Eicosapentaenoic acid ablates valproate-induced liver oxidative stress and cellular derangement without altering its clearance rate: dynamic synergy and therapeutic utility.

The omega-3 fatty acid eicosapentaenoic acid (EPA) is a superb nature's medicine, with still unfolding health benefits. Because hepatotoxicity is a prominent adverse drug reaction, we currently attempted a new approach in which EPA was challenged to both alleviate hepatotoxicity and provide synergy with anticonvulsant effects of valproate (VPA). Besides, we verified whether EPA may kinetically modulate the clearance rate of VPA. VPA (500mg/kg p.o., for 2weeks) caused rat hepatotoxicity that was manifested as notable (2- to 4-fold) rise in serum liver enzymes (GGT, ALT, and ALP), increased hepatic levels of lipid peroxides and TNF-α (3- and 7-fold) and activity of myeloperoxidase (MPO, 4-fold), lowering of serum albumin (42%), and depletion of liver reduced glutathione (GSH, 36%). Furthermore, histopathologic examination revealed hepatocellular degeneration, focal pericentral necrosis, infiltration of inflammatory cells, and steatosis. Joint treatment with EPA (300mg/kg) blunted the oxidative stress, TNF-α levels and MPO activity, while enhanced levels of serum albumin and hepatic GSH. EPA also ameliorated most of the hepatocellular anomalies evoked by VPA. Additionally, in a mouse PTZ convulsion model, EPA markedly augmented the anticonvulsant effects of VPA far beyond their single responses. On the other hand, pharmacokinetic analyses revealed that joint EPA administration had no effect on serum VPA concentrations. Collectively, results demonstrate for the first time that the ω-3 FA (EPA) markedly alleviated VPA-induced hepatotoxicity, oxidative stress, and inflammation, while enhanced its anticonvulsant effects without altering its clearance. Therapeutically, these protective and synergy profiles for EPA foster a more safe and efficacious drug combination regimen than VPA.

Evaluation of renal protective effects of the green-tea (EGCG) and red grape resveratrol: role of oxidative stress and inflammatory cytokines.

Epigallocatechin-gallate (EGCG) and resveratrol (RSVL) are two of the most promising natural medicines. We verified their capacity to ameliorate cisplatin (CP)-induced disruption of renal glomerular filtration rate (GFR) in rats, and sought the mediatory involvement of lipid peroxidation (malondialdehyde [MDA]-level) and inflammatory cytokine (TNF-α) therein. CP (10 mg kg⁻¹), a single i.p. dose, disrupted GFR (11-fold-rise in proteinuria, 2-5-fold rise in serum creatinine/urea levels) after 7 days, and killed all animals after 10 days. Kidney-homogenates from CP-treated rats displayed higher MDA and TNF-α, but lower reduced-glutathione (GSH) levels. Rats treated with EGCG (50 mg kg⁻¹, but not 25 mg kg⁻¹) had no fatalities and showed significantly-recovered GFR; while their kidney-homogenates had markedly reduced MDA, TNF-α and enhanced GSH levels at 7 days. Conversely, RSVL or quercetin (25, 50 mg kg⁻¹) neither improved GFR nor reduced (MDA)/TNF-α levels after 7 days. Resuming treatment with 50 mg kg⁻¹ for 10 days rescued only 25% of animals (p > 0.05). Correlation studies showed a significant association between creatinine level, and each of MDA (r = 0.91), GSH (r = -0.87), and TNF-α (0.91). The study showed for the first time that EGCG, unlike RSVL, can protect against CP-induced nephrotoxicity. At the molecular level, CP triggers a high level of oxidative stress and systemic inflammation, events that were all abrogated with EGCG; better than RSVL or quercetin.

Novel chemotherapeutic and renal protective effects for the green tea (EGCG): role of oxidative stress and inflammatory-cytokine signaling.

