Biomedical subjects

D Gebremedhin

Publications and source records attributed to D Gebremedhin.

At least 19 recordsLinked to original sources

PubMed2000

Production of 20-HETE and its role in autoregulation of cerebral blood flow.

In the brain, pressure-induced myogenic constriction of cerebral arteriolar muscle contributes to autoregulation of cerebral blood flow (CBF). This study examined the role of 20-HETE in autoregulation of CBF in anesthetized rats. The expression of P-450 4A protein and mRNA was localized in isolated cerebral arteriolar muscle of rat by immunocytochemistry and in situ hybridization. The results of reverse transcriptase-polymerase chain reaction studies revealed that rat cerebral microvessels express cytochrome P-450 4A1, 4A2, 4A3, and 4A8 isoforms, some of which catalyze the formation of 20-HETE from arachidonic acid. Cerebral arterial microsomes incubated with [(14)C]arachidonic acid produced 20-HETE. An elevation in transmural pressure from 20 to 140 mm Hg increased 20-HETE concentration by 6-fold in cerebral arteries as measured by gas chromatography/mass spectrometry. In vivo, inhibition of vascular 20-HETE formation with N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS), or its vasoconstrictor actions using 15-HETE or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), attenuated autoregulation of CBF to elevations of arterial pressure. In vitro application of DDMS, 15-HETE, or 20-HEDE eliminated pressure-induced constriction of rat middle cerebral arteries, and 20-HEDE and 15-HETE blocked the vasoconstriction action of 20-HETE. Taken together, these data suggest an important role for 20-HETE in the autoregulation of CBF.

Amides↗

PubMed2000

Molecular mechanisms controlling nutritive blood flow: role of cytochrome P450 enzymes.

This short review summarizes the potential role of cytochrome P450 (P450) in regulating blood flow in the brain tissue and in the skeletal muscle. We provide data showing that pressure-induced myogenic activity in the brain is largely responsible for autoregulation of CBF. This myogenic response to pressure is maintained, in part, by 20-HETE formation in arterial muscle cells through a P450 omega-hydroxylase coded for by a P450 4A cDNA. Autoregulation of CBF is a hallmark of the cerebral circulation and provides adequate nutritive blood flow despite large fluctuations in arterial pressure. Given the importance of oxidative metabolism in the brain, support of neuronal activity is mediated by functional hyperaemia to active neurones providing adequate delivery of oxidative substrate. We provide data demonstrating that this functional hyperaemia in the brain is regulated by astrocytes which sense neural activity and release dilator metabolites which shunt blood flow to active neurones. One of the metabolites released by astrocytes in this regard are epoxygenated products of arachidonic acid (AA) formed by P450 enzymes. These AA metabolites of P450 enzymes are epoxyeicosatrienoic acid (EETs). One of these P450 enzymes is coded by a 2C11 cDNA present in astrocytes. Furthermore, astrocytes are capable of inducing capillary angiogenesis which appears to be mediated, in part, by P450-derived EETs.

Animals↗

PubMed2000

Peroxynitrite reversibly inhibits Ca(2+)-activated K(+) channels in rat cerebral artery smooth muscle cells.

Peroxynitrite (ONOO(-)) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO(-) bioactivity, the present study examined the effect of ONOO(-) on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K(+) current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca(2+)-activated K(+) (BK) channels, and ONOO(-) (10-100 microM) were determined. At a pipette potential of +50 mV, ONOO(-) inhibited 39% of iberiotoxin-sensitive current. ONOO(-) was selective for iberiotoxin-sensitive current, whereas decomposed ONOO(-) had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K(+) solutions. Unitary currents were sensitive to increases in internal Ca(2+) concentration, consistent with activity due to BK channels. Internal ONOO(-) dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO(-) could be overcome by reduced glutathione. Glutathione, added after ONOO(-), restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO(-) on membrane K(+) current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO(-) has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

Animals↗

PubMed1999

Cannabinoid CB1 receptor of cat cerebral arterial muscle functions to inhibit L-type Ca2+ channel current.

