Role of the cytoskeleton in the secretory response of the frog adrenal gland to calcitonin gene-related peptide

We have previously shown that calcitonin gene-related peptide (CGRP) stimulates the secretion of corticosterone and aldosterone from the frog adrenal gland in vitro. The aim of the present study was to investigate the role of cytoskeletal elements in the stimulatory effect of CGRP on corticosteroid production. Perifusion of adrenal explants with the microfilament-disrupting agent cytochalasin B (5 x 10(-5) M) induced a reversible inhibition of the spontaneous secretion of corticosterone and aldosterone, and markedly reduced the stimulatory effect of frog CGRP (3 x 10(-7) M) on corticosteroid release. In contrast, administration of the antimicrotubular agent vinblastine (10(-5) M) and the intermediate filament inhibitor beta-beta' iminodipropionitrile (10(-3) M) had no significant effect on either spontaneous or CGRP-evoked steroid secretion. Taken together, these data provide the first evidence for the involvement of microfilaments in CGRP-induced corticosteroid secretion. In contrast, microtubules and intermediate filaments are not implicated in the mechanism of action of CGRP.     

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.     

Localization, identification, and action of galanin in the frog adrenal gland

The distribution of galanin-like immunoreactivity was studied in the adrenal gland of the frog Rana ridibunda using the indirect immunofluorescence technique. A dense network of varicose fibers immunoreactive to galanin was found in the adrenal tissue. A combination of HPLC analysis and RIA detection was used to characterize galanin-like immunoreactivity in frog adrenal gland extracts. The elution profile revealed the existence of a single form of galanin exhibiting the same retention time as synthetic frog galanin. The possible involvement of galanin in the regulation of corticosteroid secretion was investigated in vitro using a perifusion system for frog adrenal slices. For concentrations ranging from 10(-9) to 3 x 10(-6) M, synthetic frog galanin induced a dose-dependent inhibition of corticosterone and aldosterone release. Repeated pulses of galanin (10(-6) M), given at 90-min intervals, resulted in a reproducible inhibition of corticosteroid secretion without any apparent tachyphylaxis. During prolonged administration of galanin (10(-6) M), the steroidogenic effect of ACTH (10(-9) M) was significantly reduced. In contrast, galanin did not attenuate the stimulation of corticosteroid secretion induced by the angiotensin II analog [Sar1,Val5]angiotensin II. These results show the occurrence of galanin in fibers innervating the frog adrenal gland. The data also demonstrate that synthetic galanin inhibits spontaneous and ACTH-induced corticosteroid release. Taken together, these findings suggest that galanin, released by nerve fibers in the adrenal tissue, can act locally as a modulator of steroid secretion.     

Endothelin-1 induces vasoconstriction and nitric oxide release via endothelin ET(B) receptors in isolated perfused rat liver

Endothelin-1 (0.1, 1 and 10 nM) induced a significant increase in portal pressure and nitric oxide (NO) release in the isolated rat liver. The endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala(11,15)]endothelin-1-(8-21)) (0.1, 1 and 10 nM) also elicited a marked increase in portal pressure and NO release. The potency of endothelin-1 was higher than that of IRL 1620. The endothelin ET(A) receptor antagonist, BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu)) (1 and 10 microM), had no effect on the endothelin-1-induced change in portal pressure and NO current. In contrast, the endothelin ET(B) receptor antagonist, BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1-++ +methoxycarbonyltryptophanyl-D-norleucine) (1 and 10 nM), attenuated the endothelin-1-induced change in portal pressure and NO current. Administration of N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, completely abolished the endothelin-1- or IRL 1620-induced NO release. L-NMMA enhanced the increase in portal pressure and decrease in O2 consumption caused by endothelin-1. These results indicated that endothelin ET(B) receptors mediate both vasoconstriction and NO release and that NO plays a significant role in stabilizing microcirculation in isolated perfused rat liver.     