BACKGROUND: The green tea catechin, epigallocatechin-gallate (EGCG) is a superb nature's medicine candidate. We evaluated the chemotherapeutic/chemoenhancing effects of EGCG in mice bearing the solid Ehrlich ascites carcinoma (EAC) tumor, and jointly monitored levels of serum C-reactive protein (CRP), lipid peroxidation (as malondialdehyde: MDA) and leukocytosis (LC). Besides, we verified whether; and how then, EGCG would protect against a devastating CP-induced nephrotoxicity in rats. In particular, renal proinflammatory (TNF-α) and oxidant stress signals have been investigated. RESULTS: (EAC)-bearing mice displayed elevated serum-LC (2-fold), -CRP (11-fold) and -MDA levels (2.7-fold). EGCG (20, 40 mg/kg) significantly shrank tumors (by 48% and 92%, respectively), and reduced LC, CRP and MDA levels. Such responses for CP were less prominent than those of EGCG (40 mg/kg). Further, EGCG (20 mg/kg) markedly augmented such functional and biochemical responses to CP. Correlation studies showed positive association between tumor size and each of CRP (r=0.97) and LC (r=0.83). Additionally; in rats, CP (10 mg/kg) caused a prominent nephrotoxicity that was manifested as deteriorated glomerular filtration rate (GFR, 2-5-fold rise in serum creatinine/urea levels) after 4 days, and unanimous animal fatalities after 7 days. Kidney homogenates from CP-treated rats showed significantly higher MDA- and TNF-α-, and -depleted GSH levels. Rats treated with EGCG (50 mg/kg, but not 25 mg/kg) devoid the nephrotoxic effects of CP and their consequences; while their homogenates had appreciably lower MDA and TNF-α, and higher GSH levels. Notable correlation was detected between serum creatinine level and each of MDA (r=0.85), TNF-α (r=0.85) and GSH (r=-0.81). CONCLUSION: This study shows remarkable cytotoxic/chemoenhancing effects for EGCG and introduces CRP as a predictor of both tumor's progression and responsiveness to chemotherapy. Further, this study is the first to reveal that EGCG can obliterate the lethal CP-induced nephrotoxicity. Mechanistically, EGCG acts by suppressing leukocytosis, systemic inflammation, oxidative stress, and their sequelae.

Prominent chemopreventive and chemoenhancing effects for resveratrol: unraveling molecular targets and the role of C-reactive protein.

BACKGROUND: Resveratrol (RSVL) claims health benefits that pertain to the consumption of red wine/grapes. We currently evaluated the chemopreventive effects of RSVL, as well as its possible chemoenhancing effects when given with cisplatin (CP), in the Ehrlich ascites carcinoma (EAC) solid tumor model. Further, we monitored concomitant changes in serum levels of C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), leukocytic count (LC) and lipid peroxidation (measured as malondialdehyde, MDA). RESULTS: EAC-bearing mice exhibited a markedly elevated LC (2 fold), CRP (11 fold) and MDA levels (2.7 fold). RSVL (20 or 40 mg/kg) elicited significant, dose-dependent reductions in tumor size (58 and 78%, respectively), as well as in LC (normalized), CRP (down to 2 fold), TNF-alpha (down to near control levels) and MDA levels (normalized). The chemopreventive effects for CP (55% reduction in cell growth) was significantly lower than that of RSVL (40 mg/kg, 79% inhibition). Interestingly, coadministration of RSVL (20 mg/kg) markedly enhanced the chemoprevention of CP. Correlation studies revealed a high degree of positive association between tumor growth and CRP (r = 0.89) and leukocytosis (r = 0.86), thus attesting to a diagnostic/prognostic role for CRP in this solid tumor. CONCLUSION: RSVL elicited remarkable cytotoxicity on its own and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels. RSVL acts prominently by reducing inflammatory cytokines, leukocytosis and oxidative stress.

Resveratrol reverses ET-1-evoked mitogenic effects in human coronary arterial cells by activating the kinase-G to inhibit ERK-enzymes.

