Oxidized LDL increases the sensitivity of the contractile apparatus in isolated resistance arteries for Ca(2+) via a rho- and rho kinase-dependent mechanism.

BACKGROUND: Oxidized LDL reduces NO-mediated and endothelium-derived hyperpolarizing factor-mediated dilations. We studied, in hamster skeletal muscle resistance arteries (213+/-8 micrometer n=51), whether an altered vascular smooth muscle (VSM) response, particularly sensitization of the VSM contractile apparatus to Ca(2+), is involved in this oxLDL effect. Methods and Results-VSM or endothelial [Ca(2+)](i) and vascular diameter were measured in response to norepinephrine (0.3 micromol/L), sodium nitroprusside (10 micromol/L), C-type natriuretic peptide (1 to 100 nmol/L), papaverine (0.1 to 10 micromol/L), or the endothelial agonist acetylcholine (ACh, 0.01 to 1 micromol/L). OxLDL significantly increased resting VSM [Ca(2+)](i) (11+/-3%), decreased diameter (8+/-2%), and enhanced norepinephrine-induced constrictions. Dilations to sodium nitroprusside and C-type natriuretic peptide were significantly reduced (by 10+/-2% and 35+/-6%), whereas dose-response curves for papaverine and ACh were shifted to the right, despite unchanged increases in endothelial Ca(2+) after ACh. OxLDL significantly shifted the Ca(2+)-diameter relation to the left, as assessed by stepwise increasing extracellular Ca(2+) (0 to 3 mmol/L) in depolarized skeletal muscle resistance arteries. This sensitization to Ca(2+) by oxLDL was abolished after inhibition of Rho (C3 transferase) or Rho kinase (Y27632). CONCLUSIONS: OxLDL reduces VSM responsiveness to vasodilators by increasing VSM Ca(2+) but preferentially by sensitizing VSM to Ca(2+) via a Rho- and Rho kinase-dependent pathway.

Intact endothelial and smooth muscle function in small resistance arteries after 48 h in vessel culture.

Long-term culture of resistance vessels allows introduction of molecular biology techniques for use in microvascular research. The aim of the present study was to establish a culture protocol that preserved vascular integrity and function in microvessels for 48 h in culture. Skeletal muscle resistance arteries were excised from the hamster gracilis muscle. Segments were assigned to immediate functional tests or to vessel culture, during which segments were perfused and superfused at a transmural pressure of 45 mmHg with Leibovitz (L15) medium containing 15% fetal calf serum and antibiotics for 48 h. Cultured and freshly isolated vessels showed similar levels of spontaneous tone, myogenic responses, changes in smooth muscle intracellular calcium (Ca(i)(2+)) (fura 2), and vascular diameter (video microscopy) in response to 0.3 M norepinephrine and similar concentration-response curves for acetylcholine (endothelium dependent, +/-N(omega)-nitro-L-arginine) and sodium nitroprusside (endothelium independent). Measurements of endothelial Ca(i)(2+) revealed similar acetylcholine-induced increases in endothelial Ca(i)(2+) in both groups. It is concluded that vascular function can be preserved while maintaining vessels in culture. Thus it is possible to utilize protocols that require long-term treatment.

Impaired conduction of vasodilation along arterioles in connexin40-deficient mice.