Endothelin-1-induced in vitro cerebral venoconstriction is mediated by endothelin ETA receptors

The in vitro effects of endothelin-1 on cerebral veins were studied using cylindrical segments, 5 mm long, from dog pial veins. Isometric responses to endothelin-1 (10(-12)-10(-7) M) and to the endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21), 10(-12) -10(-7) M), were recorded in veins under control conditions and pretreated with the endothelin ET(A) receptor antagonist, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp), 10(-8) -10(-5) M), and the endothelin ETB receptor antagonist, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(me thoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium, 10(-6) and 10(-5) M). The response to endothelin-1 was also recorded in veins pretreated with the nitric oxide synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), or the cyclooxygenase inhibitor, meclofenamate (10(-5) M), and in veins without endothelium or placed in medium without Ca2+ but with EDTA (0.1 mM). In control veins, endothelin-1 produced a concentration-dependent contraction (EC50 = 2.0 x 10(-10) M; maximal contraction = 113 +/- 6 mg) and IRL 1620 induced no effects or a small contraction only with high concentrations (10(-8) - 10(-6) M) (EC50 = 1.5 x 10 (-8) M; maximal contraction = 9 +/- 3 mg). BQ-123 shifted the response to endothelin-1 to the right in a parallel, concentration-dependent way, whereas BQ-788, L-NAME or meclofenamate did not modify the response to endothelin-1. Compared with the control, veins in a medium without Ca2+ had similar EC50 values, but a lower maximal contraction induced by endothelin-1 (57 +/- 10 mg, P < 0.05), and veins without endothelium exhibited similar EC50 values. Thus, endothelin-1 produces marked cerebral venoconstriction that could be mainly mediated by activation of endothelin ETA receptors, may be dependent on extracellular Ca2+, and may be independent of endothelium, nitric oxide and prostanoids.     

In vivo and in vitro action of endothelin-1 on goat cerebrovascular bed

This study concerned the effects and mechanisms of action of endothelin-1 on the cerebral circulation. Cerebral blood flow was electromagnetically measured in awake goats. Endothelin-1 (0.01-0.3 nmol) produced dose-dependent decreases in this flow (maximal reduction = 34%) and increases in cerebrovascular resistance (maximal increase = 74%) (P < 0.01). IRL 1620 (Suc-[Glu9, Ala11,15]endothelin-1-(8-21), agonist for endothelin ET(B) receptors, 0.01-0.3 nmol) slightly decreased cerebral blood flow. The effects of endothelin-1, but not those of IRL 1620, on cerebral blood flow were diminished by 50% during infusion of the antagonist for endothelin ET(A) receptors, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-Trp), 2 nmol min(-1)), but not affected during infusion of the antagonist for endothelin ET(B) receptors, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-Leucyl-1-(met hoxycarbonyl)-D-tryptophyl]-Dnorleucine monosodium), 2 nmol min(-1)). Intravenous administration of NW-nitro-L-arginine methyl ester (L-NAME, 47 mg kg(-1)) or NW-nitro-L-arginine (L-NNA, 47 mg kg(-1)) reduced basal cerebral blood flow by 39 and 33%, increased cerebrovascular resistance by 108 and 98% and mean arterial pressure by 23 and 17%, and decreased heart rate by 27 and 25%, respectively (all at least P < 0.05). The increases in cerebrovascular resistance (as absolute values) induced by endothelin-1 were not affected during either L-NAME or L-NNA (as absolute values and percentages). Intravenous administration of meclofenamate (5 mg kg(-1)) did not change the cerebrovascular effects of endothelin-1 and IRL 1620. In isolated goat cerebral arteries under control, resting conditions, endothelin-1 (10(-11)-10(-7) M) induced concentration-dependent contractions (EC50 = 4.78 X 10(-9) M; maximal contraction = 3177+/-129 mg), whereas IRL 1620 (10(-11)-10(-7) M) produced no effect. This contraction produced by endothelin-1 was competitively blocked by BQ-123 (10(-7)-3 X 10(-6) M), and was not affected by BQ-788 (10(-6) and 10(-5) M). L-NAME (10(-4) M), meclofenamate (10(-5) M), indomethacin (10(-5) M), L-NAME (10(-4) M) plus meclofenamate (10(-5) M) and phosphoramidon (10(-4) M) did not affect the contraction in response to endothelin-1. Endothelium removal increased the response to endothelin-1, as well as the BQ-123 antagonism against endothelin-1 (pA2 values, 7.62 vs. 6.88; P < 0.01). In both intact and de-endothelized arteries precontracted with prostaglandin F2alpha endothelin-1 induced a further contraction, and IRL 1620 caused no effect. These results suggest that: (1) endothelin-1 produces cerebral vasoconstriction by activating endothelin ET(A) receptors probably located in smooth muscle; (2) endothelin ET(B) receptors, nitric oxide and prostanoids might be not involved in the cerebrovascular action of endothelin-1, and (3) endothelium removal may increase cerebrovascular reactivity by increasing sensitivity of endothelin ET(A) receptors to endothelin-1.     