The CB1 subtype of the cannabinoid receptor is present on neurons in the brain and mediates the perceptual effects of Delta9-tetrahydrocannabinol and other cannabinoids. We found that cat cerebral arterial smooth muscle cells (VSMC) contain the protein for the CB1 receptor and express a cDNA that has >98% amino acid homology to the CB1 cDNA expressed in rat and human neurons. Activation of the CB1 cannabinoid receptor has been shown to decrease the opening of N-type voltage-gated Ca2+ channels in neurons through a pertussis toxin-sensitive GTP-binding protein. In the present study we tested the hypothesis that activation of the cannabinoid CB1 receptor in cerebral VSMC inhibits voltage-gated Ca2+ channels and results in cerebral vasodilation. The predominant Ca2+ current identified in cat cerebral VSMC is a voltage-gated, dihydropyridine-sensitive, L-type Ca2+ current. The cannabimimetic drug WIN-55,212-2 (10-100 nM) induced concentration-dependent inhibition of peak L-type Ca2+ current, which reached a maximum of 82 +/- 4% at 100 nM (n = 14). This effect was mimicked by the putative endogenous CB1-receptor agonist anandamide, which produced a concentration-related reduction of peak L-type Ca2+ current with a maximum inhibition (at 300 nM) of 39 +/- 4% (n = 12). The inhibitory effects of both ligands on peak L-type Ca2+ currents were abolished by pertussis toxin pretreatment and application of the CB1-receptor antagonist SR-141716A (100 nM, n = 5). Both WIN-55,212-2 and anandamide produced concentration-dependent relaxation of preconstricted cerebral arterial segments that was abolished by SR-141716A. These results indicate that the CB1 receptor is expressed in cat cerebral VSMC and that the cerebral vasculature is one of the targets for endogenous cannabinoids. These findings suggest that the CB1 receptor and its endogenous ligand may play a fundamental role in the regulation of cerebral arterial tone and reactivity by modulating the influx of Ca2+ through L-type Ca2+ channels.

Amino Acid Sequence↗

PubMed1998

Cat cerebral arterial smooth muscle cells express cytochrome P450 4A2 enzyme and produce the vasoconstrictor 20-HETE which enhances L-type Ca2+ current.

1. Cerebral arteries express cytochrome P450 4A enzymes (P450 4A) and produce 20- hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of pial arterioles. It is not known which cell type in the vessel wall is responsible for the formation of 20-HETE. We examined whether freshly isolated cerebral arterial muscle cells (VSMCs) express P450 4A and produce 20-HETE. We also studied the effect of 20-HETE on pressurized cerebral arteries and on whole-cell L-type Ca2+current (ICa) recorded in cat cerebral VSMCs. 2. Cat cerebral VSMCs incubated with [14C]arachidonic acid ([14C]AA) produced 20-HETE (3.9 +/- 1.1 pmol min-1 (mg protein)-1). 3. Reverse transcription-polymerase chain reaction studies revealed that cat cerebral VSMCs express mRNA for P450 4A which metabolizes AA to 20-HETE. Cloning and sequencing of the cDNA amplified from mRNA isolated from VSMCs showed > 96 % amino acid homology to the rat and human P450 4A2 and 4A3. 4. 20-HETE (1-300 nM) induced a concentration-dependent constriction of cat cerebral arteries, which was inhibited by nifedipine. 5. Addition of 10 and 100 nM 20-HETE to the bath increased peak ICa by 50 +/- 3 and 100 +/- 10 %, respectively. This effect was not influenced by altering the frequency of depolarization. 20-HETE (100 nM) failed to increase ICa in the presence of nifedipine. 6. These results demonstrate that cat cerebral VSMCs express P450 4A enzyme, and produce 20-HETE which activates L-type Ca2+ channel current to promote cerebral vasoconstriction.

Amino Acid Sequence↗

PubMed1998

A common pathway for regulation of nutritive blood flow to the brain: arterial muscle membrane potential and cytochrome P450 metabolites.

