Isoproterenol-induced FKBP12.6/12 downregulation is modulated by ETA and ETB receptors and reversed by argirhein, a derivative of rhein

Aim:

To investigate which endothelin receptors mediated isoproterenol (ISO)-induced downregulation of FKBP12.6/12 in cardiomyocytes and study whether argirhein, a novel compound containing rhein and L-arginine that has anti-inflammatory activity, could reverse the downregulation of FKBP12.6/12 induced by ISO.

Methods:

Neonatal rat cardiomyocytes were incubated with ISO to downregulate FKBP12.6/12. Then the cells were treated with a selective ET_A blocker (PD156707) and a ET_B blocker (IRL1038), a dual ET_A/ET_B antagonist (CPU0213), and argirhein, respectively. FKBP12.6/12 expression was assayed by RT-PCR, Western blot, and immunocytochemistry.

Results:

The expression of FKBP12.6 mRNA was reduced by 37.7% (P<0.01) and 28.9% (P<0.05) relative to the control by ISO 1 and 0.1 μmol/L, respectively, but no response to ISO 0.01 μmol/L was observed in vitro. FKBP12.6/12 protein expression was reduced by 47.2% (P<0.01) and 37.8% (P<0.05) by ISO 1 and 0.1 μmol/L, respectively. This decrease was reversed significantly by PD156707, or IRL1038, and CPU0213. CPU0213 was more potent than either PD156707 or IRL-1038. Argirhein 10 μmol/L blunted the downregulation of FKBP12.6/12 by ISO, as demonstrated by the rising mRNA and protein levels and by the fluorescent density of the ISO-incubated cardiomyocytes.

Conclusion:

In cardiomyocytes, the ISO induced downregulation of FKBP12.6/12 is modulated by both ET_A and ET_B. A new compound, argirein, reversed the down-regulation of FKBP12.6/12 expression in myocardial cells stimulated with ISO.     

Different role of endothelin ETA and ETB receptors and endothelial modulators in diabetes-induced hyperreactivity of the rabbit carotid artery to endothelin-1

The influence of diabetes on regulatory mechanisms and specific receptors implicated in the contractile response of isolated rabbit carotid arteries to endothelin-1 was examined. Endothelin-1 induced a concentration-dependent contraction that was greater in arteries from diabetic rabbits than in arteries from control rabbits. Endothelium removal or N(G)-nitro-L-arginine enhanced contractions in response to endothelin-1 only in control arteries, without modifying the endothelin-1 response in diabetic arteries. Indomethacin, furegrelate (thromboxane A(2) inhibitor), or cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123; endothelin ET(A) receptor antagonist) inhibited the contractions in response to endothelin-1, the inhibition being greater in diabetic arteries than in control arteries. 2,6-Dimethylpiperidinecarbonyl-gamma-methyl-Leu-N(in)-(methoxycarbonyl)-D-Trp-D-Nle (BQ-788; endothelin ET(B) receptor antagonist) enhanced the contraction elicited by endothelin-1 in control arteries and displaced to the right the contractile curve for endothelin-1 in diabetic arteries. In summary, diabetes induces hyperreactivity of the rabbit carotid artery to endothelin-1 by a mechanism that at least includes: (1) enhanced activity of muscular endothelin ET(A) receptors; (2) impairment of endothelin ET(B) receptor-mediated nitric oxide (NO) release; and (3) enhancement of the production of thromboxane A(2).     

Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries

1. Endothelin-1 (ET-1) produces constriction of the rat mesenteric vascular bed in vivo via ET_A and ET_B receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium-denuded, small mesenteric arteries, under pressure, by use of ET-1; the ET_A receptor antagonist, BQ-123; the ET_B receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ET_B receptor selective antagonist, BQ-788; and the ET_A/ET_B antagonist, TAK-044.
2. In 3rd generation mesenteric arteries, ET-1 (10⁻¹³10⁻⁷ M) produced concentration-dependent contractions (pD₂ 9.86). SRTX S6c (10⁻¹²10⁻⁷ M) also induced concentration-dependent contractions in 53% of arteries studied, although the E_max was much less than that obtained with ET-1 (10.7±2.9% vs 101.9±2.6% of the 60 mM KCl-induced contraction).
3. Neither ET_B receptor desensitization, by a supra-maximal concentration of SRTX S6c (10⁻⁷ M), nor incubation with BQ-788 (3×10⁻⁸ M), had any significant effect on the ET-1 concentration-response curve, although both treatments tended to enhance rather than inhibit responses to ET-1.
4. In the presence of BQ-123 (10⁻⁶ M), responses to low concentrations of ET-1 (up to 10⁻¹⁰ M) were unaffected but responses to concentrations of ET-1 above 10⁻¹⁰ M were significantly inhibited.
5. SRTX S6c desensitization followed by incubation with BQ-123 (10⁻⁶ M) or co-incubation with BQ-788 (3×10⁻⁸ M) and BQ-123 caused inhibition of responses to all concentrations of ET-1, resulting in a rightward shift of the ET-1 concentration-response curve. The same effect was obtained by incubation with TAK-044 (10⁻⁸ M and 3×10⁻⁷ M).
6. Thus, responses of rat small mesenteric arteries to ET-1 are mediated by both ET_A and ET_B receptors. The relative role of ET_B receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET-1-induced contraction to ET_B receptor desensitization or BQ-788. The effect of ET_B receptor desensitization or blockade is only revealed in the presence of ET_A receptor blockade, suggesting the presence of a ‘crosstalk'' mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK-044, may be required to inhibit completely constrictor responses to ET-1.

     

Effects of an endothelin B receptor agonist on secretory phospholipase A2-IIA-induced apoptosis in cortical neurons

Endothelin (ET), a vasoconstrictive peptide, acts as an anti-apoptotic factor, and endothelin receptor B (ETB receptor) is associated with neuronal survival in the brain. Human group IIA secretory phospholipase A2 (sPLA2-IIA) is expressed in the cerebral cortex after brain ischemia and causes neuronal cell death via apoptosis. In primary cultures of rat cortical neurons, we investigated the effects of an ETB receptor agonist, ET-3, on sPLA2-IIA-induced cell death. sPLA2-IIA caused neuronal cell death in a concentration- and time-dependent manner. ET-3 significantly prevented neurons from undergoing sPLA2-IIA-induced cell death. These agonists reversed sPLA2-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Before cell death, sPLA2-IIA potentiated the influx of Ca2+ into neurons. Blockers of the L-type voltage-dependent calcium channel (L-VSCC) not only suppressed the Ca2+ influx, but also exhibited neuroprotective effects. As well as L-VSCC blockers, ET-3 significantly prevented neurons from sPLA2-IIA-induced Ca2+ influx. An ETB receptor antagonist, BQ788, inhibited the effects of ET-3. The present cortical cultures contained few non-neuronal cells, indicating that the ETB receptor agonist affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that the ETB receptor agonist rescues cortical neurons from sPLA2-IIA-induced apoptosis. Furthermore, the present study suggests that the inhibition of L-VSCC contributes to the neuroprotective effects of the ETB receptor agonist.     

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist,in normal and diseased human tissues

1. We have synthesized a new low molecular weight, non-peptide radioligand, [¹²⁵I]-PD164333, an analogue of the orally active butenolide antagonists of the endothelin ET_A receptor.
2. Analysis of saturation binding assays demonstrated that [¹²⁵I]-PD164333 bound with high affinity to a single population of receptors (n⩾3 individuals ±s.e.mean) in human aorta (K_D=0.26±0.08 nM; B_max=8.8±3.95 fmol mg^-1 protein), left ventricle from the heart (K_D=0.16±0.02 nM; B_max=34.2± 3.02 fmol mg^-1 protein) and kidney (K_D=1.24±0.16 nM; B_max=125.3±35.07 fmol mg^-1 protein). In each case Hill slopes were close to unity.
3. In kinetic experiments, the binding of [¹²⁵I]-PD164333 to ET_A receptors in sections of heart was time-dependent and rapid at 23°C. The data were fitted to a one site model, with an association rate constant (K₁ of 2.66±0.213×10⁸ M^-1 min^-1, and a half-time for association of 11 min. The binding was reversible at 23°C: analysis of the data indicated [¹²⁵I]-PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031±0.0004 min^-1, a half-time for dissociation of 216 min and a K_D calculated from these kinetic data of 0.01 nM.
4. Unlabelled PD164333 inhibited the binding of [¹²⁵I]-ET-1 to left ventricle (which expresses both subtypes) in a biphasic manner with a K_DET_A of 0.99±0.32 nM and K_DET_B of 2.41±0.22 μM, giving a selectivity of 2500 fold. ET_A-selective ligands competed monophasically for [¹²⁵I]-PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, K_D=3.93 ±0.18 nM; {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 K_D=3.53±0.69 nM. In contrast, the ET_B selective agonists, BQ3020 and sarafotoxin S6c (1 μM) did not inhibit binding.
5. In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin-1 (ET-1) concentration-response curve (pA₂=8.84) and a slope of unity.
6. In the human brain, autoradiography revealed high levels of [¹²⁵I]-PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [¹²⁵I]-PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion.
7. These results show [¹²⁵I]-PD164333 is a specific, high affinity, reversible non-peptide radioligand for human ET_A receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

