ICAM-1 expression on cardiac myocytes and aortic endothelial cells via their specific endothelin receptor subtype

Endothelin-1 (ET-1) and Endothelin-3 (ET-3) increased the expression of intercellular adhesion molecule-1 (ICAM-1) on rat neonatal cultured cardiac myocytes and rat aortic endothelial cells. ET-1-induced ICAM-1 expression on cardiac myocytes was inhibited by a selective ETA receptor antagonist, S-0139, but not by a selective ETB receptor antagonist, BQ788. ET-3-induced ICAM-1 expression on endothelial cells was inhibited by BQ788 but not by S-0139. Protein kinase C (PKC) inhibitor staurosporine inhibited ETs-induced ICAM-1 expression on both cell types. Treatment of the cells with ETs increased neutrophil adhesion, which was inhibited by S-0139 and staurosporine on cardiac myocytes and by BQ788 and staurosporine on endothelial cells. These results suggest that ETs induce neutrophil adhesion to cardiac myocytes and aortic endothelial cells by increasing ICAM-1 expression, which mediate via ETA receptor on cardiac myocytes and via ETB receptor on aortic endothelial cells. ICAM-1 expression induced by activation of ETA and ETB receptors appears to be mediated through the PKC pathway.     

Path integration in dogs

1. Desensitization of ET(A) endothelin receptor (ET(A)R) was compared between the rat and guinea-pig with regard to negative chronotropic response (NC) in the right atria (RA). 2. ET-1 (100 nM) produced distinct NC in the presence of BQ788 (300 nM), and positive chronotropic response (PC) in the presence of BQ123 (1 microM) in both species, showing that ETAR and ET(B) endothelin receptor (ET(B)R) mediate NC and PC, respectively. 3. Repetitive applications of ET-1 (50 nM) desensitized PC, and the second application only induced a strong NC in both species. Later applications of ET-1 produced virtually no response in the rat RA, whereas they produced BQ123-sensitive NCs repetitively in guinea-pig RA, exhibiting marked species difference in desensitization of ETAR-mediated NC. 4. Pretreatment with staurosporine (100 nM) prevented desensitization of ET(A)R in the rat RA altogether. However, phorbol 12-myristate 13-acetate (PMA, 300 nM) failed to induce, but rather hampered, desensitization of ET(A)R. 5. Partial amino acid sequencing of ET(A)Rs, spanning from the 2nd through the 4th intracellular loops, revealed that all the potential Ser/Thr phosphorylation sites, including a protein kinase C (PKC) site, are conserved among guinea-pigs, rats, rabbits, bovines and humans. 6. In guinea pig RA, pretreatment with okadaic acid (1 microg ml(-1)) and PMA did not facilitate desensitization of ET(A)R whereas these agents successfully desensitized ETAR during combined stimulation of beta-adrenoceptor and ET(A)R by isoproterenol (300 nM) and ET-1 (100 nM). 7. These results suggest that species differences in desensitization of ET(A)R are not caused by differences in the site(s) of, but caused by differences in the environment for phosphorylation of the receptor. Desensitization of ET(A)R appears to require phosphorylation of the receptor by PKC as well as a kinase stimulated by beta-adrenoceptor activation.     

Nociceptin (ORL-1) and mu-opioid receptors mediate mitogen-activated protein kinase activation in CHO cells through a Gi-coupled signaling pathway: evidence for distinct mechanisms of agonist-mediated desensitization

The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to Gi/Go proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of beta-adrenergic receptor kinase (betaARKct), which specifically blocks Gbetagamma-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21ras, also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing mu-opioid receptor, [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO; a mu-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, betaARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and mu-opioid receptors mediate MAP kinase activation via a signaling pathway using the betagamma-subunit of Gi, a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and mu-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate mu-opioid receptor signaling in a cellular system.     