Perfusion pressure to the brain must remain relatively constant to provide rapid and efficient distribution of blood to metabolically active neurones. Both of these processes are regulated by the level of activation and tone of cerebral arterioles. The active state of cerebral arterial muscle is regulated, to a large extent, by the level of membrane potential. At physiological levels of arterial pressure, cerebral arterial muscle is maintained in an active state owing to membrane depolarization, compared with zero pressure load. As arterial pressure changes, so does membrane potential. The membrane is maintained in a relatively depolarized state because of, in part, inhibition of K+ channel activity. The activity of K+ channels, especially the large conductance Ca(2+)-activated K+ channel (KCa) is dependent upon the level of 20-HETE produced by arterial muscle. As arterial pressure increases, so does cytochrome P450 (P4504A) activity. P4504A enzymes catalyse omega-hydroxylation of arachidonic acid and formation of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent inhibitor of KCa which maintains membrane depolarization and muscle cell activation. Astrocytes also metabolize AA via P450 enzymes of the 2C11 gene family to produce epoxyeicosatrienoic acids (EETs). Epoxyeicosatrienoic acids are released from astrocytes by glutamate which 'spills over' during neuronal activity. These locally released EETs shunt blood to metabolically active neurones providing substrate to support neuronal function. This short paper will discuss the findings which support the above scenario, the purpose of which is to provide a basis for future studies on the molecular mechanisms through which cerebral blood flow matches metabolism.

Animals↗

PubMed1998

Functional hyperemia in the brain: hypothesis for astrocyte-derived vasodilator metabolites.

BACKGROUND: Cerebral blood flow is tightly coupled to neuronal metabolic activity, a phenomenon referred to as functional hyperemia. The mechanisms underlying functional hyperemia in the brain have been extensively studied, but the link between neuronal activation and nutritive blood flow has yet to be defined. Recent investigations by our laboratory and others have identified a potential role for astrocytes as an intermediary cell type in this process. SUMMARY OF REVIEW: This short review will develop the hypothesis that cytochrome P450 epoxygenase activity in astrocytes catalyzes formation of epoxyeicosatrienoic acids (EETs), which act as potent dilators of cerebral vessels and are released in response to glutamate receptor activation within astrocytes. Neuronal activity stimulates release of arachidonic acid from the phospholipid pool of astrocytic membranes. We provide evidence that the arachidonic acid released on stimulation of glutamate receptors within astrocytes is metabolized by cytochrome P450 2C11 cDNA enzymes into EETs. CONCLUSIONS: The EETs thus formed will be released and activate K+ channels, increase outward K+ current, and hyperpolarize the plasma membrane. The resulting membrane hyperpolarization inhibits voltage-gated Ca2+ channels and leads to arteriolar dilation, thereby increasing regional nutritive blood flow in response to neuronal activity.

8,11,14-Eicosatrienoic Acid↗

PubMed1997

20-Hydroxyeicosatetraenoic acid-induced vasoconstriction and inhibition of potassium current in cerebral vascular smooth muscle is dependent on activation of protein kinase C.

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 metabolite of arachidonic acid, is a potent vasoconstrictor, and has been implicated in the myogenic activation of renal and cerebral arteries. We examined the role of protein kinase C (PKC) in the signal transduction pathway by which 20-HETE induces vasoconstriction and inhibition of whole-cell K+ current in cat cerebral vascular smooth muscle. 20-HETE induced a concentration-dependent constriction in isolated pressurized cat middle cerebral arteries (-29 +/- 8% at 1 microM). However, in the presence of an N-myristoylated PKC pseudosubstrate inhibitor peptide (MyrPsiPKC-I(19-27)), 20-HETE induced a concentration-dependent vasodilation (26 +/- 4% at 1 microM). In whole-cell voltage clamp studies, application of 20-HETE inhibited whole-cell K+ current recorded in cat cerebral vascular smooth muscle cells, an effect that was attenuated by MyrPsiPKC-I(19-27). Further evidence for the role of PKC activation in response to 20-HETE is the finding that 20-HETE increased the phosphorylation of myristoylated, alanine-rich PKC substrate in cultured cat cerebral vascular smooth muscle cells in a concentration- and PKC-dependent manner. These data provide evidence that PKC is an integral part of the signal transduction pathway by which 20-HETE elicits vasoconstriction of cerebral arteries and inhibition of whole-cell K+ current in cat cerebral vascular smooth muscle.

