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 reactivity and endothelial dysfunction in carotid arteries from rats with hyperhomocysteinemia

Background and purpose:

There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries.

Experimental approach:

Vascular reactivity to ET-1 and ET_A and ET_B receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ET_A and ET_B receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [¹²⁵I]-ET-1.

Key results:

HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ET_A or ET_B receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ET_A but not of ET_B receptors abolished enhancement in HHcy tissues. ET_A and ET_B receptor gene expressions were not up-regulated. ET_A receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A₂ receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO.

Conclusions and implications:

Increased ET_A receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ET_A receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility.British Journal of Pharmacology (2009) 157, 568–580; doi:10.1111/j.1476-5381.2009.00165.x; published online 9 April 2009This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Endothelin-1 and endothelin-2 initiate and maintain contractile responses by different mechanisms in rat mesenteric and cerebral arteries

BACKGROUND AND PURPOSE

Endothelin (ET)-1 and ET-2 cause potent long-lasting vasoconstrictions by tight binding to smooth muscle ET_A receptors. We tested the hypotheses that different mechanisms mediate initiation and maintenance of arterial contractile responses to ET-1 and ET-2 and that this differs among vascular beds.

EXPERIMENTAL APPROACH

Segments of rat mesenteric resistance artery (MRA) and basilar artery (BA) were studied in wire myographs with and without functional antagonists.

KEY RESULTS

Sensitivity and maximum of MRA contractile responses to ET-1 were not, or only moderately, reduced by stimulation of soluble GC, AC or K⁺-channels and by an inhibitor of receptor-operated ion channels. However, each of these reduced maintenance of ET-1 effects and relaxed ET-1-induced contractions in MRA. A calcium channel antagonist did not alter sensitivity, maximum and maintenance of ET-1 effects, but relaxed ET-1-induced contractions in MRA. A PLC inhibitor prevented contractile responses to ET-1 and ET-2 in MRA and BA, and relaxed ET-1- and ET-2-induced responses in MRA and ET-1 effects in BA. A Rho-kinase inhibitor did not modify sensitivity, maximum and maintenance of responses to both peptides in both arteries but relaxed ET-2, but not ET-1, effects in MRA and ET-1 effects in BA.

CONCLUSIONS AND IMPLICATIONS

PLC played a key role in arterial contractile responses to ETs, but ET-1 and ET-2 initiated and maintained vasoconstriction through different mechanisms, and these differed between MRA and BA. Selective functional antagonism may be considered for agonist- and vascular bed selective pharmacotherapy of ET-related diseases.     

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.

     

Chronic ethanol intake modulates vascular levels of endothelin-1 receptor and enhances the pressor response to endothelin-1 in anaesthetized rats

BACKGROUND AND PURPOSE: The contribution of endothelin-1 (ET-1) to vascular hyper-reactivity associated with chronic ethanol intake, a major risk factor in several cardiovascular diseases, remains to be investigated. EXPERIMENTAL APPROACH: The biphasic haemodynamic responses to ET-1 (0.01-0.1 nmol kg(-1), i.v.) or to the selective ETB agonist, IRL1620 (0.001-1.0 nmol kg(-1), i.v.), with or without ETA or ETB antagonists (BQ123 (c(DTrp-Dasp-Pro-Dval-Leu)) at 1 and 2.5 mg kg(-1) and BQ788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-gamma-methylleucyl1-D-1methoxycarbonyltryptophanyl-D-norleucine) at 0.25 mg kg(-1), respectively) were tested in anaesthetized rats, after 2 weeks' chronic ethanol treatment. Hepatic parameters and ET receptor protein levels were also determined. KEY RESULTS: The initial hypotensive responses to ET-1 or IRL1620 were unaffected by chronic ethanol intake, whereas the subsequent pressor effects induced by ET-1, but not by IRL1620, were potentiated. BQ123 at 2.5 but not 1 mg kg(-1) reduced the pressor responses to ET-1 in ethanol-treated rats. Conversely, BQ788 (0.25 mg kg(-1)) potentiated ET-1-induced increases in mean arterial blood pressure in control as well as in ethanol-treated rats. Interestingly, in the latter group, increases in heart rate, induced by ET-1 at a dose of 0.025 mg kg(-1) were enhanced following ETB receptor blockade. Finally, we observed higher levels of ETA receptor in the heart and mesenteric artery and a reduction of ETB receptor protein levels in the aorta and kidney from rats chronically treated with ethanol. CONCLUSIONS AND IMPLICATIONS: Increased vascular reactivity to ET-1 and altered protein levels of ETA and ETB receptors could play a role in the pathogenesis of cardiovascular complications associated with chronic ethanol consumption.     