In human coronary smooth muscle cells (HCSMC), treatment with the vascular mitogen; endothelin-1 (ET-1), induced cell proliferation and stimulated ERK-1/2 phosphorylation at active sites. Pretreatment with the MEK-ERK inhibitor (PD98059) appreciably reversed the mitogenic effects of ET-1. On the other hand, pretreatment with the polyphenolic stilbene resveratrol (RSVL, 1-100 microM) triggered more prominent inhibition of ET-1-evoked cell proliferation and ERK1/2 activation. Besides, RSVL also markedly (2-3 fold) and rapidly enhanced cGMP formation, but had no effect on cAMP levels. This RSVL-evoked upregulation of cGMP was insensitive to pretreatment with the soluble guanylyl cyclase (sGC)-inhibitor (ODQ, 10 microM), but was ablated with an inhibitor of pGC (PMA, 0.1 microM). Further, pretreatment with the specific cGMP-phosphodiesterase inhibitor, zaprinast (10 microM) appreciably augmented RSVL-evoked cGMP formation, ERK inhibition, and cytostatic response. Moreover, the RSVL-induced ERK-inhibitory effects were significantly reversed by the kinase-G inhibitor, KT-5823 (10 microM; 69%), but not by the kinase-A inhibitor (KT-5720). These results demonstrate a novel signaling pathway for RSVL that leads from activation of the pGC/kinase-G system to inhibition of ERK1/2 and their downstream nuclear targets. This pathway functions to counteract the atherogenic signaling induced by vascular mitogens.

Nongenomic activation of the GC-A enzyme by resveratrol and estradiol downstream from membrane estrogen receptors in human coronary arterial cells.

BACKGROUND AND AIM: Resveratrol (RSVL), a polyphenolic phytoestrogen in grapes, confers multifaceted cardiovascular benefits. The cellular and molecular basis of RSVL actions has been largely undefined until now. METHODS AND RESULTS: In human coronary smooth muscle cells (HCSMCs), RSVL markedly (3.2-fold) enhanced cGMP formation (t(1/2): 6.3 min, EC(50): 1.8 microM) and stimulated kinase-G activity (4-fold). By contrast, RSVL had no effect on cAMP or PKA activity in these cells. The RSVL-enhanced cGMP/kinase-G activity was not abrogated by the nitric oxide synthase-inhibitor (L-NMMA, 10 microM), or the soluble guanylyl cyclase (sGC)-inhibitor (ODQ, 10 microM). In membrane preparations from HCSMCs, RSVL activated GC in the particulate-, but not in the soluble-membrane fraction. Similar effects were due to the specific particulate-GC-A agonist atrial natriuretic peptide (ANP, 0.1-1 microM). The combined effects of RSVL and ANP were competitive. By contrast, the selective GC-B agonist (BNP) showed no response on cGMP, whereas that for GC-C (guanylin) produced only slight increases in cGMP levels. Estradiol (E2) mimicked the effects of RSVL on cGMP, but showed a 46% lower maximal response. Combining E2 with RSVL showed a competitive, rather than an additive, response. Further, cGMP formation by RSVL or E2 was significantly attenuated by the pure estrogen receptor blocker, ICI-182,780 (10 microM). CONCLUSION: These findings are the first to link RSVL with pGC/kinase-G activation downstream from membrane ERs in the vasculature, thus substantiating its coronary protective effects, even in endothelium-disrupted coronary arteries.

Overexpression of wild-type p53 gene renders MCF-7 breast cancer cells more sensitive to the antiproliferative effect of progesterone.

We have recently shown that growth inhibition of breast cancer cells by progesterone is due to the induction of cell differentiation, but not apoptosis. Because the tumor suppressor protein p53 plays a central role in normal cell growth and in tumor suppression, we have examined the effect of progesterone on the levels of this protein in MCF-7 cells. We show here that the antiproliferative effect of progesterone is accompanied with down-regulation of endogenous p53 protein. To study the effect of progesterone on cell growth in the presence of normal levels of p53 protein, we used transient transfection to overexpress p53 protein. MCF-7 cells were transfected with a p53 expressing vector that contains p53 human cDNA under the control of a cytomegalovirus promoter. Cell growth, cell viability, and apoptosis were analyzed in the transfected cells after six days of exposure to 100 nM progesterone. We show here that progesterone significantly enhances growth inhibition and apoptosis in MCF-7 cells overexpressing p53, but not in cells transfected with the control vector. These data suggest that re-establishing p53 function in MCF-7 breast cancer cells renders them more sensitive to the growth inhibitory effect of progesterone.