Connexins have been hypothesized to play an important role in intercellular communication within the vascular wall and may provide a mechanistic explanation for conduction of vasomotor responses. To test this hypothesis, we studied the transmission of vasomotor responses in the intact skeletal muscle microcirculation of connexin40-deficient mice (Cx40(-/-)). Arterioles were locally stimulated with hyperpolarizing dilators (acetylcholine [ACh] as well as bradykinin [Bk]) or depolarizing K(+) solution, and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.32 mm upstream. Arterial pressure was elevated 25% in Cx40(-/-) mice (94+/-5 versus 75+/-4 mm Hg). Vessels selected for study had equivalent basal diameter and vasomotor tone in both genotypes of mice. Vasomotion was present in small arterioles of both genotypes, but its intensity was exaggerated in Cx40(-/-) mice. ACh and Bk induced dilation (33% and 53%, respectively, of maximal response) at the site of application that was of similar magnitude in both genotypes. These dilations were observed to spread upstream within <1 second without significant attenuation in Cx40(+/+) mice. However, spreading was severely attenuated in Cx40(-/-) animals (11+/-4% versus 35+/-7% with ACh and 38+/-5% versus 60+/-7% with Bk in Cx40(-/-) and Cx40(+/+), respectively; P<0.05). In contrast, conducted vasoconstrictions, induced by K(+) solution decreased equally with distance in both genotypes. These results support a significant role for Cx40 in vascular intercellular communication. Our observations indicate that Cx40 is required for normal transmission of endothelium-dependent vasodilator responses and may underlie altered vasomotion patterns.

Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.

Using a novel vessel culture technique in combination with antisense oligonucleotide transfection, we tested whether the endothelium-derived hyperpolarizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arteries from hamster gracilis muscle (n=19) were perfused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in endothelial cells. Thereafter, NO- and prostaglandin-independent, EDHF-mediated vascular responses associated with hyperpolarization [i.e., decrease in smooth muscle calcium (Fura 2) and vasodilation] were studied after the application of acetylcholine (ACh). These EDHF-mediated responses were markedly attenuated (by 70%) by As- but not by S- or Scr-oligonucleotide treatment. However, the responses to norepinephrine (0.3 micromol/l), the NO donor sodium nitroprusside (1 micromol/l), and the K(Ca) channel activator NS1619 (100 micromol/l) were unaltered. As treatment, which specifically targeted the endothelial layer (as assessed by confocal microscopy), had no inhibitory effect on increases in endothelial calcium to ACh. It is concluded that a CYP2C8/9-related isoform functions as an EDHF synthase in hamster resistance arteries and that a product of this enzyme is an EDHF, or at least an integral part of the signaling cascade leading to EDHF-mediated responses.-Bolz, S.-S., Fisslthaler, B., Pieperhoff, S., de Wit, C., Fleming, I., Busse, R., Pohl, U. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.

Interaction of endothelial autacoids in microvascular control.

The endothelium releases dilating autacoids, such as nitric oxide (NO), prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF). Different mechanisms of interaction between these autacoids have been identified and are briefly reviewed here. NO, which dilates resistance vessels via the second messenger cGMP, also amplifies the responses to vasodilators, which act by elevation of cAMP (e.g., prostaglandins), in a synergistic manner. This amplification is most likely due to a cGMP-dependent inhibition of the breakdown of cAMP. An interaction of these autacoids can not only be found in vascular smooth muscle cells but also in the endothelium. A closer look towards the site of production reveals that prostacyclin attenuates the release of NO, which is achieved by a decrease of the intracellular calcium levels in endothelial cells. This feedback mechanism leads to enhanced NO formation after inhibition of cyclooxygenase. While NO does not seem to modulate prostaglandin synthesis, it has been shown recently in coronary arteries that NO attenuates the release of EDHF. Under conditions of compromised NO formation EDHF might act via this mechanism as a second line of defense. This could help in maintaining endothelium-dependent vasodilation unless an altered smooth muscle responsiveness generally reduces the efficacy of endothelial vasodilators (including EDHF). Convincing experimental evidence for such an impaired relaxation of smooth muscle has been presented recently following exposure to elevated oxLDL levels.

Endothelium-derived hyperpolarizing factor but not NO reduces smooth muscle Ca2+ during acetylcholine-induced dilation of microvessels.