Involvement of ETA and ETB receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

1. This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [³²P]phosphatidylbutanol in [³²P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol.
2. The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal effects evoked by the ET_B-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selective agonist ET-1.
3. The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET_A receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2±3.5% of the ET-1 response) was defined at low (up to 1 μM) concentrations of BQ-123, yielding an estimated K_i value for BQ-123 of 21.3±2.5 nM. In addition, the presence of 1 μM BQ-123 significantly reduced the maximal response to ET-1 but did not change the pD₂ value.
4. Increasing concentrations of the ET_B selective antagonist BQ-788 inhibited the S6c response with a K_i of 17.8±0.8 nM. BQ-788 also inhibited the effect of ET-1, although, in this case, two components were defined, accounting for approximately 50% of the response, and showing K_i values of 20.9±5.1 nM and 439±110 nM, respectively. The ET-1 concentration-response curve was shifted to the right by 1 μM BQ-788, also revealing two components. Only one of them, corresponding to 69.8±4.4% of the response, was sensitive to BQ-788 which showed a K_i value of 28.8±8.9 nM.
5. Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither ET-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123.
6. In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET_A and ET_B receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET_A and ET_B receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET_B receptors were not blocked, and 30–50% and 50–70%, respectively, when ET_B receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.

     

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).     

Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric arteries

1. We have studied the effect of endothelin-1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist.
2. Endothelin-1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration-dependent (1–100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α-toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin-1-induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin A1.
3. In intact small arteries tyrphostin A23 inhibited the force developed to endothelin-1 at all concentrations studied; at higher concentrations (10 and 100 nM) the profile of contraction was altered from a sustained to a transient response. Tyrphostin A1 inhibited the contractile response to endothelin-1 at all concentrations except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endothelin-1 (100 nM) in the absence of extracellular calcium.
4. In rat α-toxin permeabilized mesenteric arteries endothelin-1 caused a concentration-dependent increase in force in the presence of 10 μM GTP and low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin A1 did not affect endothelin-1-induced calcium sensitization of force.
5. We conclude that increased tyrosine phosphorylation is important in the contractile response induced by endothelin-1 in intact small mesenteric arteries. Furthermore our data implicate activation of this signalling pathway in the tonic phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.

     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

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.     

Agonist-dependent modulation of arterial endothelinA receptor function

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) causes long-lasting vasoconstrictions. These can be prevented by ET(A) receptor antagonists but are only poorly reversed by these drugs. We tested the hypothesis that endothelin ET(A) receptors are susceptible to allosteric modulation by endogenous agonists and exogenous ligands. EXPERIMENTAL APPROACH Rat isolated mesenteric resistance arteries were pretreated with capsaicin and studied in wire myographs, in the presence of L-NAME and indomethacin to concentrate on arterial smooth muscle responses. KEY RESULTS Endothelins caused contractions with equal maximum but differing potency (ET-1 = ET-2 > ET-3). ET-1(1-15) neither mimicked nor antagonized these effects in the absence and presence of ET(16-21). 4(Ala) ET-1 (ET(B) agonist) and BQ788 (ET(B) antagonist) were without effects. BQ123 (peptide ET(A) antagonist) reduced the sensitivity and relaxed the contractile responses to endothelins. Both effects depended on the agonist (pK(B): ET-3 = ET-1 > ET-2; % relaxation: ET-3 = ET-2 > ET-1). Also, with PD156707 (non-peptide ET(A) antagonist) agonist-dependence and a discrepancy between preventive and inhibitory effects were observed. The latter was even more marked with bulky analogues of BQ123 and PD156707. CONCLUSIONS AND IMPLICATIONS These findings indicate allosteric modulation of arterial smooth muscle ET(A) receptor function by endogenous agonists and by exogenous endothelin receptor antagonists. This may have consequences for the diagnosis and pharmacotherapy of diseases involving endothelins.     