Synthesis, spectroscopic characterization, stability assessment and DNA-binding of new 2,6-piperidinedione derivatives.

This work reports on structural characterization of new antineoplaston (ANP) representatives, namely 3-(benzoylamino)-2,6-piperidinedione (BPD), 3-(4-methoxybenzoylamino)-2,6-piperidinedione (MPD) and 3-(p-nitrobenzoylamino)-2,6-piperidinedione (NPD). These compounds were prepared by reacting N-(4-substituted benzoyl)-glutamines with N-hydroxysuccinimide to afford the corresponding esters, which were heated to produce the corresponding 2,6-piperidinedione (PD) compounds. Non-destructive analytical procedures such as 1H NMR and NIR analyses confirmed the postulated chemical structures of these PD compounds. HPLC chromatograms at an ambient temperature or from solutions preheated at 30, 40 or 60 degrees C displayed only a single peak for each compound. Combination of heat with pH modification had virtually no effect on the obtained peaks, thus attesting to the stability and purity of these compounds. MS analysis displayed molecular mass ions indicative of BPD, MPD and NPD at m/z 233.4, 263.2 and 278.3, respectively. The fragmentation patterns using MS/MS analyses conformed to the structural and molecular formulae of the prepared compounds. Furthermore, preliminary biological assessments showed the capacity of these compounds to bind to the DNA. NPD, but not BMP or MPD, had a superior affinity to the DNA than the prototype ANP-A10.

ET(B) receptor activates adenylyl cyclase via a c-PLA(2)-dependent mechanism: a novel counterregulatory mechanism of ET-induced contraction in airway smooth muscle.

Endothelin-1 (ET-1) contracted the rabbit tracheal smooth muscle (RTSM), yielding a bell-shaped tension-concentration curve. Moreover, ET-1 induced concentration- and time-dependent increases in cAMP concentrations in RTSM (EC(50), 58 nM; t(1/2), 2.4 min). Pretreatment with the AC inhibitors, SQ-22536, or 2'-5'-dideoxyadenosine, enhanced contraction to ET-1 and converted its bell-shaped tension curve into a sigmoidal one, but left contraction to carbachol and KCl unaltered. The potent ET(B)-receptor agonists, ET-3 or sarafotoxin-c, mimicked ET-1's effects on cAMP levels (EC(50) values 55 and 50 nM). Further, cAMP formation by ETs was inhibited by BQ-788 (selective ET(B) receptor blocker; IC(50), 8 nM), but not by BQ-610 (selective ET(A) receptor blocker). Removal of the epithelium did not prevent ET-induced increases in cAMP levels. Unlike isoproterenol, ETs failed to activate AC in membrane fractions from RTSM. In intact RTSM, the c-PLA(2) inhibitor, AACOCF3, and the cyclooxygenase inhibitor, indomethacin, blocked ET-induced increases in cAMP levels. These findings reveal a novel, nonepithelial, c-PLA(2)-mediated, regulatory mechanism downstream from ET(B) receptors.

H(2)O(2) opens BK(Ca) channels via the PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle.