1. We hypothesized that nitric oxide (NO) and the endothelium-dependent hyperpolarizing factor (EDHF) may dilate microvessels by different cellular mechanisms, namely Ca2+-desensitization versus decrease in intracellular free calcium. 2. Effects of acetylcholine (ACh) and the NO donors sodium nitroprusside (SNP, 0.1 - 10 micromol l(-1)) and S-Nitroso-N-acetyl-D, L-penicillamine (SNAP, 0.01 - 10 micromol l-1) on intracellular calcium ([Ca2+]i, fura 2) and vascular diameter (videomicroscopy) were studied in isolated resistance arteries from hamster gracilis muscle (194+/-12 microm) pretreated with indomethacin and norepinephrine. Membrane potential changes were determined using 1, 3-dibutylbarbituric acid trimethineoxonol (DiBAC4(3)). 3. ACh (0.1 and 1 micromol l-1)-induced dilations were associated with a [Ca2+]i decrease (by 13+/-3 and 32+/-4%) and hyperpolarization of vascular smooth muscle (VSM, by 12+/-1% at 1 micromol l-1 ACh). Nomega-nitro-L-arginine (L-NA, 30 micromol l(-1)) partially inhibited the dilation but did not affect VSM [Ca2+]i decreases or hyperpolarization. In contrast, the KCa channel inhibitors tetrabutylammonium (TBA, 1 mmol l(-1)) and charybdotoxin (ChTX, 1 micromol l(-1)) abolished the ACh-induced [Ca2+]i decrease and the hyperpolarization in VSM while a significant dilation remained (25 and 40%). This remaining dilation was abolished by L-NA. ChTX did not affect [Ca2+]i increase and hyperpolarization in endothelial cells. SNP- or SNAP-induced dilations were not associated with decreases in VSM [Ca2+]i or hyperpolarization although minor transient decreases in VSM [Ca2+]i were observed at high concentrations. 4. These data suggest that ACh-induced dilations in microvessels are predominantly mediated by a factor different from NO and PGI2, presumably EDHF. EDHF exerts dilation by activation of KCa channels and a subsequent decrease in VSM [Ca2+]i, NO dilates the microvessels in a calcium-independent manner.

Pentobarbital-sensitive EDHF comediates ACh-induced arteriolar dilation in the hamster microcirculation.

It is unclear to what extent the endothelium-derived hyperpolarizing factor (EDHF) contributes to the control of microcirculatory blood flow in vivo. We analyzed, by intravital microscopy in hamster muscles, the potential role of EDHF along the vascular tree under stimulated (ACh) or basal conditions. Experiments were performed in conscious as well as anesthetized (pentobarbital, urethan) animals. Additionally, cellular effects of the potential EDHF were studied in isolated small arteries. In pentobarbital-anesthetized animals, treatment with Nomega-nitro-L-arginine (L-NNA; 30 micromol/l) and indomethacin (3 micromol/l) reduced the dilation in response to 10 micromol/l ACh from 60 +/- 6 to 20 +/- 4%. This nitric oxide/prostaglandin-independent dilation (NPID), which was of a similar magnitude in large and small arterioles, was abolished by potassium depolarization or charybdotoxin (ChTX, 1 micromol/l) but not by glibenclamide. In conscious animals, NPID amounted to 33 +/- 3%. The inhibitor of the P-450 monooxygenase 17-octadecynoic acid (ODYA) reduced NPID further to 9 +/- 4%. ChTX abolished the NPID and also reduced basal diameters (by -11 +/- 3%). The induction of anesthesia with pentobarbital reduced NPID (to 12 +/- 6%), whereas urethan anesthesia was without effect. Pentobarbital also reduced the ACh-induced hyperpolarization of vascular smooth muscle in isolated arteries, whereas ChTX abolished it. This study suggests that a considerable part of the ACh dilation in the microcirculation is mediated by EDHF, which also contributes to the control of basal tone in conscious animals. The direct inhibitory effect of pentobarbital and ODYA supports the idea that "microcirculatory" EDHF is a product of the cytochrome P-450 pathway. The role of EDHF might be underestimated in pentobarbital-anesthetized animals.

Myogenic effects enhance norepinephrine constriction: inhibition by nitric oxide and felodipine.