     

Essential role for endothelin ETB receptors in fever induced by LPS (E. coli) in rats

1. The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and endothelin-1 (ET-1) was assessed in conscious rats.
2. Intravenous (i.v.) LPS (5.0 μg kg⁻¹) markedly increased rectal temperature to a peak of 1.30°C over baseline at 2.5 h. Pretreatment with the mixed endothelin ET_A/ET_B receptor antagonist bosentan (10 mg kg⁻¹, i.v.) or the selective endothelin ET_B receptor antagonist BQ-788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-γ-methylleucyl-D-1-methoxycarboyl-D-norleucine; 3 pmol, into a lateral cerebral ventricle–i.c.v.) reduced the peak response to LPS to 0.90 and 0.75°C, respectively. The selective endothelin ET_A receptor antagonist BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]; 3 pmol, i.c.v.) was ineffective.
3. Increases in temperature caused by IL-1β (180 fmol, i.c.v.), TNF-α (14.4 pmol, i.c.v.) or IL-1β (150 pmol kg⁻¹, i.v.) were unaffected by BQ-788 (3 pmol, i.c.v.).
4. Central injection of endothelin-1 (0.1 to 3 fmol, i.c.v.) caused slowly-developing and long-lasting increases in rectal temperature (starting 2 h after administration and peaking at 4–6 h between 0.90 and 1.15°C) which were not clearly dose-dependent. The response to endothelin-1 (1 fmol, i.c.v.) was prevented by BQ-788, but not by BQ-123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo-oxygenase inhibitor indomethacin (2 mg kg⁻¹), which partially reduced LPS-induced fever, did not modify the hyperthermic response to endothelin-1 (3 fmol, i.c.v.).
5. Therefore, central endothelin(s) participates importantly in the development of LPS-induced fever, via activation of a prostanoid-independent endothelin ET_B receptor-mediated mechanism possibly not situated downstream from IL-1β or TNF-α in the fever cascade.

     

Role of the endothelin axis and its antagonists in the treatment of cancer

The endothelins (ET) are a group of proteins that act through G-protein coupled receptors. Endothelin-1 (ET-1) was initially identified as a potent vasoconstrictor and dysregulation of the ET axis contributes to pathological processes responsible for cardiovascular disease states. More recently, the ET axis, in particular ET-1 acting through the endothelin A receptor (ET(A) ), has been implicated in the development of several cancers through activation of pathways involved in cell proliferation, migration, invasion, epithelial-mesenchymal transition, osteogenesis and angiogenesis. The endothelin B receptor (ET(B) ) may counter tumour progression by promoting apoptosis and clearing ET-1; however, it has recently been implicated in the development of some tumour types including melanomas and oligodendrogliomas. Here, we review emerging preclinical and clinical data outlining the role of the ET axis in cancer, and its antagonism as an attractive and challenging approach to improve clinical cancer management. Clinical data of ET(A) antagonists in patients with prostate cancer are encouraging and provide promise for new ET(A) antagonist-based treatment strategies. Given the unexpected opportunities to affect pleiotrophic tumorigenic signals by targeting ET(A)-mediated pathways in a number of cancers, the evaluation of ET-targeted therapy in cancer warrants further investigation.     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured.
2. ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg.
3. The selective ET_B receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET_B receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively.
4. Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1.
5. These results demonstrate that in the isolated guinea-pig trachea activation of ET_B receptors results in a contractile response, whereas activation of ET_A receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