H(2)O(2) is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H(2)O(2) opens the large-conductance, calcium- and voltage-activated (BK(Ca)) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H(2)O(2) on coronary smooth muscle involves the phospholipase A(2) (PLA(2))/arachidonic acid (AA) signaling cascades. H(2)O(2) stimulation of BK(Ca) channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA(2). Furthermore, H(2)O(2) stimulated release of [(3)H]AA from coronary myocytes, and exogenous AA mimicked the effect of H(2)O(2) on BK(Ca) channels. Inhibitors of protein kinase C activity attenuated the effect of H(2)O(2) on BK(Ca) channels, [(3)H]AA release, or intact coronary arteries. In addition, the effect of H(2)O(2) or AA on BK(Ca) channels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H(2)O(2) relaxes coronary arteries by stimulating BK(Ca) channels via the PLA(2)/AA signaling cascade and that lipoxygenase metabolites mediate this response.

cAMP-dependent vasodilators cross-activate the cGMP-dependent protein kinase to stimulate BK(Ca) channel activity in coronary artery smooth muscle cells.

cAMP-dependent vasodilators are used to treat a variety of cardiovascular disorders; however, the signal transduction pathways and effector mechanisms stimulated by these agents are not fully understood. In the present study we demonstrate that cAMP-stimulating agents enhance the activity of the large-conductance, calcium-activated potassium (BK(Ca)) channel in single myocytes from coronary arteries by "cross-activation" of the cGMP-dependent protein kinase (protein kinase G, PKG). Single-channel patch-clamp data revealed that 10 micromol/L isoproterenol, forskolin, or dopamine opens BK(Ca) channels in coronary myocytes and that this effect is attenuated by inhibitors of PKG (KT5823; Rp-8-pCPT-cGMPS), but not by inhibiting the cAMP-dependent protein kinase (protein kinase A, PKA). In addition, a membrane-permeable analog, CPT-cAMP, also opened BK(Ca) channels in these myocytes, and this effect was reversed by KT5823. Direct biochemical measurement confirmed that dopamine or forskolin stimulates PKG activity in coronary arteries but does not elevate cGMP. Finally, the stimulatory effect of cAMP on BK(Ca) channels was reconstituted in a cell-free, inside-out patch by addition of purified PKG activated by either cGMP or cAMP. In contrast, channel gating was unaffected by exposure to the purified catalytic subunit of PKA. In summary, findings from on-cell and cell-free patch-clamp experiments provide direct evidence that cAMP-dependent vasodilators open BK(Ca) channels in coronary myocytes by cross-activation of PKG (but not via PKA). Biochemical assay confirmed this cross-activation mechanism of cAMP action in these arteries. This signaling pathway is a novel mechanism for regulation of potassium channel activity in vascular smooth muscle and other cells.

Novel piperidinedione analogs as inhibitors of breast cancer cell growth.

We previously reported the utility of antineoplaston-A10 (3-phenylacetylamino-2,6-piperidinedione) as an endogenous cancer protector and immune modulator in breast cancer patients (Cancer Lett., 2000, 157, 57). In this study, four new piperidinedione A10 analogs were synthesized and tested for their antimitotic activity on a human breast cancer cell line against the prototype A10 and the antibreast cancer drug tamoxifen. Moreover, the DNA binding capacity of such compounds was evaluated against A10, (E)-3-(4-Nitrocinnamoylamino)-2,6-piperidinedione "3B" and (E)-3-(4-hydroxycinnamoylamino)-2,6-piperidinedione "3D" were several-fold more potent antiproliferative agents than A10 and tamoxifen. They also had significantly higher capacity to bind DNA than A10. Conversely, (E)-3-(cinnamoylamino)-2,6-piperidinedione "3A" and (E)-3-(4-methoxycinnamoylamino)-2,6-piperidinedione) "3C" had weaker biological profiles than the lead compound A10. Detailed synthetic, spectroscopic, and biological data are reported.

Resveratrol inhibits MAPK activity and nuclear translocation in coronary artery smooth muscle: reversal of endothelin-1 stimulatory effects.

