J. Robert McNeill

Heterogeneity in Vascular Smooth Muscle Responsiveness to Angiotensin II: Role of Endothelin

We compared the role of endothelium and of endothelin in mediating the vasoconstrictor responses to angiotensin II (Ang II) in three vascular smooth muscle preparations--aorta, mesenteric artery, and tail artery--isolated from adult male Sprague-Dawley rats. The vasoconstrictor potency for Ang II in blood vessels with endothelium varied in the following rank order: aorta > mesenteric artery > tail artery. Although the maximal tension responses to Ang II were similar for mesenteric and tail arteries, it was significantly lower in aorta. Endothelium removal led to a leftward shift in the concentration-response curves to Ang II in the aorta but a rightward shift in the mesenteric artery. Strikingly, Ang II failed to evoke tension responses in tail artery in the absence of endothelium. The endothelin-A (ETA)-selective antagonist BQ-123 blocked the responses to Ang II in a noncompetitive manner, with partial and complete attenuation of responses in the endothelium-intact mesenteric and tail artery preparations, respectively. In contrast, BQ-123 did not affect the responses to Ang II in the aorta. BQ-123 also failed to affect the responses to Ang II in endothelium-denuded mesenteric artery rings. The Ang II type 1 (AT1) receptor-selective antagonist losartan competitively blocked the responses to Ang II in the three tissues (pA2, 8.3 to 8.7) when endothelium was present. These data suggest that there are endothelium-dependent regional variations in vascular tissue sensitivity to Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of an Endothelin Antagonist on Hemodynamic Responses to Angiotensin II

We determined changes in blood pressure, cardiac output, and total peripheral conductance evoked by intravenous infusions of angiotensin II (Ang II) in conscious, unrestrained normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) before and after pretreatment with bosentan, a nonselective endothelin antagonist. Blood pressure was recorded by radiotelemetry and cardiac output by ultrasonic transit-time flow probes. Bosentan per se failed to affect basal blood pressure and evoked only small changes in cardiac output and total peripheral conductance in both strains. The pressor effects of Ang II were exaggerated in SHR compared with WKY. Strikingly, bosentan pretreatment blunted the increases in blood pressure, the fall in cardiac output, and the decreases in conductance evoked by lower doses of Ang II but not higher doses of the peptide. This effect was observed in both rat strains but was more pronounced in SHR. These data suggest that endothelin contributes to the hemodynamic effects of Ang II in both SHR and WKY and that endothelin may contribute to the exaggerated pressor responsiveness of SHR to Ang II.

Role of oxidative stress and nitric oxide in regulation of spontaneous tone in aorta of DOCA-salt hypertensive rats

The roles of nitric oxide (NO), superoxide anion (O2−), and hydrogen peroxide (H2O2) in the modulation of spontaneous tone were investigated in isolated aorta from deoxycorticosterone acetate (DOCA)‐salt hypertensive rats. Increases in preload from 1 to 5 g were accompanied by increases in spontaneous tone in aortic rings from DOCA‐salt hypertensive rats but not from SHAM‐normotensive rats. Tone was higher in endothelium‐denuded aortic rings than in endothelium‐intact vessels. Inhibition of nitric oxide synthase (NOS) with 300 μM NG‐nitro‐L‐arginine methyl ester (L‐NAME) increased spontaneous tone. Basal O2− generation was higher in aortic rings from DOCA‐salt hypertensive rats than in those from SHAM‐normotensive rats. Stretch increased O2− levels even further in the DOCA‐salt group. In rings isolated from DOCA‐salt hypertensive rats, administration of the O2− scavenger, superoxide dismutase (SOD, 150 U ml−1), or the nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase inhibitor, apocynin (100 μM), completely abolished the development of spontaneous tone in endothelium‐intact aortic rings but not in endothelium‐denuded or in L‐NAME‐treated rings. SOD and apocynin decreased the generation of O2− in endothelium‐intact, endothelium‐denuded, and L‐NAME‐treated aortic rings. Oral treatment of DOCA‐salt hypertensive rats with the O2− scavengers, tempol or tiron, or with apocynin for 3 weeks prevented the development of hypertension and abolished the increases in O2− generation and spontaneous tone. Administration of catalase (1000 U ml−1) to aortic rings increased spontaneous tone in vessels from DOCA‐salt hypertensive rats. Administration of the cyclooxygenase (COX) inhibitor, valeroyl salicylate, or the thromboxane/prostaglandin antagonist, SQ 29548, to aortic rings abolished tone. The results suggest that NO plays a major role in preventing the generation of spontaneous tone in isolated aortic rings from DOCA‐salt hypertensive rats. NADPH‐oxidase‐derived O2− enhanced spontaneous tone by inactivating NO. Endogenous H2O2 appears to mitigate the increase in tone. In addition, a COX component may also contribute to spontaneous tone.

Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat

OBJECTIVE Earlier, we reported that high insulin incubation in vitro leads to increased ETA receptor expression in cultured rat aortic smooth muscle cells (Diabetes 1998, 47: 934-944). Our later observation of enhanced endothelin-1 evoked vasoconstriction in aorta from the hyperinsulinemic obese Zucker rat indicated that this interaction might also be relevant in vivo. To further examine the relationship between insulinemia and endothelin, we characterized endothelin receptor expression and endothelin-1 peptide levels in vascular tissues and plasma from young and old obese Zucker rats. METHODS 12 and 40-week-old Zucker obese and lean rats were used. Plasma endothelin-1 levels and endothelin-1 peptide content in the mesenteric artery and in the thoracic aorta were examined by radioimmunoassay. Messenger RNA levels of endothelin-1 peptide and ETA and ETB receptors were examined in the aortic and mesenteric vessels using RT-PCR. RESULTS Obese rats from both age groups had significantly higher plasma levels of insulin (4-10 fold), total cholesterol (2-3 fold), triglycerides (10-fold), and glucose (approximately 1.5 fold) than their lean counterparts. There was a trend toward worsening lipoproteinemia and glycemia, but improved insulinemia with age in the obese rats. In association with these changes, obese rats exhibited attenuated endothelin-1 peptide and preproET-1 mRNA levels, but conversely elevated ETA and ETB receptor mRNA levels in both aortic and mesenteric vessels. CONCLUSION These data suggest that vascular tissue from the metabolically dysregulated obese Zucker rat exhibits attenuated endothelin-1 peptide production and elevated endothelin receptor levels. Since elevated insulin levels have been linked to increased endothelin receptor expression, it is plausible that hyperinsulinemia upregulates endothelin receptors contributing to elevated vasoconstrictor responses to endothelin-1 in this model of obesity and hypertension.

Altered vascular reactivity and KATP channel currents in vascular smooth muscle cells from deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

This study was designed to evaluate the contribution of ATP-dependent potassium (KATP) channels to the changes in vascular reactivity and spontaneous tone observed in vessels isolated from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In phenylephrine preconstricted aortic rings, cromakalim induced concentration-dependent, glibenclamide-sensitive relaxation. The concentration response curve to cromakalim was shifted to the right in DOCA-salt hypertensive rats (EC50: 0.850 ± 0.100 μM) compared with SHAM-normotensive rats (0.108 ± 0.005 μM), and the maximum relaxation (Emax) evoked by cromakalim was significantly lower in aortic rings from the DOCA group (68 ± 2%) compared with the SHAM group (108 ± 5%). The results were similar in endothelium-denuded rings. Spontaneous tone was observed in aortic rings (5 g preload) from DOCA-salt but not SHAM rats. Cromakalim abolished spontaneous tone and the effect was blocked by glibencamide. In whole cell patch clamp studies, increasing extracellular K+ concentrations from 5.4 to 140 mM and the administration of cromakalim evoked dramatic increases in KATP channel currents in aortic cells isolated from SHAM rats. In contrast, in aortic cells from DOCA-salt hypertensive rats, KATP channel currents were either absent or weak in response to challenges by elevated extracellular K+ and by cromakalim. These findings suggest that the function of KATP channels is impaired in smooth muscle cells from aorta of DOCA-salt hypertensive rats, which may contribute to the impaired vasodilatation and spontaneous tone observed in these rats.

Insulin-induced biphasic responses in rat mesenteric vascular bed: role of endothelin.