     

Elevated plasma endothelin-1 level in streptozotocin-induced diabetic rats and responsiveness of the mesenteric arterial bed to endothelin-1

1. Both the plasma endothelin-1 (ET-1) levels and the plasma glucose levels were markedly elevated in streptozotocin (STZ)-induced diabetic rats.
2. The maximum contractile response of the mesenteric arterial bed to ET-1 was significantly reduced, and the vasodilatation induced by the ET_B-receptor agonist IRL-1620 in the mesenteric arterial bed was significantly reduced in STZ-induced diabetic rats.
3. ET-1 (10⁻⁸ M) caused a transient vasodilatation followed by a marked vasoconstriction in methoxamine-preconstricted mesenteric arterial beds. The ET-1-induced vasodilatation was significantly larger in beds from diabetic rats than in those from age-matched controls. By contrast, the ET-1-induced vasoconstriction was significantly smaller in STZ-induced diabetic rats than in the controls.
4. Both removal of the endothelium with Triton X-100 and preincubation with BQ-788 (10⁻⁶ M) (ET_B-receptor antagonist) abolished the ET-1-induced vasodilatation. Preincubation with BQ-485 (10⁻⁶ M) or BQ-123 (3×10⁻⁶) (ET_A-receptor antagonist) significantly augmented the ET-1-induced vasodilatation in control mesenteric arterial beds, but not that in beds from diabetic rats.
5. These results demonstrate that marked increases not only in plasma glucose, but also in plasma ET-1 occur in STZ-induced diabetic rats. We suggest that the decreased contractile response and the increased vasodilator response of the mesenteric arterial bed to ET-1 may both be due to desensitization of ET_A receptors, though ET_B receptors may also be desensitized. This desensitization may result from the elevation of the plasma ET-1 levels seen in STZ-induced diabetic rats.

     

Increased endothelin-1 vasoconstriction in mesenteric resistance arteries after superior mesenteric ischaemia-reperfusion

BACKGROUND AND PURPOSE

Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1.

EXPERIMENTAL APPROACH

SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24 h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O₂^•−) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively.

KEY RESULTS

I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ET_B mRNA expression, and down-regulated ET_A mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ET_B receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ET_B receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ET_B receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, N^ω-nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O₂^•− production by increasing NADPH oxidase 1 (NOX-1) and p^47phox mRNA.

CONCLUSIONS AND IMPLICATIONS

Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ET_B receptors and decrease in NO bioavailability.     

Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.     

Participation of endogenous endothelin and ETA receptor in premicturition contractions in rats with bladder outlet obstruction

A relationship between endogenous endothelins and bladder overactivity has recently been suggested, but the related endothelin receptor subtype has not been identified. Here, to evaluate the involvement of endothelin-1 and its receptors in bladder overactivity, we investigated endothelin-1 levels and the expression of its receptors in the bladder of rats with bladder outlet obstruction (BOO), a model for bladder overactivity. We also investigated the effects of a selective endothelin ET_A receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2′-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on bladder functions in conscious BOO rats. Partial obstruction of the urethra led to a progressive increase in bladder weight from weeks 1 to 6. Binding assays performed using plasma membranes prepared from these bladders to estimate endothelin receptor density from the maximum [¹²⁵I]endothelin-1 binding showed increased endothelin receptor density (about double) at 1, 2, and 6 weeks after the operation in the BOO bladder. The densities of endothelin ET_A receptors in the bladder of sham-operated and BOO rats at 2 weeks after operation were about 3.5 and 5 times those of endothelin ET_B receptors respectively. Furthermore, the endothelin-1 level was also increased in the BOO bladder. Two weeks after operation, BOO rats showed an increase in maximum bladder capacity and micturition volume and the generation of premicturition contractions. The frequency of premicturition contractions was dose-dependently reduced by YM598 (0.1–3 mg/kg, i.v.) without any effect on other voiding parameters in BOO rats. These data suggest that endothelin-1 and endothelin ET_A receptors might be involved in the generation of premicturition contractions in BOO rats, and that endothelin ET_A receptor antagonists such as YM598 may have ameliorating effects in patients with bladder overactivity associated with BOO.     