Animals↗

PubMed1996

20-HETE is an endogenous inhibitor of the large-conductance Ca(2+)-activated K+ channel in renal arterioles.

The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

Animals↗

PubMed1996

Coexistence of two types of Ca(2+)-activated K+ channels in rat renal arterioles.

Single-channel K+ currents were characterized in vascular smooth muscle cells freshly isolated from preglomerular arterioles (15-40 microns OD) of the rat. Under conditions of symmetrical K+ (145 mM), two types of single-channel K+ currents with unitary slope conductances of 68 +/- 2.6 and 251 +/- 4.9 pS were recorded from excised inside-out membrane patches. The open state probability (NPo) of these two types of K+ channels was voltage sensitive and the channels were highly selective for K+ over Na+. Elevation of intracellular calcium concentration ([Ca2+]i) from 0.1 to 0.5 microM on the cytoplasmic face of inside-out patches increased the frequency of opening and the NPo of both the 68-pS and the 251-pS K+ channels. Application of ATP (0.1-1 mM) to the internal surface of inside-out patches had no effect on the activities of both channel types. Internally applied Ba2+ (1 mM) blocked both of these channels. Externally applied tetraethylammonium (0.1-0.3 mM) or charybdotoxin (50 nM) blocked both the 68-pS and the 251-pS K+ channels. Externally applied apamin (50 nM), however, selectively blocked the 68-pS K+ channel but had no effect on the frequency of opening of the 251-pS K+ channel. Apamin also reduced macroscopic K+ current recorded from voltage-clamped rat renal arteriolar muscle cells by 25-30%. These results indicate the coexistence of two types of Ca(2+)-activated K+ channels in the membranes of vascular smooth muscle cell isolated from renal preglomerular arterioles of the rat that differ in unitary conductances and pharmacological properties.

Animals↗

PubMed1996

Stereospecific effects of epoxyeicosatrienoic acids on renal vascular tone and K(+)-channel activity.

The present study examined the effects of 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on the diameter of small renal arteries of the rat and assessed their action on K(+)-channel activity in vascular smooth muscle (VSM) cells isolated from these vessels. The R,S-isomer of 11,12-EET (1, 10, and 100 nM) increased the diameter of small renal arteries preconstricted with phenylephrine; however, the S,R-isomer was inactive. Both the R,S- and S,R-isomers of 14,15-EET had little effect on the diameter of these vessels even at a high concentration (100 nM). The vasodilator effect of 11(R),12(S)-EET was attenuated by tetraethylammonium (TEA, 1 mM) and iberiotoxin (100 nM), selective inhibitors of the large-conductance Ca(2+)-activated K+ (KCa) channel. In contrast, apamin (100 nM) and 4-aminopyridine (2 mM), which are inhibitors of other types of K+ channels, had no effect on the vasodilatory effect of 11,12-EET. In patch-clamp experiments, 100 nM racemic 11,12-EET increased outward K+ currents in VSM cells. Addition of the R,S-isomer or racemic 11,12-EET (1-100 nM), but not the S,R-isomer, increased the activity of KCa channel recorded from renal VSM cells with cell-attached patches. However, racemic EET had no effect on this channel when added to the internal (inside-out) or external (outside-out) face of excised membrane patches. These results suggest that 11,12-EET is a potent dilator of small renal arteries and that the R,S-isomer is the active enantiomer. The vasodilator effect of 11,12-EET appears to involve activation of KCa channel.

8,11,14-Eicosatrienoic Acid↗

PubMed1996

Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors.