The mechanism of myometrial contractions induced by endothelin-1 in rat

Experiments were performed to characterize endothelin-1-induced contractions and the role of endothelin (ET) receptor subtypes in rat myometrium. The binding sites of [(125)I]-ET-1 were saturable with high affinity. Scatchard plot analysis revealed that ET-1 binding sites in the myometrium constituted a single population. The dissociation equilibrium constant (Kd) and the maximum binding sites (Bmax) were determined to be 48.9+/-3.0 pM and 1364.0+/-210.3 fmol/mg protein respectively. Specific [(125)I]-ET-1 binding was inhibited completely by unlabelled ET-1 and Ro 46-2005 (mixed-type ET receptor antagonist), but not fully (90.7+/-1.4%) by BQ 123 (a selective ETA receptor antagonist), and not at all by RES 701-1 (a selective ETB receptor antagonist). ET-1 induced myometrial contractions were composed of two types, an increase in resting tone and rhythmic contractions. These contractions were inhibited by BQ 123 and Ro 46-2005, but not by RES 701-1. ET-1-induced contractions were greatly reduced in Ca2+-free Krebs' solution. Nifedipine abolished the rhythmic contractions without affecting the increase in resting tone. These results suggest that ETA receptors are predominantly localized in rat myometrium and that excitation of ETA receptors evokes two types of contractions by increasing the cytoplasmic Ca2+ concentration.     

Characterization of endothelin receptors in the human umbilical artery and vein

The aim of the present study was to characterize pharmacologically endothelin receptors that are present in human umbilical vessels. 2. Endothelin-1 (ET-1) and endothelin-2 (ET-2) are potent stimulants of both the human umbilical artery (pEC50 7.9 and 7.5) and vein (pEC50 8.1 and 8.0). Endothelin-3 (ET-3) is inactive on the artery but contracts the vein (pEC50 7.6). IRL1620 is inactive in both vessels. The order of potency of agonists is suggestive of a typical ET(A) receptor in the artery (ET-1 = ET-2 > > ET-3) and a mixture of ET(A) and ET(B) receptors in the vein (ET-1 = ET-2 > or = ET-3). 3. The selective ET(A) receptor antagonist, BQ123, competitively inhibits the effect of ET-1 in the human umbilical artery (pA2 6.9), while in the vein, only a mixture of BQ123 and BQ788 (a selective ET(B) antagonist) weakly displaces to the right of the cumulative concentration-response curve to ET-1. Contractions induced by ET-3 in the vein are inhibited by BQ788 (pA2 7.6), but not by BQ123. 4. Inhibition of Ca2+ channels by nifedipine (0.1 microM) is accompanied by a significant decrease of the maximal response to ET-1 by 40% in the artery and by 30% in the vein. The response of the vein to ET-3 is almost abolished by nifedipine. 5. The results indicate that: (i) endothelins contract the human isolated umbilical artery via stimulation of an ET(A) receptor type; (ii) the contraction induced by ET-1 in the vein is mediated by both ET(A) and ET(B) receptors, while ET-3 stimulates the ET(B) receptor; (iii) the contribution of Ca2+ channels to the contraction mediated by the ET(B) receptor appears to be more important than to that mediated by the ET(A) receptor.     

Endothelin ET(B) receptors counteract venoconstrictor effects of endothelin-1 in anesthetized rats

The inhibitory effects of BQ 788 (3 mg/kg, i.v., ET(B)-receptor antagonist) on endothelin-1 (ET-1)- or IRL 1620 (ET(B)-receptor agonist)-induced changes in mean arterial pressure (MAP), mean circulatory filling pressure (MCFP, driving force of venous return), arterial resistance (RA), venous resistance (RV) and cardiac output (CO) were characterized in 6 groups of pentobarbital-anesthetized rats. ET-1 or IRL 1620 (0.5, 1 and 2 nmol/kg, i.v.) dose-dependently increased MAP, RA, RV and MCFP and decreased CO. Maximum changes in RA, RV and CO elicited by ET-1 were greater than those by IRL 1620. Equimolar doses of ET-1 and IRL 1620 also caused similar initial transient decreases in MAP. BQ 788 alone slightly elevated MCFP, but did not alter other variables. The ET(B)-blocker abolished all changes elicited by IRL 1620, but only partially inhibited its responses on MCFP, showing the presence of BQ 788-insensitive receptors. BQ 788 also abolished ET-1's depressor response, partially inhibited its effect on MCFP, and markedly augmented its effects on RA, RV and CO. Thus, ET(B)-receptors counteract the sustained constrictor effects of ET-1 on arterial and venous resistance vessels Our results indicate a substantial arterial and venous dilator role for ET(B) receptors.     