In porcine coronary arteries, short-term treatment with resveratrol (RSVL) substantially inhibited MAPK activity (IC50 = 37 microM); and immunoblot analyses revealed consistent reduction in the phosphorylation of ERK-1/-2, JNK-1 and p38, at active sites. Endothelin-1 (ET-1), a primary antecedent in coronary heart diseases, enhanced MAPK activity, phosphorylation and nuclear translocation in a concentration-responsive but RSVL-sensitive manner. RSVL had no effect on basal or forskolin-stimulated cAMP levels, a known downregulator of the MAPK cascade. Likewise, inhibition of MAPK by RSVL was not reversed by the estrogen receptor blockers tamoxifen and ICI-182,780. Conversely, RSVL remarkably attenuated basal and ET-1-evoked protein tyrosine phosphorylation. Because MAPKs promote smooth muscle proliferation and contraction, their current inhibition may contribute to the putative protection by RSVL against coronary heart diseases. These effects apparently do not involve interaction with estrogen receptors.

Evidence for a tyrosine kinase-dependent activation of the adenylyl Cyclase/PKA cascade downstream from the G-protein-linked endothelin ETA receptor in vascular smooth muscle.

Endothelin (ET-1), a contractor and mitogen in the vasculature, enhanced cAMP production (t1/2, 2.2 min; EC50, 89 +/- 6.3 nM) and stimulated activity of the cAMP-dependent protein kinase (PKA) in pig coronary arteries. These responses were blunted by the protein tyrosine kinase (PTK) inhibitors genistein and herbimycin-A, but not by inhibitors of protein kinase C or cyclooxygenase. In contrast, forskolin-stimulated cAMP production was unaffected by PTK inhibition. Immunoblot analysis revealed that ET-1 induced a concentration-dependent protein tyrosine (PT) phosphorylation. Sarafotoxin-c, a selective ETB receptor agonist, had no effect on either cAMP levels or PT phosphorylation. Moreover, pervanadate (PV), a potent inhibitor of PT phosphatases, enhanced both cAMP formation and PT phosphorylation, both of which were blocked by PTK inhibitors. The effects of ET-1 and PV were not additive, suggesting a similar mode of activation, whereas responses to ET-1 and forskolin were synergistic. These findings indicate that AC and PKA are activatable via a nonreceptor PTK-dependent pathway downstream from the G-protein-linked ETA receptor. Because cAMP is a dilator and antimitogen in smooth muscle, stimulation of AC activity may be a negative feedback mechanism regulating ET-1-induced vasoconstriction and/or mitogenesis.

Role of endogenous endothelin-1 in stress-induced gastric mucosal damage and acid secretion in rats.

In rats subjected to 8 h water-immersion stress, gastric and duodenal mucosal hemorrhage and erosions were detected by macroscopic and histopathological examination. Moreover, plasma and gastric mucosal endothelin-1 (ET-1) levels rose appreciably in a time-related manner during water immersion, with a higher concentration detected in gastric mucosa. Thus, the percentage increases in plasma (gastric mucosal) ET-1, relative to basal levels, after 1, 4 and 8 h of water immersion were 86(172), 169(322) and 210(391)%, respectively. Likewise, a marked increase of gastric acid output was demonstrated 30 min after water immersion and lasted for 3 h. Pretreatment with the endothelin ET(A)/ET(B) receptor blocker, bosentan (30 and 100 mg kg(-1)), orally, dose-dependently antagonized gastric and duodenal mucosal damage as indicated by reductions in lesion lengths of 67 and 80%, respectively. Similar protective effects on mucosa were observed when bosentan was given by the intramuscular route. On the other hand, bosentan suppressed the rate of acid output by 30.3+/-2.1% in the stressed rats, but had no such effect in non-stressed animals. Taken together, results from this study implicate the endogenous peptide, ET-1, as a powerful mediator of stress-evoked gastro-duodenal mucosal damage and, moreover, present bosentan as a potential protector against hyperacidity and mucosal erosion that occur as a consequence of stress.

Non-epithelial endothelin-A receptors activate adenylate cyclase in rat trachea: biochemical mechanisms and physiological implications.