Renal protective effect of YM598, a selective endothelin ET(A) receptor antagonist,against diabetic nephropathy in OLETF rats

We have investigated the effect of potassium (E)-N-[6-methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]-2-phenylenthenesulfonamidate (YM598), a selective endothelin ET(A) receptor antagonist, on renal function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type II diabetes. YM598 (0.1 or 1 mg kg(-1)), enalapril (5 mg kg(-1)), an angiotensin-converting enzyme inhibitor, or vehicle was administered once daily by gastric gavage to 22-week-old male Otsuka Long-Evans Tokushima Fatty rats for 32 weeks. Enalapril but not YM598 mildly lowered blood pressure in the diabetic rats. YM598 blunted the development of albuminuria in a dose-dependent manner. High dose of YM598 reduced albuminuria comparable to enalapril. Urinary endothelin-1 excretion was greater in the diabetic than in the control rats, and was not substantially influenced by the agents. These data suggest that endothelin is involved in the progression of diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty rats, and an endothelin ET(A) receptor antagonist may be useful for the treatment of diabetic nephropathy.     

Role of endothelin ET(B) receptors in the renal hemodynamic and excretory responses to big endothelin-1

We determined the role of endothelin ET(B) receptor in the renal hemodynamic and excretory responses to big endothelin-1, using A-192621, a selective endothelin ET(B) receptor antagonist and the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the endothelin ET(B) receptor gene. An intravenous injection of big endothelin-1 produced a hypertensive effect, which is greater in wild-type (+/+) rats pretreated with A-192621 and in homozygous (sl/sl) rats. Big endothelin-1 markedly increased urine flow, urinary excretion of sodium and fractional excretion of sodium in wild-type rats treated with the vehicle. These excretory responses to big endothelin-1 were markedly reduced by pharmacological endothelin ET(B) receptor blockade. On the other hand, big endothelin-1 injection to the endothelin ET(B) receptor-deficient homozygous animals resulted in a small diuretic effect. When renal perfusion pressure was protected from big endothelin-1-induced hypertension by an aortic clamp, the excretory responses in vehicle-treated wild-type rats were markedly attenuated. In homozygous or A-192621-treated wild-type rats, there was a small but significant decreasing effect in urine flow. In addition, big endothelin-1 significantly elevated nitric oxide (NO) metabolite production in the kidney of wild-type rats but not in the homozygous rats. We suggest that the diuretic and natriuretic responses to big endothelin-1 consist of pressure-dependent and pressure-independent effects and that the increased NO production via the activation of endothelin ET(B) receptors in the kidney is closely related to the big endothelin-1-induced excretory responses.     

Noradrenaline release from rat sympathetic neurones triggered by activation of B2 bradykinin receptors

1. The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [³H]-noradrenaline and the outflow of radioactivity was determined under continuous superfusion.
2. Bradykinin (100 nmol l⁻¹ applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 μmol l⁻¹) and nicotine (10 μmol l⁻¹) caused elevations in ³H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm⁻¹, 1.0 Hz).
3. When bradykinin was applied for periods of 2 min, the evoked ³H overflow was half-maximal at 12 nmol l⁻¹ and reached a maximum of 2.3% of cellular radioactivity. The preferential B₁ receptor agonist des-Arg⁹-bradykinin failed to alter ³H outflow. The B₂ receptor antagonists, [D-Phe⁷]-bradykinin (1 μmol l⁻¹) and Hoe 140 (10 nmol l⁻¹), per se did not alter ³H outflow, but shifted the concentration-response curve for bradykinin-evoked ³H overflow to the right by a factor of 7.9 and 4.3, respectively.
4. Bradykinin-induced overflow was abolished in the absence of extracellular Ca²⁺ and in the presence of either 1 μmol l⁻¹ tetrodotoxin or 300 μmol l⁻¹ Cd²⁺, as was electrically-induced overflow. Activation of α₂-adrenoceptors by 1 μmol l⁻¹ UK 14,304 reduced both bradykinin- and electrically-triggered overflow. The Ca²⁺-ATPase inhibitor thapsigargin (0.3 μmol l⁻¹) failed to alter either type of stimulated overflow. Caffeine (10 mmol l⁻¹) enhanced bradykinin-induced overflow, but reduced overflow triggered by electrical field stimulation.
5. Inclusion of Ba²⁺ (0.1 to 1 mmol l⁻¹) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin-triggered overflow by almost 400%. Application of 1 mmol l⁻¹ Ba²⁺ for periods of 2 min triggered ³H overflow, and this overflow was abolished by 1μmol l⁻¹ tetrodotoxin and enhanced by 10 mmol l⁻¹ caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l⁻¹) in the superfusion buffer caused similar increases of bradykinin- and electrically evoked ³H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter ³H outflow.
6. Treatment of cultures with 100 ng ml⁻¹ pertussis toxin caused a significant increase in bradykinin-, but not in electrically-, evoked tritium overflow. Treatment with 100 ng ml⁻¹ cholera toxin reduced both types of stimulated ³H overflow.
7. These data reveal bradykinin as a potent stimulant of action potential-mediated and Ca²⁺-dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B₂-receptors, presumably linked to pertussis- and cholera toxin-insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K⁺ (K_M) channels, like caffeine and Ba²⁺, indicate that the secretagogue action of bradykinin probably involves inhibition of these K⁺ channels.

