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Biomedical subjects

I Fleming

Publications and source records attributed to I Fleming.

At least 37 records · Page 2Linked to original sources
PubMed2000

Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis).

Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 microm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.

Animals↗
PubMed2000

EDHF: a cytochrome P450 metabolite in coronary arteries.

Endothelium-dependent relaxation cannot be fully attributed to the release of nitric oxide or prostacyclin (PGI2). In resistance-sized vessels and coronary arteries a high proportion of endothelium-dependent relaxation, in response to agonist-induced or mechanical stimulation of endothelial cells, can be attributed to the release of 1 or more endothelium-derived hyperpolarizing factor (EDHF). In coronary arteries EDHF has been pharmacologically characterized as a cytochrome P450 (CYP)-derived metabolite of arachidonic acid. We show here that a CYP 2C arachidonic acid epoxygenase, homologous to CYP 2C8/9, is expressed in cultured human endothelial cells and native porcine coronary artery endothelial cells. Down-regulation of CYP 2C protein by transfection of porcine coronary arteries with anti-sense oligonucleotides decreased EDHF-mediated vascular responses while EDHF-mediated hyperpolarisation and relaxation were potentiated by the CYP-inducing compound beta-naphthoflavone. Thus, CYP 2C appears to play a crucial role in the generation of EDHF-mediated responses in porcine coronary arteries.

Animals↗
PubMed2000

Cytochrome P450 2C is an EDHF synthase in coronary arteries.

Studies designed to elucidate the identity of the potent vasodilator autacoid endothelium-derived hyperpolarizing factor (EDHF) in the coronary vascular bed have highlighted a role for vascular cytochrome P450 (CYP) enzymes in cardiovascular homeostasis. Not only is there strong evidence suggesting that the putative coronary EDHF synthase is an endothelially expressed CYP epoxygenase, but CYP products such as epoxyeicosatrienoic acids and reactive oxygen species have been implicated in the regulation of intracellular signalling cascades and vascular cell proliferation.

Animals↗
PubMed2000

Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress.

Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress-induced activation of eNOS apparently Ca2+-independent.

Amino Acid Sequence↗
PubMed2000

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.

Acetylcholine↗
PubMed2000

Nifedipine increases cytochrome P4502C expression and endothelium-derived hyperpolarizing factor-mediated responses in coronary arteries.

In addition to NO and prostacyclin, endothelial cells release a factor that elicits vasodilatation by hyperpolarizing the underlying vascular smooth muscle cells. In some vascular beds, this so-called endothelium-derived hyperpolarizing factor (EDHF) displays the characteristics of a cytochrome P450 (CYP)-derived arachidonic acid metabolite, such as an epoxyeicosatrienoic acid. Native porcine and cultured human coronary artery endothelial cells were screened for CYP epoxygenases, and CYP2B, CYP2C, and CYP2J were detected with reverse transcription-polymerase chain reaction. The CYP inducer beta-naphthoflavone and the Ca(2+) antagonist nifedipine significantly increased CYP2C mRNA but did not change the expression of CYP2J or CYP2B. To determine the relationship between CYP2C expression and EDHF production in native endothelial cells, we incubated porcine coronary arteries with nifedipine. Nifedipine enhanced endothelial CYP2C protein expression, as well as the generation of 11,12-epoxyeicosatrienoic acid. In organ bath experiments, pretreatment with nifedipine enhanced bradykinin-induced, EDHF-mediated relaxations as well as the concomitant hyperpolarization of smooth muscle cells. The specific CYP2C9 inhibitor sulfaphenazole, on the other hand, significantly attenuated EDHF-mediated hyperpolarization and relaxation. These results demonstrate that in porcine coronary arteries, the elevated expression of a CYP epoxygenase, homologous to CYP2C8/9, is associated with enhanced EDHF-mediated hyperpolarization in response to bradykinin. Therefore, we propose that an isozyme of CYP2C is the most likely candidate for the CYP-dependent EDHF synthase in porcine coronary arteries.

8,11,14-Eicosatrienoic Acid↗
PubMed1999

Cytochrome P450 2C is an EDHF synthase in coronary arteries.

In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.

8,11,14-Eicosatrienoic Acid↗
PubMed1999

Signal transduction of eNOS activation.