Abstract—The vasodilatory capacity of insulin has been widely reported, yet some investigators have not noted this effect. Because insulin has been shown to enhance endothelin release, we speculated that endothelin could be attenuating insulin-evoked vasodilation. We examined the effect of ex vivo insulin perfusion on vascular resistance by using the Sprague-Dawley rat mesenteric vascular bed. In methoxamine-preconstricted preparations, insulin (3.0 pmol/L to 10 nmol/L) evoked a concentration-dependent decrease in perfusion pressure (PP) with a maximal response of 42.0±9.2%, whereas continuous exposure to 10 nmol/L insulin induced a 51.8±3.5% relaxation. Further exposure to 10 nmol/L insulin resulted in the generation of endothelin and a subsequent loss of the vasodilatory response. Indomethacin had no effect on vascular responses. The vasodilatory response was significantly inhibited by nitric oxide synthase inhibition (20.5±4.2%;P <0.01) and calcium-activated potassium channel blockade (28.5±3.7%;P <0.05). Endothelial denudation attenuated the vasodilatory component (20.3±7.1%;P <0.01) and altered the biphasic pattern of the response. The decline in insulin-evoked vasodilation was significantly prevented by an endothelin-A antagonist (BQ123), an endothelin-B antagonist (BQ788), and nonselective endothelin blockade with both BQ123 and BQ788. These results demonstrate that the endothelium is intimately involved in regulating the vascular response to insulin. Insulin promotes the release of nitric oxide and endothelium-derived hyperpolarizing factor. During sustained exposure to higher concentrations, this vasodilatory effect is countered by the pathological generation of endothelin. Endothelin receptor blockade facilitates the maintenance of vasodilation despite high insulin concentrations.

Differential effects of phosphoramidon on contractile responses to angiotensin II in rat blood vessels

1 Cumulative concentration‐tension response (C‐R) curves to angiotensin II (AII), big endothelin‐1 (big ET‐1), ET‐1 and arginine vasopressin (AVP) were determined in endothelium intact‐ring preparations of aorta, mesenteric artery and tail artery isolated from adult male Sprague‐Dawley rats in the presence or absence of the neutral metalloprotease inhibitor, phosphoramidon. 2 The order of sensitivity of the three rat vascular smooth muscle preparations to AII, big ET‐1 and ET‐1 was aorta> mesenteric artery> tail artery whereas that for AVP was reversed, namely, tail artery> mesenteric artery> aorta. 3 Phosphoramidon blocked the responses to AII in a concentration‐dependent manner, whereas even very high concentrations of phosphoramidon (100 μm) failed to affect the tension responses evoked by ET‐1 and AVP in all three preparations. Low concentrations of phosphoramidon (10 μm) produced significant increases in EC50 values for AII in tail artery (P<0.01) and mesenteric artery (P<0.05) but not in aorta. The rank order of sensitivity to the inhibition by phosphoramidon was tail artery< mesenteric artery< aorta. Phosphoramidon‐evoked rightward shifts in the C‐R curves to AII were much higher than those to big ET‐1 in both mesenteric artery and tail artery. 4 In endothelium‐denuded preparations, AII failed to evoke any increases in tension in tail artery while the responsiveness of the mesenteric artery to AII was reduced significantly relative to endothelium‐intact tissues with a rightward shift in the C‐R curve and a decrease in the maximal response. On the other hand, the C‐R curve to AII was shifted to the left in aorta following removal of the endothelium. Importantly, ET‐1 and AVP evoked vasoconstrictor responses were unaffected by the inclusion of a high concentration of phosphoramidon (100 μm) in endothelium‐denuded aorta and mesenteric artery. 5 The results suggest that AII‐evoked tension responses of blood vessels such as tail artery are completely endothelium‐dependent; in relatively larger blood vessels such as mesenteric artery they are partially endothelium‐dependent while in much bigger conduit type blood vessel such as aorta, they are endothelium‐independent. It is concluded that the vasoconstrictor responses to AII in mesenteric artery and tail artery may be mediated by the release of endothelins from the endothelium by increased formation from big ET, an effect that is blocked by phosphoramidon.

Role of endothelin and vasopressin in DOCA-salt hypertension.