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.     

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.     

The effect of endothelin-2 (ET-2) on migration and changes in cytosolic free calcium of neutrophils

The effect of endothelin-2 (ET-2) on neutrophil migration and intracellular calcium was studied. Depending on the concentration, ET-2 enhanced or inhibited neutrophil migration. At low concentrations ET-2 caused a chemotactic stimulation of migration, in contrast with endothelin-1 (ET-1) which caused a chemokinetic stimulation of migration. At higher concentrations ET-2 inhibited formyl-methionylleucyl-phenylalanine(fMLP)-activated migration. Both activation and inhibition by ET-2 were completely dependent on extracellular Ca²⁺. Unlike ET-1 which caused an increase in cytosolic free Ca²⁺ at a concentration which stimulated migration, ET-2 caused a measurable increase of cytosolic free Ca²⁺ at a concentration which did not stimulate migration. This strongly suggests that there is no correlation between maximal stimulation of cytoplasmic free calcium, and maximal stimulation of migration. Influx of extracellular Ca²⁺ was required for both activation of migration and change in cytosolic free Ca⁺, because no effect was observed in the absence of extracellular Ca⁺, and because blockers of Ca²⁺-influx inhibited ET-2-activated migration. The ET_A-receptor antagonist cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), and the ET_B-receptor antagonist [Cys¹¹-Cys¹⁵]-endothelin-1(11–21) (IRL1038) antagonized the stimulatory effect of ET-2 on migration, and the inhibitory effect of high concentrations of ET-2 on fMLP-activated chemotaxis. This suggests that both the ET_A-receptor and the ET_B-receptor are involved in the stimulatory effect of low concentrations of ET-2, and in the inhibitory effect of high concentrations of ET-2.     

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).     

Impairment of both nitric oxide-mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes

BACKGROUND AND PURPOSE

To investigate whether diabetes affects either or both nitric oxide (NO)-mediated and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in endothelium-dependent relaxation of mesenteric arteries from streptozotocin-induced diabetic rats.

EXPERIMENTAL APPROACH

Wire myography was employed to examine endothelial function of mesenteric arteries. Superoxide levels were measured by L-012 and lucigenin-enhanced chemiluminescence. Western blotting was used to quantify protein expression levels.

KEY RESULTS

Superoxide levels were significantly increased in diabetic mesenteric arteries compared with normal arteries. Diabetes significantly reduced the sensitivity to the endothelium-dependent relaxant, acetylcholine (ACh) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA) + a soluble guanylate cyclase inhibitor (ODQ), the sensitivity to ACh was significantly decreased in the diabetic arteries compared with normal arteries, indicating an impaired EDHF-type relaxation despite increased expression of intermediate- and small-conductance calcium-activated potassium channels. Conversely, when the contribution of EDHF was inhibited with TRAM-34 + apamin + iberiotoxin, maximum relaxations to ACh were significantly decreased in diabetic compared with normal arteries, suggesting that the contribution of NO was also impaired by diabetes. Basal levels of NO release, indicated by contraction to L-NNA, were also significantly decreased in diabetic arteries. Western blot analysis demonstrated that diabetic arteries had an increased expression of Nox2, decreased pSer⁴⁷³Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling.