Endothelial cells release several compounds, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF), that mediate the vascular effects of vasoactive hormones. The identity of EDHF remains unknown. Since arachidonic acid causes endothelium-dependent relaxations of coronary arteries through its metabolism to epoxyeicosatrienoic acids (EETs) by cytochrome P450, we wondered if the EETs represent EDHFs. Precontracted bovine coronary arteries relaxed in an endothelium-dependent manner to methacholine. The cytochrome P450 inhibitors, SKF 525A and miconazole, significantly attenuated these relaxations. They were also inhibited by tetraethylammonium (TEA),an inhibitor of Ca2+-activated K+ channels, and by high [K+]0 (20 mmol/L). Methacholine also caused hyperpolarization of coronary smooth muscle (-27 +/- 3.9 versus -40 +/- 5.1 mV), which was completely blocked by SKF 525A and miconazole. In vessels prelabeled with [3H] arachidonic acid, methacholine stimulated the release of 6-ketoprostaglandin F1alpha, 12-HETE, and the EETs. Arachidonic acid relaxed precontracted coronary arteries, which were also blocked by TEA, charybdotoxin, another Ca2+-activated K+ channel inhibitor, and high [K+]0. 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET relaxed precontracted coronary vessels (EC50, 1 X 10(-6) mol/L). The four regioisomers were equally active. TEA, charybdotoxin, and high [K+]0 attenuated the EET relaxations. 11,12-EET hyperpolarized coronary smooth muscle cells from -37 +/- 0.2 to -59 +/- 0.3 mV. In the cell-attached mode of patch clamp, both 14,15-EET and 11,12-EET increased the open-state probability of a Ca2+-activated K+ channel in coronary smooth muscle cells. This effect was blocked by TEA and charybdotoxin. These data support the hypothesis that the EETs are EDHFs.

8,11,14-Eicosatrienoic Acid↗

PubMed1996

Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes.

BACKGROUND AND PURPOSE: Brain parenchymal tissue metabolizes arachidonic acid (AA) via the cytochrome P450 (P450) epoxygenase to epoxyeicosatrienoic acids (EETs). EETs dilate cerebral arterioles and enhance K+ current in vascular smooth muscle cells from large cerebral arteries. Because of the close association between astrocytes and the cerebral microcirculation, we hypothesized that brain epoxygenase activity originates from astrocytes. This study was designed to identify and localize an AA epoxygenase in rat brain astrocytes. We also tested the effect of EETs on whole-cell K+ current in rat cerebral microvascular smooth muscle cells. METHODS: A functional assay was used to demonstrate endogenous epoxygenase activity of intact astrocytes in culture. Oligonucleotide primers derived from the sequence of a known hepatic epoxygenase, P450 2C11, were used in reverse transcription/polymerase chain reaction of RNA isolated from cultured rat astrocytes. The appropriate size reverse transcription/polymerase chain reaction product was cloned into a plasmid vector and sequenced. A polyclonal peptide antibody was raised against P450 2C11 and used in Western blotting and immunocytochemical staining of cultured astrocytes. A voltage-clamp technique was used to test the effect of EETs on whole-cell K+ current recorded from rat cerebral microvascular muscle cells. RESULTS: Based on elution time of known standards and inhibition by miconazole, an inhibitor of P450 AA epoxygenase, cultured astrocytes produce 11,12- and 14,15-EETs when incubated with AA. The sequence of a cDNA derived from RNA isolated from cultured rat astrocytes was 100% identical to P450 2C11. Immunoreactivity to glial fibrillary acidic protein, a marker for astrocytes, colocalized with 2C11 immunoreactivity in double immunochemical staining of cultured astrocytes. EETs enhanced outward K+ current in muscle cells from rat brain microvessels. CONCLUSIONS: Our results demonstrate that a P450 2C11 mRNA is expressed in astrocytes and may be responsible for astrocyte epoxygenase activity. Given the vasodilatory effect of EETs, our findings suggest a role for astrocytes in the control of cerebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral microvessels may involve activation of K+ channels.