Myogenic, pressure-induced vasoconstriction may amplify the effects of circulating vasoconstrictors. Through intravital microscopy in cremaster arterioles (31 to 115 microm diameter), the relative contribution of myogenic responses (MR) to norepinephrine (NE)-induced constriction and the inhibitor potency of nitric oxide (NO) or a Ca2+ entry blocker (CEB), felodipine (F), were examined. In 24 anesthetized hamsters, a vessel occluder was placed around the aorta to control cremaster vessel inflow pressure (IP). NE infusion increased blood pressure (by 50 +/- 2 mm Hg) and induced significant constriction (24% +/- 9%) in small arterioles (< 65 microm) only. The constriction, which was not altered by adrenergic blockade, was dependent on the actual IP and was abolished when the IP increase was blocked. NO synthase (NOS) blockade unmasked a significant MR in large arterioles. F inhibited the MR predominantly in large vessels. In isolated microvessels, F completely blocked the pressure-induced Ca2+ increase and MR. We conclude that circulating NE constricts muscle arterioles mainly by a myogenic mechanism. NO effectively opposes MR in larger arterioles, thus restricting MR and vasoconstrictor reinforcement to a small section of the vasculature being tightly controlled by metabolic signals. MR, which otherwise would impair adjustment of peripheral resistance, is reduced by CEB predominantly in larger arterioles, similar to NO.

Nitric oxide opposes myogenic pressure responses predominantly in large arterioles in vivo.

A myogenic vasoconstriction may amplify the effects of circulating vasoconstrictors. In cremaster arterioles, the contribution of a myogenic component to the constriction on intravenous infusion of norepinephrine (NE) or angiotensin II (Ang II) was studied. Second, the role of endothelium-derived nitric oxide (NO) in the control of these myogenic constrictions and its site of action in the resistance vascular bed was investigated. In 30 anesthetized (pentobarbital) hamsters, the cremaster was prepared for intravital microscopy, and a pneumatic vessel occluder was placed around the aorta to vary blood pressure in the hindquarter of the animal. Intravenous infusion of NE (0.5 nmol/min) increased the systemic blood pressure by 52+/-2 mm Hg. Simultaneously, constrictions of up to 33+/-6% were observed in the small arterioles (SAs; maximal inner diameter, 36 to 65 microm). The constrictions were not significantly altered by a local adrenergic blockade but were abolished when the pressure elevation in the cremaster arterioles was blocked by partial occlusion of the abdominal aorta. Diameters in large arterioles (LAs; maximal inner diameter, 65 to 127 microm), however, did not change significantly on NE infusion. Similar responses in the arterioles were observed when the local pressure was increased stepwise from 60 to 120 mm Hg by partial opening of the aortic occluder. However, after treatment of the cremaster tissue with the inhibitor of the NO synthase, N(G)-nitro-L-arginine (L-NNA, 30 micromol/L), a significant pressure-induced constriction of up to 16+/-3% occurred in LAs, whereas the magnitude of the constriction in SAs remained unchanged. L-NNA also abolished the increases in blood flow that were observed with increments in pressure in control animals. Similar results were obtained when Ang II was used to increase blood pressure. We conclude that a myogenic constriction of SAs contributes markedly to the overall response of cremaster arterioles to circulating vasoconstrictors. NO effectively opposes the myogenic response in LAs, thus preventing myogenic constrictions in a vascular region where constriction cannot be fully controlled by metabolic dilation. If this attenuating effect of NO on myogenic constriction also takes place in other organs, it might be a decisive mechanism in controlling changes of total peripheral vascular resistance elicited by vasoconstrictors.

Elevation of plasma viscosity induces sustained NO-mediated dilation in the hamster cremaster microcirculation in vivo.