In the present study, we investigated the mechanisms underlying the differential effects of endothelins (ETs) in the rat trachea. Sarafotoxin-S6c (SRTX-c) and ET-3 were more potent spasmogens to rat tracheal strips than ET-1. The EC50 values were 12, 14.1 and 89.1 nM, respectively. Tension responses to ET-1 and ET-3, but not to SRTX-c, were enhanced by either indomethacin or the ET(A) blocker, BQ-610 (1 microM). In epithelium-intact tracheal rings, both ET-1 and ET-3 activated adenylate cyclase (AC) in a concentration-dependent manner. The activation by ET-1 of AC was significantly higher than that of ET-3. Thus, EC50 values for ET-1 and ET-3 were 71 and 200 nM, and maximal cAMP increments were 196% and 62% above baseline, respectively. SRTX-c, up to 1 microM, did not alter basal cAMP level. Mechanical removal of the epithelium neither had an effect on AC activation by ET-1 or ET-3, nor did it alter the inability of SRTX-c to modulate AC activity. Conversely, pre-incubation of tracheal strips with indomethacin (1 microM) virtually ablated the increments in cAMP by the ETs. Likewise, BQ-610 attenuated AC activation, concentration-dependently (IC50=28.2 nM). Taken together, the present study suggests that ET(A) receptors, from non-epithelial source, are functionally-linked to AC activation via a prostanoid-dependent pathway. This ET(A)-initiated cascade acts to negatively regulate muscle contraction. Such a cross-talk between ET signals most likely accounts for variation of tension responses to ET homologs.

Endothelins-induce cyclicAMP formation in the guinea-pig trachea through an ETA receptor- and cyclooxygenase-dependent mechanism.

1. The non-selective endothelin agonist, endothelin-1 (ET-1), and the selective ETB receptor agonist, sarafotoxin-S6c (SRTX-c), contracted guinea-pig isolated trachea in a concentration-dependent manner. The EC50 value for ET-1 (11 +/- 2.1 nM) was significantly higher than that of SRTX-c (3.2 +/- 0.21 nM) and the maximal developed tension due to SRTX-c was 42.8 +/- 2.3% higher than that produced by ET-1 (P < 0.05). 2. Pretreatment with the ETA antagonist, BQ-610, appreciably enhanced the developed tension due to ET-1 but not SRTX-c. Likewise, the cyclo-oxygenase inhibitor, indomethacin, markedly potentiated the contractile responses to ET-1, but not to SRTX-c. Combining BQ-610 with indomethacin was not more effective than either of them in augmenting ET-1-evoked tension. 3. ET-1 significantly increased cyclic AMP formation in the trachea in concentration- and time-dependent manners. A t1/2 value of 4.3 min, an EC50 value of 20 +/- 3 nM and a maximal cyclic AMP increment of 124% above the basal level, were obtained for ET-1. Similarly but less effectively, ET-3 (0.1 microM) increased cyclic AMP level (35 +/- 3.7% compared to 94 +/- 7.8% for the same concentration of ET-1). By contrast, SRTX-c did not alter the cyclicAMP level when applied in concentrations up to 1 microM. 4. Pre-incubation of the trachea with BQ-610 (1 microM) or indomethacin (1 microM) prevented cyclicAMP formation by either ET-1 or ET-3. 5. The results of the present study indicate a negative regulatory role mediated by the ETA receptor on the ETB-triggered mechanical response. This effect is likely to be mediated by activation of adenylate cyclase through a cyclo-oxygenase-dependent mechanism.

Prostaglandins mediate the stimulatory effects of endothelin-1 on cyclic adenosine monophosphate accumulation in ciliary smooth muscle isolated from bovine, cat, and other mammalian species.