Consistent with its classification as a Ca2+/calmodulin-dependent enzyme the constitutive endothelial nitric oxide (NO) synthase (eNOS) can be activated by receptor-dependent and -independent agonists as a consequence of an increase in the intracellular concentration of free Ca2+ ([Ca2+]i) and the association of the Ca2+/calmodulin complex with eNOS. Additional post-translational mechanisms regulate the activity of eNOS, including the interaction of eNOS with caveolin-1, heat shock protein 90 (Hsp90), or membrane phospholipids, as well as enzyme translocation and phosphorylation. In response to fluid shear stress the maintained production of NO by native and cultured endothelial cells is associated with only a transient increase in [Ca2+]i. In the absence of extracellular Ca2+ and in the presence of calmodulin antagonists, shear stress stimulates a maintained production of NO which is insensitive to the removal of extracellular Ca2+, but sensitive to tyrosine kinase inhibitors, Hsp90-binding proteins and phosphatidylinositol 3-kinase inhibitors. A pharmacologically identical activation of eNOS can be induced by protein tyrosine phosphatase inhibitors suggesting that the phosphorylation of eNOS, and possibly that of an associated regulatory protein(s), is crucial for its Ca(2+)-independent activation.

Animals↗
PubMed1999

Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.

Androstadienes↗
PubMed1999

Angiotensin-converting enzyme inhibitor ramiprilat interferes with the sequestration of the B2 kinin receptor within the plasma membrane of native endothelial cells.

BACKGROUND: ACE (kininase II) inhibitors have been shown to exert their beneficial cardiovascular effects via the inhibition of both angiotensin II formation and bradykinin breakdown. Because recent evidence suggests that ACE inhibitors may also interfere with B2 kinin receptor signaling and thus enhance the vascular response to bradykinin, we examined whether the distribution of B2 kinin receptors within the plasma membrane of native endothelial cells is affected by an ACE inhibitor. METHODS AND RESULTS: Localization of the B2 kinin receptor in membranes prepared from native porcine aortic endothelial cells was evaluated by means of specific [3H]bradykinin binding and immunoprecipitation of the B2 receptor from isolated membranes. Effects of bradykinin and ramiprilat on intracellular signaling were determined by monitoring the activation of the extracellularly regulated kinases Erk1 and Erk2 as well as [Ca2+]i increases in fura 2-loaded endothelial cells. Stimulation of native endothelial cells with bradykinin 100 nmol/L resulted in the time-dependent sequestration of the B2 receptor to caveolin-rich (CR) membranes, which was maximal after 5 minutes. Pretreatment with ramiprilat 100 nmol/L for 15 minutes significantly attenuated the recovery of B2 kinin receptors in CR membranes while increasing that from membranes lacking caveolin. This effect was not due to the inhibition of bradykinin degradation, because no effect was seen in the presence of an inhibitory concentration of the synthetic ACE substrate hippuryl-L-histidyl-L-leucine. Ramiprilat also decreased [3H]bradykinin binding to CR membranes when applied either before or after bradykinin stimulation. Moreover, ramiprilat resulted in reactivation of the B2 receptor in bradykinin-stimulated cells and induced a second peak in [Ca2+]i and reactivation of Erk1/2. CONCLUSIONS: The ACE inhibitor ramiprilat interferes with the targeting of the B2 kinin receptor to CR membrane domains in native endothelial cells. Therefore, effects other than the inhibition of kininase II may account for the effects of ramiprilat and other ACE inhibitors on the vascular system.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Isometric contraction induces the Ca2+-independent activation of the endothelial nitric oxide synthase.

Shear stress and tyrosine phosphatase inhibitors have been shown to activate the endothelial NO synthase (eNOS) in a Ca2+/calmodulin-independent manner. We report here that isometric contraction of rabbit aorta activates eNOS by a pharmacologically identical pathway. Endothelium-intact aortic rings were precontracted under isometric conditions up to 60% of the maximal phenylephrine-induced tone. The NO synthase inhibitor NGnitro-L-arginine (L-NA) and the soluble guanylyl cyclase inhibitor NS 2028 induced an additional contraction, the amplitude of which depended on the level of precontraction. The maximal production of NO by isometrically contracted aortic rings (as estimated by the increase in cGMP in detector smooth muscle cells in a superfusion bioassay) was observed during the initial phase of isometric contraction and was greater than that detected following the application of acetylcholine. The supplementary L-NA-induced increase in vascular tone was inhibited by the nonselective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A. Another tyrosine kinase inhibitor, genistein, the calmodulin antagonist calmidazolium, and the selective protein kinase C inhibitor, Ro 31-8220, had no effect. Coincident with the enhanced NO formation during isometric contraction was an increase in the tyrosine phosphorylation of endothelial proteins, which also correlated with the level of precontraction. Thus, isometric contraction activates eNOS via a Ca2+-independent, tyrosine kinase inhibitor-sensitive pathway and, like shear stress, seems to be an independent determinant of mechanically induced NO formation.

1-Methyl-3-isobutylxanthine↗
PubMed1999

NO: the primary EDRF.