8,11,14-Eicosatrienoic Acid↗

PubMed1995

Shear activated channels in cell-attached patches of cultured bovine aortic endothelial cells.

We investigated the response of inward rectifier K+ (IRK) currents in bovine aortic endothelial cells (BAECs) to shear stress. Shear evoked reversible hyperpolarization in current clamped BAECs. Voltage clamped BAECs exhibited large inward and small outward whole cell K+ currents blocked by cesium and increased in amplitude by exposure to shear stress. The open state probability of IRK channels in cell-attached membrane patches was increased within minutes of exposure to shear stress. IRK channels in inside-out patches were activated by increases in [Ca2+]i from 10(-7) to 10(-6) mM. We demonstrate that shear stress induces hyperpolarization and gating of single channel and whole cell IRK currents in BAECs.

Animals↗

PubMed1995

Halothane and isoflurane decrease the open state probability of K+ channels in dog cerebral arterial muscle cells.

BACKGROUND: Both halothane and isoflurane evoke cerebral vasodilation. One of the potential mechanisms for arterial vasodilation is enhanced K+ efflux resulting from an increased opening frequency of membrane K+ channels. The current study was designed to determine the effects of volatile anesthetics on K+ channel current in single vascular smooth muscle cells isolated from dog cerebral arteries. METHODS: Patch clamp recording techniques were used to investigate the effects of volatile anesthetics on macroscopic and microscopic K+ channel currents. RESULTS: In the whole-cell patch-clamp mode, in cells dialyzed with pipette solution containing 2.5 mM EGTA and 1.8 mM CaCl2, depolarizing pulses from -60 to +60 mV elicited an outward K+ current that was blocked 65 +/- 5% by 3 mM tetraethylammonium (TEA). Halothane (0.4 and 0.9 mM) depressed the amplitude of this current by 18 +/- 4% and 34 +/- 6%, respectively. When 10 mM EGTA was used in the pipette solution to strongly buffer intracellular free Ca2+, an outward K+ current insensitive to 3 mM TEA was elicited. This K+ current, which was reduced 51 +/- 4% by 1 mM 4-aminopyridine, was also depressed by 17 +/- 5% and 29 +/- 7% with application of 0.4 and 0.9 mM halothane, respectively. In cell-attached patches using 145 mM KCl in the pipette solution and 5.2 mM KCl in the bath, the unitary conductance of the predominant channel type detected was 99 pS. External application of TEA (0.1 to 3 mM) reduced the unitary current amplitude of the 99 pS K+ channel in a concentration-dependent manner. The open state probability of this 99 pS K+ channel was increased by 1 microM Ca2+ ionophore (A23187). These findings indicate that the 99 pS channel measured in cell-attached patches was a TEA-sensitive, Ca(2+)-activated K+ channel. Halothane and isoflurane reversibly decreased the open state probability (NPo), mean open time, and frequency of opening of this 99 pS K+ channel without affecting single channel amplitude or the slope of the current-voltage relationship. CONCLUSIONS: Halothane and isoflurane suppress the activity of K+ channels in canine cerebral arterial cells. These results suggest that mechanisms other than K+ channel opening likely mediate volatile anesthetic-induced vasodilation.

Animals↗

PubMed1994

Hypoxia increases the activity of Ca(2+)-sensitive K+ channels in cat cerebral arterial muscle cell membranes.

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O2, PO2, on the predominant K+ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K(+)-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NPo) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]i) between 0 and 100 microM, but was significantly increased upon elevation of intracellular free Ca2+ concentration ([Ca2+]i). Low concentrations of external tetraethylammonium (0.1-3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O2 from 21% to < 2% reversibly increased the NPo, mean open time, and event frequency of the Ca(2+)-sensitive, high-conductance single-channel K+ current recorded at a patch potential of +20 mV. A similar reduction in PO2 also produced a transient increase in the activity of the 215-pS K+ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca2+]i or intracellular pH (pHi) of cat cerebral arterial muscle cells, as measured using Ca(2+)- or pH-sensitive fluorescent probes. Reduced PO2 caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pretreatment with 1 mM tetraethylammonium. These results suggest that reduced PO2 increases the activity of a high-conductance, Ca(2+)-sensitive K+ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca2+]i and pHi.