We studied whether a flow-independent increase of luminal wall shear stress (WSS) could dilate hamster arterioles in vivo and which endothelial mediators are potentially involved. To this end the plasma viscosity was elevated by exchanging blood for dextran-erythrocyte solution thereby augmenting WSS. Diameters of small and large arterioles as well as red blood cell velocities were measured before and after exchange of blood for solutions of identical haematocrit containing either high- (HMWD) or low-molecular weight dextran (LMWD). The potential role of endothelial autacoids was investigated by local application of the NO-synthase inhibitor NG-nitro-L-arginine (L-NNA), the inhibitor of cyclooxygenase, indomethacin (3 microM), or the K+-channel blocker, tetrabutylammonium (TBA, 0.1 mM) to assess the potential effects of EDHF. HMWD (n = 11 animals) increased plasma viscosity by 64 +/- 3% and dilated arterioles of all branching orders (A1-A4) significantly [by 24 +/- 3% (A1-A2) and 32 +/- 3% (A3-A4)]. This dilation compensated fully for the calculated initial increase of WSS. LMWD (n = 6) did not affect plasma viscosity or arteriolar diameters. Tissue treatment with L-NNA (30-300 microM, n = 12) substantially diminished the HMWD-induced dilation in small arterioles (A3-A4; to 13 +/- 3%; P<<0.05) and virtually abolished it in large ones (A1-A2). Consequently, the calculated WSS increased significantly in these arterioles (by 31 +/- 5%). TBA combined with L-NNA (n = 4) did not reduce further the remaining dilation. Indomethacin (n = 6) had no effect on HMWD-induced dilation. We conclude that an increase of WSS induces a mainly NO-mediated arteriolar dilation. This dilation occurs in all arteriolar branching orders and is of sufficient magnitude to compensate for the initial WSS-increase. Thus, any elevations of WSS fulfil the requirement for a signal to change diameter along the arteriolar tree in a coordinated manner. The fully compensating dilation which we observed indicates that WSS is a controlled variable. It does, however, raise questions as to its role as a continuous endothelial stimulus.

Indomethacin enhances endothelial NO release--evidence for a role of PGI2 in the autocrine control of calcium-dependent autacoid production.

OBJECTIVE: We studied whether NO or prostacyclin (PGI2), which are continuously released by endothelial cells, have autocrine/paracrine effects on the calcium-dependent autacoid production by modulating the intracellular Ca2+ concentration ([Ca2+]i). METHODS: Histamine(His)-induced [Ca2+]i increases (Fura 2-method) and NO-dependent cGMP increase were measured in human umbilical vein endothelial cell (HUVECs) before and after cyclooxygenase inhibition or application of cAMP- and cGMP-elevating drugs. RESULTS: 0.3 microM His increased endothelial [Ca2+]i from 77 +/- 2 nM to 418 +/- 59 nM. The His-induced [Ca2+]i increases were significantly attenuated following treatment with PGI2 (by 23%) and forskolin (by 33%), both increasing the cAMP release from HUVECs (by 49% and 66%). The His-induced [Ca2+]i increases were inhibited by the protein kinase A-activator cBIMPS (by 61%) which also abolished the His-induced PGI2 release. Conversely, inhibition of the PGI2 production with indomethacin significantly augmented the His-induced [Ca2+]i increases (by 32%), resulting in a significantly augmented NO production as indicated by an enhanced LNNA-sensitive cGMP increase in HUVECs. In contrast, neither increases of cGMP (basal 0.4 +/- 0.1 pmol/mg) elicited by 10 microM SNP (21 +/- 2 pmol/mg) or 10 microM C-type natriuretic peptide (CNP, 4.6 +/- 1.6 pmol/mg) nor its reduction by 30 microM LNNA had any effect on the His-induced [Ca2+]i increases. CONCLUSION: PGI2 attenuates agonist-induced [Ca2+]i increases by a cAMP-dependent mechanism, thereby modulating not only its own synthesis via a negative feedback but also that of NO. Consequently, reduced PGI2 levels result in an increased NO production. NO which does not cause a negative feedback control by cGMP might therefore compensate for the lack of PGI2.