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.     

Effects of recombinant human erythropoietin (rHuEpo) on the interactions between glomerular endothelial cells and mesangial cells: regulation of cell proliferation via endothelin (ET)-1

To elucidate the effects of recombinant human erythropoietin (rHuEpo) on the interactions between glomerular endothelial cells (GENs) and mesangial cells (GMCs), we investigated whether or not cultured bovine GENs alter endothelin (ET)-1 secretion from bovine MCs and MC proliferate under basal or rHuEpo-stimulated conditions. Incubation for 24 hours with synthetic ET-1 stimulated MC in a dose-dependent manner. In addition, 100 pg/ml of ET-1 significantly stimulated MC proliferation after more than 12 hours incubation. Moreover, rHuEpo stimulated ET-1 secretion from GENs in a dose-dependent manner and showed less stimulation of ET-1 secretion from GMCs than GENs. DNA synthesis in both GENs and GMCs was significantly stimulated with more than 5 U/ml of rHuEpo. ET-1 secretion from GENs co-cultured with GMCs was higher than that from cultured GENs only. Conditioned medium, obtained from co-culture of GENs and GMCs, stimulated the proliferation of GMCs that were significantly inhibited with 10(-6) M approximately 10(-5) M BQ-123, a ETA receptor antagonist. These results suggest that rHuEpo directly stimulates the proliferation of GENs and GMCs, and this stimulatory effect is in part due to ET-1 secreted from these cells, especially GENs.     

Effects of endothelin-1 on the rat pituitary-adrenocortical axis under basal and stressful conditions

Endothelins (ETs) and their receptor subtypes A and B (ETA and ETB) are expressed in the various components of the mammalian hypothalamo-pituitary-adrenal (HPA) axis, but their involvement in the functional regulation of HPA is controversial. To gain insight into this topic, we have investigated the effects of ET-1 and/or the specific antagonists of ETA and ETB receptors (BQ-123 and BQ-788, respectively) on the plasma concentrations of ACTH, corticosterone and aldosterone of non-stressed (control) and ether- or cold-stressed rats. The study of the effects of the administration of the two ET-receptor antagonists alone could provide informations about the possible action of endogenous ETs on the HPA axis. Exogenous ET-1 increased ACTH, corticosterone and aldosterone blood levels in control rats, as well as evoked a sizable enhancement of the HPA axis response to ether stress and a marked depression of the response to cold stress. BQ-123 and BQ-788 did not prevent the stimulatory effect of exogenous ET-1 in control rats, but when administered alone, raised the plasma concentrations of ACTH, corticosterone and aldosterone. Both ET-receptor antagonists magnified the HPA axis response to ether and cold stresses, but their effect was not counteracted by exogenous ET-1. Although very difficult to interpret, our present findings allow us to conclude that endogenous ETs play a role in the maintenance of the basal activity of rat HPA axis acting through ETA and ETB receptor subtypes, which are partially insensitive to BQ-123 and BQ-788. Conversely, the involvement of ETs in the modulation of the HPA axis responses to various stresses is very doubtful.     

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.     

Cloning of Drosophila MCM homologs and analysis of their requirement during embryogenesis

Modification of blood flow by endothelin-1 (ET-1) was examined in the s.c. HSN fibrosarcoma and compared to normal tissues of anaesthetised CBH/CBi rats. The ET receptor subtypes involved in the response were investigated using the ET(A) and ET(B) receptor antagonists BQ-610 and BQ-788, respectively. Blood flow and vascular resistance were determined using the uptake of radiolabelled iodo-antipyrine (125I-IAP). BQ-610 or BQ-788 was infused for 30 min prior to blood flow determination. ET-1 was administered 15 min into the infusion time. BQ-610 and BQ-788 infused alone did not modify any vascular parameters. Tumour blood flow increased slightly following ET-1, contrasting with most normal tissues, in which blood flow was reduced. Vascular resistance increased in all tissues, including the tumour. Neither antagonist significantly modified the ET-1-induced changes in tumour blood flow or vascular resistance, whereas in the majority of normal tissues BQ-610 attenuated and BQ-788 potentiated the vascular resonse to ET-1. Our results show that the HSN tumour vasculature is only weakly responsive to ET- 1 and antagonism of its effects by BQ-610 and BQ-788. This contrasts with the majority of normal tissues, in which ET- 1 induces an intense vasoconstriction.     