The relative roles of endothelin (ET) and vasopressin (AVP) in the regulation of blood pressure (BP), cardiac output (CO) and total peripheral resistance (TPR) were investigated in the early stages (24 – 31 days) of development of hypertension in the conscious deoxycorticosterone acetate (DOCA)‐salt hypertensive rat model. BP was recorded with radiotelemetry devices and CO with ultrasonic transit‐time probes. TPR was calculated from the BP and CO recordings. The contributions of endogenous ET and AVP were studied by infusing [d(CH2)51,O‐Me_Tyr2,Arg8]‐vasopressin, a V1‐receptor antagonist, and bosentan, a mixed ETA/ETB receptor antagonist (Study 1). Vascular responsiveness was estimated from the changes in TPR evoked by i.v. infusions of ET‐1 and AVP (Study 2). In study 1, infusion of bosentan reduced TPR and BP dramatically in DOCA‐salt hypertensive rats but not in SHAM control rats, and this effect was greater when the AVP system had been blocked. In contrast, the V1 receptor antagonist alone failed to change TPR and BP in DOCA‐salt hypertensive rats. However, subsequent infusion of the V1 receptor antagonist during the plateau phase of the response in bosentan pretreated DOCA‐salt hypertensive rats led to significant decreases in both BP and TPR. In study 2, TPR and BP responses to ET‐1, but not AVP, were greater in DOCA‐salt rats than in control rats. CO responses to ET‐1 or AVP were similar in the two groups. The results suggest that both ET and AVP play a role in the maintenance of TPR and BP; when one system is blocked the other compensates. However, the magnitude of the contribution to the hypertensive state appears greater for ET than for AVP. Enhanced vascular responses to ET appear to contribute to this greater role.

Vasopressin accelerates protein synthesis in neonatal rat cardiomyocytes

Arginine vasopressin (AVP) has been shown to promote vascular smooth muscle cell hypertrophy and hyperplasia of fibroblasts. The present study examines the effect of AVP and endothelin-1 (ET-1) on protein, DNA, and RNA synthesis in primary cultures of serum deprived neonatal rat cardiomyocytes (RC) as assessed by changes in [3H] phenylalanine, [3H] thymidine, and [14C] uridine incorporation respectively. Both AVP and ET-1 evoked significant increases in protein synthesis in RC of 36 ± 12% (p < 0.05) and 53 ± 22% (p < 0.01) respectively. The stimulating action of AVP on [3H] phenylalanine incorporation was abolished by pretreatment with 2-nitro-4carboxyphenyl-N, N-diphenylcarbamate (NCDC), a phospholipase C (PLC) inhibitor. [14C] uridine incorporation was significantly higher in cells incubated with ET-1 (95 ± 12%) but not AVP (9 ± 11%). Neither AVP nor ET-1 significantly affected cell number or [3H] thymidine incorporation, suggesting a lack of a hyperplastic effect. AVP evoked an increase in [Ca2+]i levels (162 ± 12 nmol/L from a basal value of 77 ± 6 nmol/L) which was completely abolished by pretreatment with either NCDC or cyclopiazonic acid (sarcoplasmic reticulum (SR) Ca2+ pump inhibitor) but unaffected by ryanodine (ryanodine sensitive SR Ca2+ store depletor). Taken together, these data suggest that AVP, in a PLC dependent manner, stimulates both protein synthesis and augments [Ca2+]i release in RC from ryanodine insensitive (IP3 sensitive) Ca2+ stores. Thus, AVP may promote cardiac hypertrophy via direct effects on cardiomyocyte protein synthesis secondary to IP3 mediated [Ca2+]i release.

Angiotensin II elevates cytosolic free calcium in human lung adenocarcinoma cells via activation of AT1 receptors

Angiotensin II (Ang II), bradykinin (BK), and endothelin-1 (ET-1) evoked alterations in cytosolic free calcium, [Ca2+]i, levels were determined using fura-2 fluorescence methodology in a human lung adenocarcinoma cell line (A549), a non-neoplastic lung cell line and a small cell lung carcinoma cell (SCLC) line. Ang II and BK evoked a rapid, concentration-dependent transient increase in [Ca2+]i in A549 cells. The peak [Ca2+]i increases attained with Ang II (1 microM) and BK (1 microM) were 3- and 4-fold higher, respectively (P < 0.01) than the basal [Ca2+]i values. This effect of Ang II was completely abolished by inclusion of losartan (DuP 753), an AT1 subtype selective antagonist. Removal of extracellular Ca2+ from the incubation medium led to significant diminution of the peak [Ca2+]i response to Ang II but not to BK. In contrast to Ang II and BK, ET-1 failed to evoke an increase in [Ca2+]i levels in A549 cells. Neither Ang II nor ET-1 evoked any appreciable increase in [Ca2+]i levels of non-neoplastic lung cell and SCLC cell lines. These data confirm that the human non-small cell lung cancer cells (A549) selectively express AT1 subtype receptors for Ang II that are functionally coupled to Ca2+ mobilization from both extra and intracellular sources.