Endothelin-1 stimulates deoxyribonucleic acid synthesis and contraction in testicular peritubular myoid cells

In the testis, endothelin-1 (ET-1) is produced by Sertoli cells, and it has been proposed to be a paracrine factor participating in the regulation of tubular and interstitial function. The response of purified testicular peritubular myoid cells (TPMC) to ET-1 was investigated in the present study. TPMC expressed a single class of high-affinity receptors that were shown by competitive binding experiments with sarafotoxin-6c to belong to the ETA subtype. The binding of ET-1 to TPMC was followed by rapid internalization of the receptor-ligand complex. ET-1 induced a prompt rise in intracellular Ca2+ concentration that was blunted in Ca(2+)-free medium and in the presence of Mn2+ or of voltage-operated-calcium-channel (VOC) blockers, indicating that both Ca2+ mobilization from intracellular stores and extracellular Ca2+ influx were involved. Thymidine uptake was promoted by ET-1 in a time-dependent manner, and the use of cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp] (BQ123) reduced the incorporation of thymidine. Protein kinase C (PKC) inhibition (100 nM calphostin C) abolished the ET-1 mitogenic effect. ET-1 also promoted TPMC contraction, as evaluated in collagen lattices, in a dose-related manner, with the half-maximal response observed at 1 nM. As in the case of mitogenesis, BQ123 blunted ET-1-induced contraction. PKC inhibition abolished ET-1-induced contraction. These findings indicate that ET-1 promotes DNA synthesis and contraction of TPMC and that both effects are mediated by PKC; they suggest as well that ET-1 may have a physiological role in the interaction between Sertoli cells and TPMC.     

The interaction of endothelin receptor responses in the isolated perfused rat lung

To gain more insight into the complex pulmonary interactions of endothelins (ET), we studied airway and vascular responses to endothelins in isolated perfused rat lungs in the presence of the novel ET(B)-receptor antagonist BQ788. In particular we focused on airway responses and on prostacyclin release. The effectiveness of BQ788 in our system was shown by its ability to concentration-dependently prevent vasoconstriction (IC50 0.1 microM), bronchoconstriction (IC50 0.1 microM) and prostacyclin production (IC50 < 0.1 microM) induced by the ET(B)-receptor agonist IRL1620 (1 nmol). Airway responses to ET-1: ET-1-induced bronchoconstriction was aggravated by BQ123 (1 or 8 microM), while BQ788 pretreatment (1 or 8 microM) showed no significant effect. Simultaneous treatment with 8 microM BQ123 and BQ788 attenuated the ET-1-induced bronchoconstriction. Vascular responses to ET-1: ET-1 (1 nmol)-induced vasoconstriction was potentiated by BQ788 (1 or 8 microM), but attenuated by the ET(A)-receptor antagonist BQ123 (1 microM). In the presence of BQ788 diminished amounts of the stable prostacyclin metabolite 6-keto-PGF1alpha were detected in the perfusate. Simultaneous treatment with 8 microM BQ123 and BQ788 completely prevented the ET-1-induced vasoconstriction. Conclusions: Both ET(A)- and ET(B)-receptors contribute to ET-1-induced vasoconstriction and bronchoconstriction. The ET-1-induced vasoconstriction is attenuated by stimulation of ET(B)-receptors, a response that is partly mediated by prostacyclin. Due to the mutual interactions between ET(A)- and ET(B)-receptors, simultaneous inhibition of both receptors is required to prevent the deleterious effects of ET-1 on lung functions.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium

The effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.     

