in the murine hearts after monoamineoxidase inhibition and injection of L-DOPA indicated the preservation of the synthesis and overtaking of catecholamines by the heart muscle. The increase of the metabolic turnover and shortening of the time of the metabolic turnover of NA and A have pointed that the increased NA content in the hearts of the traumatized animals is the consequence of the increased requirements of these catecholamines by the heart muscle in the early posttraumatic phase.

[Effects of non-lethal scalding on the density and affinity of beta adrenoreceptors in cardiac muscle in rats]. / Effets de l'échaudage non létal sur la densité et l'affinité des beta-adrénocepteurs dans le muscle cardiaque du rat.

Our results indicate changes in both Bmax and KD of the beta-adrenergic receptors in the ventricular heart muscle of the rat, submitted to non-lethal scalding, even in the early posttraumatic period. The changes of heart, plasma and urine noradrenaline and adrenaline contents indicate significant increase in sympatho-adrenal activity after non-lethal scalding. The parallel changes of the beta-adrenergic receptor characteristics, which depend on the changes of catecholamines in the heart muscle and plasma only at certain time intervals after trauma, tend to preserve normal-control values.