Animals↗

PubMed1994

Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels.

The purpose of this study was to determine whether arachidonic acid can be converted to 20-hydroxyeicosatetraenoic acid (HETE) by P-450 enzymes in cat cerebral microvasculature, to identify the P-450 isoforms responsible for the formation of this metabolite, and to characterize the vasoactive effects of 20-HETE on these vessels. Cerebral microvessels were isolated by filling them with a suspension of magnetized iron oxide (particle size = 10 microns) and separated from minced cerebral cortical tissue using a magnet. Cat cerebral microvessels were homogenized and incubated with [14C]arachidonic acid (AA), and cytochrome P-450-dependent metabolites of AA were separated by reverse-phase high-pressure liquid chromatography. A major metabolite that coeluted with synthetic 20-HETE was identified. The formation of this metabolite was dependent on NADPH and was inhibited by 17-octadecynoic acid (ODYA), a specific suicide-substrate inhibitor of the omega-hydroxylation of AA by P-450 enzymes. Western blot analysis confirmed the presence of a P-450 enzyme of the 4A gene family in cat cerebral microvessels. Gas chromatography/mass spectrometry analysis revealed that this metabolite has an identical mass-to-charge ratio (391 m/z) as that of standard 20-HETE. Exogenous 20-HETE constricted pressurized cat pial arteries in a concentration-dependent manner with a threshold concentration of < 1.0 nM. 20-HETE (1 nM) inhibited the activity of a 217-pS K+ channel recorded in cell-attached patches of isolated cat cerebral microvascular muscle cells. Blockade of endogenous P-450 activity with 17-ODYA markedly increased the activity of the 217 pS K+ channel in these cells, an action that was completely reversed by a nanomolar concentration of 20-HETE, suggesting that 20-HETE might be an endogenous modulator of the 217 pS K+ channel in cerebral arterial muscle cells. These results demonstrate the presence of P-450 4A enzyme activity in the cerebral microvasculature of the cat that converts AA to 20-HETE. The potent vasoconstrictor effects of 20-HETE on cerebral vessels suggests that metabolites of P-450 enzymes of the 4A gene family could play an important role in regulating cerebral microvascular tone.

Animals↗

PubMed1993

Modulation of vascular tone in renal microcirculation by erythrocytes: role of EDRF.

The effect of erythrocytes (red blood cells, RBC) on vascular tone in the renal microcirculation was examined using the juxtamedullary nephron microvascular preparation perfused in vitro with a physiological salt solution containing 5% albumin. The basal diameters of the arcuate, interlobular, proximal, and distal afferent arterioles averaged 444 +/- 24, 74 +/- 3, 29 +/- 1, and 19 +/- 1 micron, respectively, when perfused with a cell-free solution at a pressure of 80 mmHg. The diameters of the arcuate and interlobular arteries increased by 14 +/- 4 and 13 +/- 4%, respectively, whereas the diameter of the proximal and distal portions of the afferent arterioles decreased by 7 +/- 2% when perfusion pressure was elevated from 80 to 160 mmHg. The addition of RBC to the perfusate reduced the basal diameters of interlobular and afferent arterioles by 11 +/- 4 and 15 +/- 3%, respectively. The maximal vasoconstrictor response was seen after the addition of only 1% RBC to the perfusate. Removal of platelets did not block the vasoconstrictor response to addition of RBC to the perfusate. The role of endothelium-derived relaxing factor (EDRF) in the vasoconstrictor response to RBC was studied by addition of nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NNA, 100 microM) to the perfusate. L-NNA reduced the basal diameters of interlobular and afferent arterioles by 7 +/- 3 and 9 +/- 3%, respectively, and abolished the vasoconstrictor response to RBC. L-NNA had no effect on the pressure-diameter relationships of the preglomerular vasculature when added to perfusates already containing RBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