ET(B) receptor and nitric oxide synthase blockade induce BQ-123-sensitive pressor effects in the rabbit

Endothelin-1 (0.25 nmol/kg, injected into the left cardiac ventricle) induces a protracted increase of mean arterial pressure that is significantly reduced by the selective ET(A) receptor antagonist BQ-123 (1 and 10 mg/kg) in the anesthetized rabbit. The sole administration of the selective ET(B) antagonist BQ-788 (0.25 mg/kg) induces a pressor response abolished by BQ-123 (1 mg/kg). Concomitant to the increase in mean arterial pressure, BQ-788 induces a significant increase in plasma levels of endothelin-1 and its precursor big endothelin-1. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) also increases arterial blood pressure, and the response is reduced dose-dependently by BQ-123 (1 and 10 mg/kg). In addition, the administration of BQ-788 in the presence of L-NAME induced a further increase in arterial blood pressure. The duration of the pressor response to L-NAME is also significantly reduced by an endothelin-converting enzyme inhibitor, phosphoramidon (10 mg/kg). Finally, L-NAME induces an increase in plasma levels of big endothelin-1 but not endothelin-1. Our results illustrate that blockade of either nitric oxide synthase or ET(B) receptors triggers a raise in plasma levels of endothelin-1 or its precursor. These later moieties are suggested to be significantly involved, through the activation of ET(A) receptors, in the pressor effects of L-NAME and BQ-788 in the anesthetized rabbit.     

Effect of various detergents on the interaction between BK virus and susceptible cells

The effects of neuropeptide Y (NPY1-36), of two analogs (Leu31-Pro34 NPY and NPY18-36) and of Peptide YY (PYY) on aldosterone and corticosterone secretions by freshly isolated rat adrenal capsule/zona glomerulosa preparations were investigated in vitro. NPY-related peptides (NPY1-36, Leu31-Pro34 NPY, NPY18-36), but not PYY, induced a dose-dependent release of aldosterone at concentrations ranging from 10(-8) to 10(-6) M. All the investigated peptides failed to significantly affect corticosterone secretion in concentrations ranging from 10(-10) to 10(-6) M (NPY1-36, NPY18-36), 10(-11) to 10(-6) M (Leu31-Pro34 NPY) or 10(-9) to 10(-6) M (PYY). Aldosterone secretion by this preparation of isolated adrenal capsule/zona glomerulosa was also significantly stimulated by high potassium levels (55 mEq) or by angiotensin II (AII) in concentrations ranging from 10(-8) to 10(-6) M. Moreover, NPY and Y1 or Y2 receptor agonists were positive aldosterone releasing agents as potent as AII. The present data support the existence of: (1) NPY binding sites of the Y3-like subtype, on rat adrenal capsule/zona glomerulosa. (2) A stimulatory effect of NPY on aldosterone production. So that the NPYergic innervation of the rat adrenal capsule/zona glomerulosa could be implicated in the multifactorial control of aldosterone production.     

Effect of endothelin receptor antagonists (BQ-485, Ro 47-0203) on collagen deposition during the development of bleomycin-induced pulmonary fibrosis in rats

Previous evidence suggests a role for endothelin-1 (ET-1) in the pathogenesis of pulmonary fibrosis. To determine if ET-1 regulates collagen deposition in pulmonary fibrosis, we examined the effect of the non-selective ETA and ETB receptor antagonist bosentan (Ro 47-0203), and a selective ETA receptor antagonist, BQ-485, on collagen deposition during the development of bleomycin-induced pulmonary fibrosis in rats. Lung collagen content, derived from measurements of hydroxyproline and expressed as mg collagen/lung, was increased in the bleomycin-treated animals by day 7 (bleomycin, 22.88+/-1.46; control 18.50+/-0.98; P<0.05), continued to increase up to day 14 (bleomycin, 38.80+/-2.17; control 22.57+/-0.77; P<0.001) and then remained constant to 21 days. Daily treatment by gavage with bosentan (100 mg/kg) did not prevent the increase in collagen deposition induced by instillation of bleomycin at any of the times measured. Continuous administration of BQ-485, by subcutaneously implanted minipump (7.5 mg/day), also failed to prevent the bleomycin-induced collagen deposition at 14 days. These findings suggest that ET-1 does not modulate collagen deposition during the development of bleomycin-induced pulmonary fibrosis. Further studies are required to assess whether endothelin receptor antagonists modulate other components of the fibrotic response or play a role in man.     

