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D R Harder

Publications and source records attributed to D R Harder.

120 records · Page 7Linked to original sources
PubMedDate not supplied

Bioassay of an endothelium-derived hyperpolarizing factor from bovine coronary arteries: role of a cytochrome P450 metabolite.

An endothelium-derived hyperpolarizing factor (EDHF) mediates a part of the vasodilatory action of bradykinin. A bioassay method was developed to investigate the properties of EDHF on bovine coronary arterial smooth muscle cells. Cannulated bovine coronary arteries with an intact endothelium that were treated with indomethacin and NG-nitro-L-arginine methyl ester served as the EDHF donor. The effect of the donor vessel perfusate was examined on a 240 pS single-channel calcium (Ca2+)-activated potassium (K+) current (KCa) and resting membrane potential in recipient coronary arterial smooth muscle cells. The open state probability (NPo) of the channel averaged 0.011 +/- 0.003 during basal perfusate flow. After stimulation of the donor vessels with bradykinin (10(-10)-10(-6) M), the perfusate induced a 1.2- to 5-fold increase in the NPo (n = 7, p < 0.001). This increase in channel activity was attenuated by either removing the endothelium of the donor arterial segment or upon inhibition of cytochrome P450 in the donor arterial segment with the combination of 17-octadecynoic acid and miconazole. The resting cell membrane averaged -60 +/- 2 mV, and hyperpolarized to -69 +/- 1.5 mV (n = 6, p < 0.05) in response to the perfusate following stimulation of the donor vessel with bradykinin. Addition of 14, 15-epoxyeicosatrienoic acid mimicked the effects of the perfusate and increased the NPo of the KCa channel from 0.01 +/- 0.001 to 0.05 +/- 0.001. These findings suggest that bradykinin stimulates the release of a transferable endothelial factor that activates KCa channels and hyperpolarizes coronary arterial smooth muscle cell membranes. These findings support the hypothesis that coronary arteries release an EDHF which is a cytochrome P450 metabolite of arachidonic acid.

8,11,14-Eicosatrienoic Acid↗
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Role of cytochrome P-450 in elevating renal vascular tone in spontaneously hypertensive rats.

The contribution of cytochrome P-450 metabolites of arachidonic acid in elevating vascular tone in the kidneys of adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was examined using a juxtamedullary nephron microvascular preparation perfused in vitro with a physiological salt solution containing 5% albumin. At 80 mm Hg, the basal internal diameters of arcuate and interlobular arteries and proximal and distal afferent arterioles of the SHR averaged 341 +/- 15.81 +/- 5.24 +/- 0.5 and 19 +/- 0.3 micron, respectively. These diameters were 5-29% smaller (p < 0.05) than those measured in corresponding vessels in WKY rats. Addition of the P-450 inhibitors, ketoconazole (100 microM) or 17-octadecynoic acid (17-ODYA, 20 microM), to the perfusate and bath increased the diameters of the preglomerular vasculature of the SHR by 6-29%, but by only 3-13% in WKY rats and reduced significantly the differences in pressure-diameter relations between the two groups. The rate of formation of 20-hydroxyeicosatetraenoic acid (20-HETE) by renal cortical microsomes was similar in SHR and WKY rats. However, the production of epoxyeicosatrienoic acids (EETs) by cortical microsomes was 87% lower and dihydroxyeicosatrienoic acids (DHETs) 70% higher in the SHR than WKY rats. Ketoconazole (100 microM) reduced the formation of 20-HETE by 80%, and EETs and DHETs production by more than 98% in SHR and WKY rats. 17-ODYA (20 microM) reduced the formation of 20-HETE, EETs and DHETs by more than 98% in both groups. These results suggest that cytochrome P-450 metabolites of arachidonic acid contribute to the elevated renal vascular tone in adult SHR. This may be due to an enhanced vascular responsiveness to vasoconstrictor P-450 metabolites in SHR or an elevated local production of vasoconstrictor eicosanoids in the renal vasculature of SHR rather than in renal cortical tissue.

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PubMedDate not supplied

Role of cytochrome P-450 enzymes and metabolites of arachidonic acid in the control of vascular tone.