CONCLUSION AND IMPLICATIONS

The contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer⁴⁷³Akt and/or eNOS uncoupling.     

Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery

1. The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves.
2. Vasopressin (10⁻¹⁰–10⁻⁷ M) caused concentration-dependent contractions (pD₂=8.36±0.09). The V₁-receptor antagonist d(CH₂)₅Tyr(Me)AVP (10⁻⁹–10⁻⁸ M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA₂ value of 9.83 with a slope of 1.10±0.14.
3. Vasopressin (3×10 ⁻¹⁰ and 10⁻⁹ M) caused concentration-dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration-response curve for noradrenaline. The V₁-receptor antagonist induced concentration-dependent inhibitions of potentiation induced by vasopressin. The selective V₁-receptor agonist [Phe^*, Orn⁸]-vasotocin (3×10 ⁻¹⁰ and 10⁻⁹ M) induced potentiation of electrical stimulation-evoked responses which was also inhibited in the presence of the V₁ antagonist (10⁻⁸ M). In contrast, the V₂-receptor agonist deamino-8-D-arginine vasopressin (desmopressin 10⁻⁸–10⁻⁷ M) did not modify the electrical stimulation-induced responses and the V₂-receptor antagonist [d(CH₂)₅, D-Ile^*, Ile⁴, Arg⁸]-vasopressin (10⁻⁸–10⁻⁷ M) did not affect the potentiation evoked by vasopressin.
4. In artery rings contracted by 10⁻⁶ M noradrenaline in the presence of 10⁻⁶ M guanethidine and 10⁻⁶ M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency-dependent relaxations which were unaffected by 10⁻⁹ M vasopressin but abolished by 10⁻⁶ M tetrodotoxin.
5. Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl₂ to KCl depolarized vessels. The augmenting effects were inhibited by the V₁ antagonist.
6. In the presence of the calcium antagonist nifedipine (10⁻⁶ M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl.
7. The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V₁ receptor stimulation which brings about an increase in calcium entry through dihydropyridine-sensitive calcium channels.

     

Myoendothelial coupling in the mesenteric arterial bed; segmental differences and interplay between nitric oxide and endothelin-1

Background and purpose:

We tested the hypothesis that activated arterial smooth muscle (ASM) stimulates endothelial vasomotor influences via gap junctions and that the significance of this myoendothelial coupling increases with decreasing arterial diameter.

Experimental approach:

From WKY rats, first-, second-, third-and fourth-order branches of the superior mesenteric artery (MA1, MA2, MA3 and MA4 respectively) were isolated and mounted in wire-myographs to record vasomotor responses to 0.16–20 µmol·L⁻¹ phenylephrine.

Key results:

Removal of endothelium increased the sensitivity (pEC₅₀) to phenylephrine in all arteries. The nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) (100 µmol·L⁻¹) did not modify pEC₅₀ to phenylephrine in all denuded arteries, and increased it in intact MA1, MA2 and MA3 to the same extent as denudation. However, in intact MA4, the effect of L-NAME was significantly larger (ΔpEC₅₀ 0.57 ± 0.02) than the effect of endothelium removal (ΔpEC₅₀ 0.20 ± 0.06). This endothelium-dependent effect of L-NAME in MA4 was inhibited by (i) steroidal and peptidergic uncouplers of gap junctions; (ii) a low concentration of the NO donor sodium nitroprusside; and (iii) by the endothelin-receptor antagonist bosentan. It was also observed during contractions induced by (i) calcium channel activation (BayK 8644, 0.001–1 µmol·L⁻¹); (ii) depolarization (10–40 mmol·L⁻¹ K⁺); and (iii) sympathetic nerve stimulation (0.25–32 Hz).

Conclusions and implications:

These pharmacological observations indicated feedback control by endothelium of ASM reactivity involving gap junctions and a balance between endothelium-derived NO and endothelin-1. This myoendothelial coupling was most prominent in distal resistance arteries.