EndothelinB receptors in rabbit pulmonary resistance arteries: effect of left ventricular dysfunction

The endothelin (ET) receptor that mediates vasoconstriction of the isolated rabbit pulmonary resistance artery was characterized using selective ET receptor agonists and antagonists. We also examined changes in ET-induced vasoconstriction brought about by left ventricular dysfunction using the rabbit coronary ligation model. The rank order of potency for contraction was sarafotoxin S6c (S6c) > ET-1 = ET-3, which is characteristic of an ETB-like receptor. The combined ETA/ETB receptor antagonist SB209670 (1 microM) antagonized responses to ET-1 and S6c with estimated pKb values of 6.8 +/- 0.2 and 7.8 +/- 0.2, respectively. BQ788 (1 microM) antagonized responses to S6c and ET-3 (but not ET-1) with estimated pKb values of 7.1 +/- 0.2 and 6.6 +/- 0.1, respectively. The ETA receptor antagonist FR139317 (1 microM), either alone or in combination with BQ788, did not inhibit responses to ET-1. The profile of the ET-1 response was not altered by left ventricular dysfunction. In control rabbits, the inhibitor of nitric oxide synthase N omega-nitro-L-arginine methyl ester (100 microM) had no significant effect on the potency of either ET-1 or S6c. In the coronary-ligated rabbits, however, it significantly increased the potency (10-15-fold) of both ET-1 and S6c. These results suggest that the ET receptor that mediates contraction in rabbit pulmonary resistance arteries has the characteristics of an ETB-like receptor. The responses to ET-1 are not altered by LVD but may be modified by increased release of nitric oxide.     

Health of older women

We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC50=8 nM) and time-dependent (t1/2=1.2 min) manner. Cytosolic phospholipase A2 (cPLA2), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF3, quinacrine and manoalide, PLA2 inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCalpha and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. G?-6976, a compound that inhibits PKCalpha and beta specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 microM), a specific activator of PKCalpha, beta, and gamma induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCalpha, but not PKCbeta, from cytosol to the particulate fraction. These results suggest that PKCalpha plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA2, p42mapk and p44mapk in the CISM cells. The data presented are consistent with a role for PKCalpha, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA2 phosphorylation and cPLA2 activity, but had no inhibitory effect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhibitor, inhibited ET-1-stimulated MAPK activity but had no inhibitory effect on AA release in the ET-1-stimulated cells. We conclude that in CISM cells, ET-1 activates PKCalpha, which activates cPLA2, which liberates AA for prostaglandin synthesis.     

Evidence that angiotensin II, endothelins and nitric oxide regulate mitogen-activated protein kinase activity in rat aorta

We measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aortic strips with or without endothelium, and examined effects of angiotensin receptor antagonists, endothelin receptor antagonists and nitric oxide (NO)-related agents. Endothelium removal produced an activation of MAP kinase activity in the strips, whereas the enzyme activity was not affected in the adventitia. The MAP kinase activation was inhibited by either the angiotensin AT1 receptor antagonist losartan or the endothelin ETA receptor antagonist BQ 123. The combination of both antagonists caused an additive inhibition. The angiotensin AT2 receptor antagonist PD 123,319 and the endothelin ETB receptor antagonist BQ 788 did not affect the MAP kinase activation. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) caused an activation of MAP kinase in the endothelium-intact aorta and the MAP kinase activation was inhibited by losartan or BQ123. The NO releaser nitroprusside inhibited the MAP kinase activation induced by endothelium removal or angiotensin II. These results suggest that even in isolated arteries, NO of endothelial origin tonically exert MAP kinase-inhibiting effects and endogenous angiotensin II and endothelins in the media are tonically released to cause MAP kinase-stimulating effects in medial smooth muscle.     

Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells.     