Since the discovery of an endothelium-derived relaxing factor (EDRF) by Furchgott and Zawadzki (Furchgott and Zawadzki. 1980), which was later identified as nitric oxide (NO) (Ignarro et al., 1987; Palmer et al., 1987; Furchgott, 1988), it has become clear that there are a number of additional endothelium-derived vasodilator and vasoconstrictor autacoids (endothelin-1, prostaglandin H2, and the endothelium-derived hyperpolarizing factor: EDHF). None of these autacoids play such a central role in the regulation of vascular tone and homeostasis as the primary EDRF, the free radical NO, which is generated via a live-electron oxidation of a guanidino nitrogen from L-arginine by an NO synthase (NOS).

Animals↗
PubMed1999

Chronic selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in rats.

OBJECTIVE/METHODS: In order to investigate whether selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in the rat hindlimb, rats were selectively bred to establish two strains, one with a pronounced hypertriglyceridemia (HT) and the other with normal plasma levels of triglycerides (LT). RESULTS: Carotid arteries and aortae removed from 3, 6, 9 and 12 month old LT- and HT-rats exhibited a normal morphology. However, marked morphological differences were observed between vessels from 18-20 month old HT- and LT-rats. The endothelium-dependent vasodilator acetylcholine (2 to 50 micrograms/kg), administered into the iliac artery, elicited a concentration-dependent increase in hindlimb blood flow which was not different in 3, 6 and 9 month old LT- or HT-rats but was impaired in 12 and 18-20 month old HT-rats. In contrast the endothelium-independent vasodilator sodium nitroprusside enhanced blood flow in both strains to a similar extent. Neither administration of the nitric oxide (NO) synthase (NOS) substrate, L-arginine, nor the NOS inhibitor NGnitro-L-arginine, affected the responsiveness to endothelium-dependent vasodilators in 12 month old HT-rats. These attenuated responses could not be attributed to a decrease in endothelial NOS expression as Western blot analysis revealed identical levels of this enzyme in the aortae and carotid arteries from LT- and HT-rats. Determination of superoxide anion (O2-) formation however, demonstrated a markedly elevated production of O2- in aortae from HT-rats. CONCLUSION: We conclude that chronic selective hypertriglyceridemia, an independent risk factor in the development and progression of atherosclerosis, leads to an endothelial dysfunction which is associated with an increased vascular O2- production and a subsequent decrease in bioavailable NO.

Acetylcholine↗
PubMed1999

Inhibition of the production of endothelium-derived hyperpolarizing factor by cannabinoid receptor agonists.

1. The endogenous cannabinoid, anandamide, has been reported to induce an 'endothelium-derived hyperpolarizing factor (EDHF)-like' relaxation in vitro. We therefore investigated the effects of cannabinoid CB1 receptor agonists; HU 210, delta9-tetrahydrocannabinol (delta9-THC) and anandamide, and a CB1 antagonist/inverse agonist, SR 141716A, on nitric oxide (NO) and EDHF-mediated relaxation in precontracted rings of porcine coronary, rabbit carotid and mesenteric arteries. 2. In rings of mesenteric artery HU 210 and delta9-THC induced endothelium- and cyclo-oxygenase-independent relaxations which were sensitive to SR 141716A. Anandamide (0.03-30 microM) induced a slowly developing, endothelium-independent relaxation which was abolished by diclofenac and was therefore mediated by cyclo-oxygenase product(s). None of the CB1 agonists tested affected the tone of precontracted rings of rabbit carotid or porcine coronary artery. 3. In endothelium-intact segments, HU 210, delta9-THC and anandamide did not affect NO-mediated responses but under conditions of continuous NO synthase/cyclo-oxygenase blockade, significantly inhibited acetylcholine and bradykinin-induced relaxations which are attributed to the production of EDHF. The effects of HU 210 and delta9-THC were not observed when experiments were performed in the presence of SR 141716A suggesting the involvement of the CB1 receptor. 4. In a patch clamp bioassay of EDHF production, HU 210 decreased the EDHF-mediated hyperpolarization of detector smooth muscle cells when applied to the donor segment but was without effect on the membrane potential of detector cells. The inhibition of EDHF production was unrelated to alterations in Ca2+ -signalling or cytochrome P450 activity. 5. These results suggest that the activation of endothelial CB1 receptors appears to be negatively coupled to the production of EDHF.

Animals↗
PubMed1998

Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation.