The metabolism of arachidonic acid (AA) into vasoactive products by cyclooxygenase and lipoxygenase enzymes has been well described, as has their biological relevance. Recently, a number of studies have demonstrated the ability of cytochrome P-450 (P450) enzymes to metabolize AA into biologically important regulators of vascular tone. There are two categories of vasoactive P450 metabolites, namely those catalyzed by epoxygenase enzymes which generate epoxyeicosatrienoic acids (EETs) and those enzymes which generate hydroxyeicosatetraenoic acids (HETEs). Except for 20-HETE, P450 metabolites of AA occur as stereo- and regioisomers which determine, to some extent, their biological activity. 5,6-, 8,9-, 11,12- and 14,15-EETs are generally potent dilators in a number of vascular beds with a sensitivity which appears to increase as the vasculature decreases in size toward capillaries. HETEs, such as 12R- and 20-HETE, can be potent activators of vascular tissue with 20-HETE contracting cerebral and renal microvessels at concentrations of < 10(-10) M. Both EETs and HETEs can be made by vascular and extravascular tissue. Available data suggests that EETs are formed by endothelial and parenchymal tissue while HETEs can be endogenously formed in arterial muscle where they appear to act as second messengers. This review will discuss the molecular biology, stereochemistry, biological activity and importance of P450 metabolites of AA as para- and autocrine controllers of organ blood flow. We will also discuss the large diversity of P450 enzyme isoforms and how such diversity can provide for precise physiological control of vascular tone.

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Cytochrome P450 metabolites of arachidonic acid as intracellular signaling molecules in vascular tissue.

Recent studies from our laboratory have indicated that vascular smooth muscle cells (VSMC) metabolize arachidonic acid via a P4504A-dependent pathway to 20-hydroxyeicosatetraenoic acid (20-HETE), and that this system serves as a novel signal transduction pathway that plays a central role in the regulation of vascular tone. The major metabolite of arachidonic acid formed in cerebral and renal arteries is 20-HETE. The mRNA and protein for P4504A enzymes, which produce 20-HETE, have been localized in VSMC. 20-HETE is a potent vasoconstrictor, that acts in part by inhibition of the opening of the large conductance, calcium-activated potassium channel, and depolarizes VSMC membrane. A preliminary study also indicated that 20-HETE activates the L-type calcium current in cerebral arterial smooth muscle. Inhibition of the endogenous production of 20-HETE in renal and cerebral arterioles attenuates pressure-dependent myogenic tone in vitro, as well as autoregulation of renal and cerebral blood flow in vivo. There is also evidence that indicates that nitric oxide regulates the formation of 20-HETE by binding and inactivating the P450 heme moiety, thus providing a negative feedback control mechanism for this system. The data outlined suggest that 20-HETE could act as a intracellular second messenger that plays an integral role in the signal transduction processes underlying the development of pressure-dependent myogenic tone.

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Membrane mechanisms in arterial hypertension.

The purpose of this review is to focus on alterations in vascular muscle membrane potentials (Em), ionic permeabilities, and ionic transport systems which may either contribute to or be a consequence of the hypertensive state. Three models of hypertension are discussed: 1) deoxycorticosterone-salt (DOCA-salt)-induced hypertension; 2) low-renin (presumably volume expanded) renal hypertension (LRRH); and 3) the spontaneously hypertensive rat (SHR) of the Okamoto-Aoki Kyoto-Wistar strain and its normotensive genetic control (WKY). The importance of studying all possible mechanisms of increased contraction in vascular smooth muscle is stressed.

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Cellular actions of halothane on cat cerebral arterial muscle.

The effects of halothane on intracellular membrane potential (Em) and force development in cat MCA were studied. Halothane (0.07-0.14 mM/1) relaxed isolated MCA which had developed myogenic tone. Measurement of Em showed that halothane depolarized this preparation in a dose-dependent fashion in the face of vessel relaxation, demonstrating uncoupling of electrical and mechanical activity. Halothane markedly inhibited the contractile effects of histamine and serotonin suggesting that, apart from its direct action on cerebral arterial tone, it also blunts the action of vasoactive agents. When this preparation is partially depolarized from -62 to -50 mV with excess K+, halothane, while having only a small (1.2 mV) additional depolarizing effect, consistently elicits contraction rather than relaxation. Thus, the action of this particular volatile anesthetic on cerebral arteries can depend upon the resting level of Em. These studies indicate that halothane relaxes myogenic tone in cat MCA by an intracellular mechanism, but that the direction of its effect (i.e., relaxation vs. contraction) may depend upon the prior level of Em and muscle cell activation.

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Altered membrane properties of cerebral vascular smooth muscle following subarachnoid hemorrhage: an electrophysiological study. I. Changes in resting membrane potential (Em) and effect on the electrogenic pump potential contribution to Em.