Cross-talk between receptor-mediated phospholipase C-beta and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes

Phospholipase C-beta (PLC-beta) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as alpha1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) induce hypertrophy of myocytes. The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PKC activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10(-8)M), phenylephrine (PHE, 10(-5)M) or Ang II (10(-7)M) resulted in a rapid (0-10 min) activation of PLC-beta to an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10(-6)M) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0-5 min) translocation of PKC- immunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-alpha, -delta and -xi immunoreactivities was observed. PMA caused translocation of PKC-alpha, PKC-epsilon as well as PKC-delta. Cellular redistribution of PKC activity measured by [32P]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation. Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10(-4)M) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10(-6)M) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-beta and after a lag-phase PLD, whereas PHE and Ang II activate only PLC-beta. PLC-beta stimulated by ET-1, may cross-talk with PLD via translocation of PKC-epsilon. These signals are possibly linked to the hypertrophic response.     

Endothelin-1 regulates tone of isolated small arteries in the rat: effect of hyperendothelinemia

Chronic elevation of plasma endothelin-1 (ET-1) levels has been reported in several pathological conditions. To investigate the consequences of increased circulating ET-1 on vascular responsiveness, Sprague-Dawley rats (n=16) were chronically instrumented with a minipump delivering ET-1 at a constant dose for 7 days. Plasma ET-1 levels were more than doubled in treated (0.98+/-0.09 pmol/L; P<.05) versus untreated sham-operated rats (0.43+/-0.04 pmol/L), whereas systolic arterial blood pressure increased (139+/-5 versus 128+/-4 mm Hg in untreated rats; P<.05). After rats were killed, segments of middle cerebral (MCA) and mesenteric (MES) arteries were mounted on an isometric myograph. ET-induced contraction was shifted to the right in ET-1-treated animals and not modified by BQ123 (an ET(A) receptor antagonist); bosentan (ET(A/B) receptor antagonist) prevented ET-1-induced contraction in both groups. After inhibition of nitric oxide synthase with N(omega)-nitro-L-arginine (L-NNA), both phenylephrine and oxymetazoline (an alpha2-adrenoceptor agonist) induced MCA contraction. The sensitivity to phenylephrine was decreased in ET-1-treated compared with control rats (P<.05). Sensitivity to phenylephrine-induced contraction was decreased by BQ123 in control rats only. In contrast, L-NNA revealed greater oxymetazoline-induced contractions in treated compared with control MCA rings (P<.05); this potentiation was blunted by bosentan but unaffected by BQ123. Removal of the endothelium revealed a direct constrictor effect of oxymetazoline that was insensitive to L-NNA alone or combined with bosentan; however, oxymetazoline induced significantly lower constriction in treated rat MCA segments. Responses to oxymetazoline were also blunted in treated compared with untreated denuded MES arteries. In conclusion, chronic elevated plasmatic ET-1 decreases smooth muscle cell sensitivity to contractile agonists both in MCA and MES rings. In cerebral vessels, endothelial alpha2-adrenoceptor-dependent stimulation induced greater contractile responses in treated rats which were sensitive to bosentan, suggesting that oxymetazoline stimulates ET-1 release from the endothelium. This may represent a compensatory mechanism for the loss of smooth muscle sensitivity.     

Teratocarcinosarcoma of the paranasal sinuses: a clinicopathologic and immunohistochemical study

We observed endothelin (ET)-induced contractile responses on prostatic and epididymal segments, as well as the facilitation of an electrically stimulated tone on prostatic segments of isolated rat vas deferens. In both segments, the selective ET(B)-receptor agonists, IRL 1620 and sarafotoxin S6c, produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of contraction potencies (pD2 value) was ET-1 = ET-2 > ET-3 > sarafotoxin S6c = IRL 1620. The maximum responses of ET-induced contractions in the prostatic segments were larger than those in the epididymal segments. The contractile response to ET-3 was antagonized by pretreatment for 30 min with BQ-123 (10 nM), a selective ET(A) receptor antagonist, and BQ-788 (1 microM), a selective ET(B) receptor antagonist. The contractile responses to ET-1 were antagonized by pretreatment with BQ-123 (10 microM), but not with BQ-788 (1 microM). The ET-3-induced facilitation on the twitch response to electrical stimulation in the prostatic segment of the vas deferens was antagonized by BQ-123 (0.1 microM) and BQ-788 (1 microM). The ET-1-induced facilitation was antagonized by pretreatment with BQ-123 (3 microM), but not with BQ-788 (10 microM). These results suggest that in rat vas deferens the ET(A) receptors are divided into BQ-123-sensitive ET(A1) and BQ-123-insensitive ET(A2) subtypes, and the production of a contractile response of smooth muscle as well as the facilitation of neurotransmission are accomplished through mediation by ET(A1)- and ET(A2)-subtypes.     