In this study we first established, by immunoblotting with specific antibodies, the temporal changes in cellular levels of protein kinase C (PKC) isoforms during differentiation of 3T3-F442A pre-adipocytes. Both pre-adipocyte and adipocyte 3T3-F442A cells were found to express PKC-alpha, -gamma, -delta, -epsilon, -zeta and -mu. However we were unable to detect PKC-beta, -eta or -theta. The same PKC isoform expression profile was found in rat adipocytes. The alpha, delta and gamma isoforms displayed similar temporal patterns of expression during differentiation of 3T3-F442A cells; all increased rapidly, peaking at day 2 of differentiation. Subsequently, the expression of these isoforms decreased, resulting in lower levels in fully differentiated adipocytes than in pre-adipocytes. The expression of PKC-epsilon increased steadily during differentiation, resulting in markedly elevated levels in adipocytes. Although expression of PKC-mu increased during differentiation, this was attributable to prolonged confluence rather than to the differentiation process itself. No change was observed in PKC-zeta levels during adipocyte development. Anti-sense oligodeoxynucleotides (ODNs) were used to deplete selectively the individual PKC subtypes. Each of the ODNs used effectively depleted the specific isoforms to undetectable levels and did not affect expression of the other PKC subtypes. This approach indicated that pre-adipocyte differentiation is not dependent upon PKC-zeta but that PKC-alpha,-delta and -mu each exert an inhibitory influence upon differentiation. Use of anti-sense ODNs to deplete PKC-epsilon and -gamma revealed that pre-adipocyte differentiation is dependent upon each of these isoforms. However, PKC-gamma, but not PKC-epsilon, appeared to be necessary for the clonal expansion of differentiating cells, suggesting that PKC-epsilon is required at a later phase in the differentiation process, when its expression is elevated, for the attainment and maintenance of the adipocyte phenotype.

3T3 Cells↗
PubMed1998

Ca2+-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress.

Fluid shear stress enhances NO formation via a Ca2+-independent tyrosine kinase inhibitor-sensitive pathway. In the present study, we investigated the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide and of fluid shear stress on endothelial NO production as well as on the membrane association and phosphorylation of the NO synthase (NOS) III. Phenylarsine oxide (10 micromol/L) induced an immediate and maintained NO-mediated relaxation of isolated rabbit carotid arteries, which was insensitive to the removal of extracellular Ca2+ and the calmodulin antagonist calmidazolium. This phenylarsine oxide-induced vasodilatation was unaffected by genistein but abrogated by the tyrosine kinase inhibitor erbstatin A. Incubation of native or cultured endothelial cells with phenylarsine oxide resulted in a time-dependent tyrosine phosphorylation of mainly Triton X-100-insoluble (cytoskeletal) proteins, along with a parallel change in the detergent solubility of NOS III, such that the enzyme was recovered in the cytoskeletal fraction. A similar, though slightly delayed, phenomenon was also observed after the application of fluid shear stress but not in response to any receptor-dependent agonist. Although Ca2+-independent NO formation was sensitive to erbstatin A, phenylarsine oxide treatment was associated with the tyrosine dephosphorylation of NOS III rather than its hyperphosphorylation. Proteins that also underwent redistribution in response to the tyrosine phosphatase inhibitor included paxillin, phospholipase C-gamma1, mitogen-activated protein kinase, and the tyrosine kinases Src and Fyn. We envisage that fluid shear stress and tyrosine phosphatase inhibitors may alter the conformation and/or protein coupling of NOS III, facilitating its interaction with specific phospholipids, proteins, and/or protein kinases that enhance/maintain its Ca2+-independent activation.

Analysis of Variance↗
PubMed1998

Pulsatile stretch in coronary arteries elicits release of endothelium-derived hyperpolarizing factor: a modulator of arterial compliance.

To date, the release of the endothelium-derived hyperpolarizing factor (EDHF) has been demonstrated only in response to receptor-dependent Ca2+-elevating agonists. Since endothelial cells in situ are continuously subjected to rhythmic distension, we investigated the effect of rhythmic stretch on the release of EDHF from isolated porcine coronary arteries. In the combined presence of diclofenac and N(G)-nitro-L-arginine (L-NNA), sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) led to simultaneous oscillations in the external diameter of coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. In order to directly demonstrate the release of EDHF, the intraluminal solution from endothelium-intact coronary segments exposed to pulsatile stretch was applied to detector rat aortic smooth muscle cells, the membrane potential of which was continuously measured using the patch-clamp technique. The hyperpolarization of detector cells induced by the intraluminal solution was proportional to the amplitude of the pressure oscillations applied to the donor artery and was attenuated by either preincubation of donor arteries with 17-octadecynoic acid or application of either tetrabutylammonium or iberiotoxin to detector cells. In contrast to the bradykinin-induced release of EDHF, the EDHF synthesized in response to pulsatile stretch did not exhibit any tachyphylaxis. These findings demonstrate for the first time that the synthesis of EDHF in coronary arteries can be mechanically stimulated by rhythmic vessel wall distension and suggest that the continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.

6-Ketoprostaglandin F1 alpha↗