PURPOSE: To examine the effects and mechanisms of endothelin-1 (ET-1) on cyclic adenosine monophosphate (cAMP) accumulation, inositol 1,4,5-trisphosphate (IP3) production, and contraction in ciliary muscle (CM) isolated from bovine, cat and other mammalian species. METHODS: Ciliary muscle was incubated in the absence and presence of ET-1 for 5 minutes. Indomethacin (Indo, 2.5 microM) and IBMX (0.1 mM) were added 10 minutes before the addition of the peptide. Cyclic AMP accumulation and prostaglandin E2 (PGE2) release were measured by radioimmunoassay, IP3 production was measured by ion-exchange chromatography, and changes in tension were recorded isometrically. RESULTS: First, ET-1 (0.1 microM) increased PGE2 release by 58% to 105% and cAMP accumulation by 98% to 393% in CMs isolated from bovine, cat, dog and human, and these effects were blocked completely by Indo (2.5 microM). Unlike any other species, in bovine CM, ET-1 increased IP3 production (EC50 = 17 nM) and contraction (EC50 = 13 nM), and these effects were not inhibited by Indo. Second, kinetic studies revealed that in bovine and cat CMs, ET-1 stimulated cAMP accumulation and PGE2 release in a time- and dose-dependent manner, and these effects were inhibited by Indo in a time- and dose-dependent manner, and PGE2 increased cAMP accumulation in a dose-dependent manner (EC50 = 0.175 microM). The stimulatory effect of ET-1 on cAMP accumulation is mediated through the ETA receptor subtype, because in contrast to ET-1, which is an ETA receptor agonist, ET-3 and Sarafotoxin-S6c, two ETB receptor agonists, had little effect on cAMP accumulation. In addition, BQ 610, an ETA receptor subtype antagonist, inhibited ET-1-induced cAMP accumulation in a dose-dependent manner (IC50s for bovine and cat were 11 and 19.5 nM, respectively). Quinacrine, a phospholipase A2 inhibitor, inhibited ET-1-induced cAMP accumulation in a dose-dependent manner (IC50s for bovine and cat were 22 and 19 microM, respectively). PGE2, but not ET-1, stimulated adenylyl cyclase activity in membranes isolated from bovine and cat CMs. CONCLUSIONS: In CMs isolated from bovine, cat, dog and human, ET-1-induced cAMP accumulation is mediated through the release of PGs. ET-1 binds to the ETA receptor subtype to activate phospholipase A2 and to release arachidonic acid for PG synthesis. PGs, such as PGE2, may interact with the EP receptor to stimulate adenylyl cyclase. Although ET-1-induced PG release could function to modulate, through cAMP, the responses to muscarinic receptor stimulation, the precise role of these effects in intraocular pressure lowering and accommodation remains to be delineated.

Characterization of iris sphincter smooth muscle endothelin receptor subtypes which are coupled to cyclic AMP formation and polyphosphoinositide hydrolysis.

In the mammalian iris sphincter smooth muscle, endothelins (ET) activate both adenylate cyclase and the polyphosphoinositide cascade, and the levels of cyclic AMP (cAMP) and inositol 1,4,5-trisphosphate (IP3) produced are species specific. Radioligand binding studies, using [125I]ET-1 and [125I]ET-3 and determination of changes in cAMP, IP3 and contraction due to the peptides revealed the existence of ETA and ETB receptor subtypes in this tissue. In rabbit sphincter, ETA receptors constitute about 80% of total ET receptor population and these are coupled to IP3 production and contraction. In bovine sphincter, ETB receptors constitute about 72% of the total ET receptors and these are coupled to cAMP formation. Thus, in rabbit sphincter: 1) ET-1 and ET-2, two potent ETA receptor agonists, induced IP3 production and contraction at a much higher rate than ET-3, a weak ETA agonist. The EC50 for contraction by ET-1, ET-2 and ET-3 were 40, 45 and 300 nM, respectively. 2) Sarafotoxin-S6c (SRTX-c), a selective ETB receptor agonist, had no effect on IP3 and contraction in this tissue. 3) D-Asp-L-Pro-D-Val-L-Leu-D-Trp (BQ-123), a selective ETA receptor antagonist, inhibited the above responses to ET. 4) ET and SRTX-c induced cAMP formation at a much lower rate than that of IP3 and contraction. In contrast, in the bovine sphincter: 1) ET and SRTX-c induced cAMP formation in a dose-dependent manner, the order of potency being SRTX-c > ET-3 congruent to ET-2 congruent to ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)