     

Stimulation of catecholamine biosynthesis via the protein kinase C pathway by endothelin-1 in PC12 rat pheochromocytoma cells

It has been reported that endothelins (ETs) stimulate catecholamine release from chromaffin cells. However, it is not known whether ETs also affect catecholamine biosynthesis. Thus, using a rat pheochromocytoma cell line, PC12, we examined the effects of ETs on catecholamine biosynthesis. The mRNA level and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis, were increased significantly by endothelin-1 (ET-1) (100nM). These stimulatory effects were inhibited completely by a blocker for the A-type endothelin receptor, BQ-123 [cyclo(D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)] (1 microM), but not by a blocker for the B-type endothelin receptor, BQ-788 (N-cis 2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (1 microM). Also, Ro-32-0432 (3-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido-[1,2-a]indol-10-yl]-4-(1-methyl-3-indolyl)-H-pyrrole-2,5-dione hydrochloride) (100nM), a protein kinase C inhibitor, completely inhibited ET-1-induced increases in TH activity and mRNA level. Furthermore, ET-1 (100nM) significantly stimulated protein kinase C activity, as well as inositol 1,4,5-triphosphate production; these stimulatory effects were abolished by BQ-123 but not by BQ-788. Moreover, ET-1 (100nM) significantly increased both the TH-protein level and the intracellular catecholamine content. By contrast to ET-1, endothelin-3 did not affect catecholamine synthesis. These results indicate that ET-1, but not ET-3, stimulates catecholamine synthesis through the PKC pathway in PC12 cells. Also, the use of selective ET receptor antagonists suggests that the effects of ET-1 on catecholamine biosynthesis are mediated through ET(A).     

Block and allosteric modulation of GABAergic currents by oenanthotoxin in rat cultured hippocampal neurons

Background and purpose:

Oenanthotoxin (OETX), a polyacetylenic alcohol from plants of the genus Oenanthe, has recently been identified as potent inhibitor of GABA-evoked currents. However, the effects of OETX on the inhibitory postsynaptic currents (IPSCs), as well as the pharmacological mechanism(s) underlying its effects on GABA_A receptors, remain unknown. The purpose of this study was to elucidate the mechanism underlying the inhibition of GABAergic currents by OETX.

Experimental approach:

Effects of OETX on GABAergic currents were studied using the patch clamp technique on rat cultured hippocampal neurons. Miniature IPSCs (mIPSCs) were recorded in the whole-cell configuration, while the current responses were elicited by ultrafast GABA applications onto the excised patches.

Key results:

OETX potently inhibited both mIPSCs and current responses, but its effect was much stronger on synaptic currents. Analysis of the effects of OETX on mIPSCs and evoked currents disclosed a complex mechanism: allosteric modulation of both GABA_A receptor binding and gating properties and a non-competitive, probably open channel block mechanism. In particular, OETX reduced the binding rate and nearly abolished receptor desensitization. A combination of rapid clearance of synaptic GABA and OETX-induced slowing of binding kinetics is proposed to underlie the potent action of OETX on mIPSCs.

Conclusions and implications:

OETX shows a complex blocking mechanism of GABA_A receptors, and the impact of this toxin is more potent on mIPSCs than on currents evoked by exogenous GABA. Such effects on GABAergic currents are compatible with the convulsions and epileptic-like activity reported for OETX.