Subarachnoid hemorrhage was produced experimentally in cats by intracisternal injection of non-heparinized autologous arterial blood obtained by cardiac puncture under ketamine and xylazine anesthesia. Cats were sacrificed at varying time intervals between 30 min and 7 days post ictus. Measurements of resting membrane potential were recorded from smooth muscle cells of the basilar artery. These measurements were obtained by impalement from the adventitial surface of isolated but otherwise intact segments of the artery using glass microelectrodes with tip sizes less than 0.1 micron. The resting membrane potential recorded in vitro from animals previously subjected to subarachnoid hemorrhage in vivo was consistently and significantly depolarized when compared to normal controls. This depolarization was present as early as 30 min post ictus. Addition of the cardiac glycoside, ouabain, in a concentration of 10(-5)M depolarized cells from both control and experimental animals. There is a significant electrogenic pump potential contribution to the resting membrane potential of vascular smooth muscle cells. Ouabain is a potent blocker of Na+, K+-ATPase, the enzyme responsible for maintaining the cation electrochemical gradients. The depolarization recorded in these cells following subarachnoid hemorrhage is not, therefore, due to impairment of the electrogenic pump. The significance and implications of these findings are discussed.

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Reversal of delayed vasospasm by TS-011 in the dual hemorrhage dog model of subarachnoid hemorrhage.

PURPOSE: Arachidonic acid is avidly metabolized to a potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE), in the cerebral circulation. 20-HETE has been reported to contribute to the acute fall in cerebral blood flow following subarachnoid hemorrhage (SAH), but its role in the development of delayed vasospasm is unknown. The present study examined whether delayed vasospasm is associated with elevations in 20-HETE in CSF in the dual hemorrhage model of SAH in dogs and if blockade of the synthesis of 20-HETE with N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011) can reverse delayed vasospasm in this model. MATERIALS AND METHODS: Delayed vasospasm was induced in 22 adult beagle dogs by dual injection of blood (0.5 mL/kg) into the cisterna magna on days 1 and 4. Sequential samples of CSF were collected before intracisternal injections of blood on days 1 and 4 and after the development of delayed vasospasm on day 7. Sequential angiograms were obtained before and after intracisternal injection of blood on days 1 and 4 and before and 1 hour after administration of TS-011 (1 mg/kg IV) on day 7. RESULTS: The dogs consistently developed delayed vasospasm, and the diameter of the basilar artery fell to 68 +/- 3% (n = 15), 3 days after the second intracisternal injection of blood. The levels of 20-HETE in CSF increased from 4 +/- 2 to 39 +/- 16 pg/mL. In 9 dogs with delayed vasospasm, acute blockade of the synthesis of 20-HETE with TS011 (1 mg/kg IV) significantly increased the diameter of the basilar artery by 39%. Chronic administration of TS-011 (1 mg/kg per day) attenuated the development of delayed vasospasm, and the diameter of the basilar artery fell by 17 +/- 1% versus the 33 +/- 3% decrease in diameter seen in control animals 3 days following the second injection of blood into the cisterna magna. CONCLUSIONS: These results indicate that the development of delayed vasospasm in dogs is associated with an increase in 20-HETE levels in CSF, and acute blockade of the synthesis of 20-HETE with TS-011 reverses delayed vasospasm in this model.

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PubMedDate not supplied

Effect of nifedipine on endothelin induced contractions of skeletal muscle arterioles of spontaneously hypertensive rats.

Endothelin is a potent vasoactive polypeptide isolated from cultured endothelial cells. However, there are relatively few studies of the action of endothelin on microvessels in vivo. To determine the effects of this compound on arterioles of normotensive and hypertensive rats, endothelin (1 x 10(-12) M to 1 x 10(-8) M) was dissolved in physiological salt solution and superfused over the cremaster muscle of 12-15 week old spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) controls. Endothelin caused about a 55% constriction of second order arterioles and complete closure of most third order arterioles and all fourth order arterioles studied. SHR arterioles tended to be more sensitive to endothelin than those of WKY, although this difference was significant for only the third order arterioles. Endothelin induced contractions were significantly inhibited by 10(-6) M nifedipine in both WKY and SHR. These studies demonstrate that endothelin is a potent constrictor of skeletal muscle arterioles, and suggest that activator Ca2+ for endothelin induced contractions of these vessels enters the vascular smooth muscle cells through dihydropyridine sensitive calcium channels.

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Functional aspects of eicosanoid hydroxylation by lung and kidney cytochromes P450. Expression of cDNAs in mammalian cells and E. coli.

A gene subfamily of cytochromes P450 with catalytic activity toward various eicosanoid substrates has been studied with a variety of techniques in this laboratory, including purification and characterization, localization at the tissue and subcellular levels, physiological function, and cloning and expression in prokaryotic and eukaryotic systems. This paper reports experiments directed toward determining the function of the cytochrome P4504A metabolite, 20-hydroxyarachidonic acid (20-hydroxyeicosatetraenoic acid; 20-HETE), in cellular ion flux, immunohistochemical localization in lung, the effects of a mechanism-based inhibitor, 12-hydroxy-16-heptadecynoic acid (12-HHDYA) on PGE1 omega-hydroxylation, and the structure-function determinants which govern the activities of the enzymes encoded by this gene subfamily.

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