Endothelin-1 as a putative modulator of gene expression and cellular physiology in cultured human corporal smooth muscle cells

PURPOSE: Increases in cytosolic calcium levels trigger smooth muscle contraction while nuclear calcium increases are thought to regulate gene expression. Endothelin-1 (ET-1) affects both. The goal of these studies was to further investigate the importance of ET-1 to corporal physiology by examining its actions on proliferation and immediate early gene (IEG) expression in cultured human corporal smooth muscle cells. MATERIALS & METHODS: Early passage (1-3) smooth muscle cells were grown in culture and exposed to either phenylephrine (PE) or ET-1 in the absence and presence of serum, the ET(A) or ET(B) selective antagonist BQ123 or IRL1038, or the L-type Ca2+ channel blocker, verapamil. Cell proliferation was assessed with a hemocytometer. The effects of ET-1 on c-myc and c-fos were evaluated using Northern blot analysis. Parametric or nonparametric statistics were used as appropriate. RESULTS: Addition of ET-1 (100 nM) to serum-starved cultured corporal smooth muscle cells was associated with a nearly 2-fold increase in cell number, as well as 2 to 6-fold increases in c-myc and c-fos levels. Cellular proliferation was inhibited by ET(A)- or ET(B)-receptor subtype blockade with BQ123 (1 microM) or IRL1038 (1 microM), respectively, or blockade of Ca2+ channels with verapamil (10 microM). PE (3 microM) had no detectable effect on smooth muscle proliferation. CONCLUSIONS: Cell proliferation was mediated by activation of the ET(A) and ET(B) receptor subtypes, dependent on transmembrane Ca2+ flux, and correlated with significant increases in c-myc and c-fos mRNA levels. These studies extend previous observations to indicate the potential pleotropic actions of this peptide in the regulation of human corporal smooth muscle physiology in vivo.     

GTP-binding protein regulates the contractile response elicited by the phorbol ester (PMA)-induced activation of protein kinase C in the isolated rat aorta

1. The aim of the present study was to investigate the involvement of GTP-binding protein in the contractile response induced by activation of protein kinase C (PKC) in isolated rat aorta. The rats were treated with islet-activating protein (IAP) for 4 days prior to the experiments. 2. In the aorta from control rats, phorbol 12-myristate 13-acetate (PMA) produced biphasic contractions; twitch contraction superimposed on the slowly developing contraction. The twitch contraction was abolished by the removal of external Ca2+ or by treatment with nicardipine. In the aorta pretreated with IAP, PMA produced only a slowly developing contraction, and no twitch contraction was induced. 3. The application of Ca2+ to aortic strips in a Ca(2+)-free solution, that had been treated with 10(-6) M PMA caused concentration-dependent contraction, and the contraction was completely inhibited by IAP. 4. Pretreatment with IAP inhibited Ca(2+)-induced contraction of the aorta in Ca(2+)-free medium in the presence of 10(-6) M clonidine, but did not affect the Ca(2+)-induced contraction in the medium treated with 10(-6) M phenylephrine and 10(-7) M nicardipine. 5. These results suggest that the activation of PKC by PMA produces biphasic contractions in the rat aorta. The twitch contraction may be induced by the activation of voltage-dependent Ca(2+)-channels and the activation may be regulated by IAP-sensitive GTP-binding protein.