Selective and non-selective ET antagonists reveal an ETB receptors mediated ET-1-induced behavioural effect in conscious rats

The injection of endothelin-1 (ET-1) (1 pmol/rat) into the dorsolateral periaqueductal gray (PAG) area of freely moving rats induced rotation along the long axis of the body (barrel-rolling). Preinjection (10 min before) of BQ-788 (an ETB receptor selective antagonist; 5 nmol) or bosentan (an ETA/ETB receptor non-selective antagonist; 10 nmol) to the PAG reduced the behavioral response to ET-1. In contrast, pretreatment with FR139317 (an ETA receptor selective antagonist; 5 nmol) did not affect the ET-1-induced barrel-rolling. These results suggest that barrel-rolling induced by microinjection of ET-1 into the PAG area is predominantly mediated via ETB-like receptors.     

Suppressive effects of the endothelin receptor (ETA) antagonist BQ-123 on ET-1-induced reduction of lipoprotein lipase activity in 3T3-L1 adipocytes

Endothelin (ET)-1 reduced heparin-releasable lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes in a concentration-dependent manner. However, a selective ETB receptor agonist, [Ala1,3,11,15]ET-1, did not act like ET-1. The ET-1-induced decrease in LPL activity was suppressed by a selective ETA receptor antagonist, BQ-123: the concentration-response curve for the ET-1 reduction of LPL activity was shifted to the right in the presence of BQ-123 in a concentration-dependent manner. This antagonistic effect of BQ-123 clarifies that the ETA receptor is responsible for the ET-1-induced reduction of LPL activity in 3T3-L1 adipocytes, which suggests that there is therapeutic potential for ETA antagonists in LPL-related lipoprotein disorders.     

Activation of phospholipase C and guanylyl cyclase by endothelins in human trabecular meshwork cells

PURPOSE: To characterize effects of endothelins on activities of phospholipase C (PLC) and nucleotide cyclases in human trabecular meshwork (TM) cells. METHODS: Cultured simian virus 40-transformed human TM (HTM-3) or non-transformed (HTM-16) cells were used. Changes in the PLC activity were determined by assaying the production of [3H] inositol phosphates. Accumulation of cyclic GMP or cyclic AMP in cell lysate was measured by radioimmunoassay. RESULTS: Endothelin-1 (ET-1; 1 microM) stimulated PLC in HTM-16 cells, but Sarafotoxin S6c (SRTX), an ET(B) receptor subtype-selective agonist (1 microM), did not. Similar results were obtained in HTM-3 cells: ET-1, but not ET-3 or SRTX, activated PLC in a dose-dependent manner, with a calculated EC50 of 646 pM. The peptide also stimulated the accumulation of cGMP in a concentration-dependent manner with an EC50 of 37.2 pM. ET-3 or SRTX was not effective except at much higher concentrations. Both the PLC and guanylyl cyclase stimulation induced by ET-1 (10 nM) were completely inhibited by pretreating the cells with BQ-123 (<10 microM), an ET(A) receptor selective antagonist, but not by BQ-788 (10 microM), an ET(B) receptor subtype-specific antagonist. Neither ET-1 nor ET-3 stimulated adenylyl cyclase activity in HTM-3 cells at concentration as high as 1 microM. CONCLUSION: ET-1 activates PLC and guanylyl cyclase in TM cells. Potency profiles of ET receptor agonists and antagonists suggest that the ET(A) receptor subtype is involved in both actions of ET-1. The effects of the ET peptides in TM cells are interesting and could be part of the mechanism of their IOP-lowering effect.     

Behavioral signs of acute pain produced by application of endothelin-1 to rat sciatic nerve

We examined whether endothelin-1 (ET-1), a potent vasoconstrictive peptide secreted in high concentration by metastatic prostate cancer cells, produces endothelin receptor-dependent pain behavior when applied to rat sciatic nerve. ET-1 (200-800 microM) applied to the epineurial surface of rat sciatic nerve produced reliable, robust, unilateral hindpaw flinching lasting 60 min. Pre-emptive systemic morphine completely blocked this effect in a naloxone-reversible manner, suggesting that this behavior was pain-related. Equipotent doses of epineurially applied epinephrine had no effect, suggesting that ET-1 effects are on tissue sites other than sciatic nerve microvessels. Prior and co-administration of BQ-123, an endothelin-A (ET(A)) receptor antagonist, also blocked ET-1-induced hindpaw flinching establishing that pain behavior induced by ET-1 application to rat sciatic nerve is ET(A) receptor mediated.