Biomedical subjects

D R Harder

Publications and source records attributed to D R Harder.

At least 37 records · Page 2Linked to original sources

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

Contribution of 20-HETE to the vasodilator actions of nitric oxide in renal arteries.

The present study examined the contribution of elevations in cGMP versus inhibition of cytochrome P-4504A enzymes and the production of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) to the vasodilator actions of NO in renal arterioles. The NO donor sodium nitroprusside (SNP) at 10(-5), 10(-4), and 10(-3) M reduced the production of 20-HETE in microsomes prepared from renal arterioles to 80 +/- 2, 43 +/- 5, and 7 +/- 1% of control, respectively (n = 4). In other experiments, the vasodilator response to SNP (10(-7) to 10(-3) M) was examined in rat renal interlobular arteries (<90 micron ID), preconstricted with phenylephrine (1 microM) under control conditions and after blockade of the cGMP and P-4504A pathways. Inhibition of guanylyl cyclase with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (10 microM, n = 6) or of cGMP-dependent protein kinase with 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT-5823, 1 microM; n = 5) attenuated the vasodilator response to SNP by 26 and 30%, respectively. In contrast, inhibition of the endogenous production of 20-HETE with a suicide substrate, irreversible inhibitor [17-octadecynoic acid (17-ODYA), 1 microM, n = 5], or a selective, competitive inhibitor of 20-HETE formation (dibromo-dodecenyl-methylsulfimide, 25 microM, n = 5) markedly impaired the vasodilator response to SNP by 76 and 78%, respectively. Similarly, when 20-HETE levels were fixed at 100 nM (n = 6), the response to SNP was attenuated by 73%. Blockade of both pathways with ODQ and 17-ODYA completely abolished the response to SNP (n = 6). These results indicate that the vasodilator response to NO is largely cGMP independent and that inhibition of 20-HETE formation contributes to the cGMP-independent effects of NO in the renal microcirculation.

Alkaloids↗

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↗

PubMed1998

Tissue sources of cytochrome P450 4A and 20-HETE synthesis in rabbit lungs.

We previously reported that 20-hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P450 (cP450) 4A metabolite of arachidonic acid (AA) in human lung tissue, and is a potent cyclooxygenase-dependent vasodilator of isolated pulmonary arteries. In the present investigations, we identified sources of cP450 4A immunospecific protein, messenger RNA (mRNA), and 20-HETE synthesis in rabbit lungs. Microsomes of peripheral lung tissue, airways, small and large vessels, and lysates of alveolar macrophages all express proteins of approximately 50 kD which cross-reacted with a primary antibody raised against rat liver cP450 4A1. Peripheral lung tissue, small and large pulmonary arteries, airways, and isolated vascular smooth muscle cells from small pulmonary arteries produced 20-HETE when incubated with AA. Expression of cP450 4A6/4A7 mRNA was readily detectable by reverse transcription-polymerase chain reaction using isoform-specific probes and 5 microg total RNA extracted from microdissected small pulmonary arteries. These data demonstrate that small pulmonary arteries express cP450 4A proteins and vascular smooth muscle cells derived from these arteries synthesize 20-HETE. Furthermore, cP450 4A appears to be widely distributed in rabbit tissue, raising the possibility that 20-HETE generated from nonvascular tissue could serve as a paracrine factor in the pulmonary circulation.

Animals↗

PubMed1997

Role of P-450 arachidonic acid epoxygenase in the response of cerebral blood flow to glutamate in rats.

BACKGROUND AND PURPOSE: Glutamate, a major excitatory neurotransmitter in the brain, has been implicated in the hyperemic response to increases in the activity of neurons, but the mechanism of glutamate-induced dilation of cerebral blood vessels is unknown. Glutamate has been shown to enhance the release of arachidonic acid (AA) in brain tissue and cultured astrocytes. We have previously shown that astrocytes metabolize AA to vasodilator products, epoxyeicostrienoic acids (EETs), and express a P-450 AA epoxygenase, P-450 2C11. We tested the hypothesis that glutamate-induced dilation of cerebral arterioles is mediated in part by changes in the formation and release of EETs by perivascular astrocytes. METHODS: Primary astrocyte cultures were prepared from 3-day-old rat pups. The cells were labeled with [14C]AA, and the effect of glutamate on the formation of EETs from [14C]AA by cultured astrocytes was studied. The expression of P-450 2C11 protein in the microsomal fractions of cultured astrocytes was assessed by Western blot. In vivo cerebral blood flow measurements were made in adult rats by laser-Doppler flowmetry after administration of glutamate into the subdural space of the rat before and after treatment with miconazole. RESULTS: Glutamate treatment (100 mumol/L for 30 minutes) induced a threefold increase in the formation of EETs from [14C]AA by cultured astrocytes, and the increase was inhibited by miconazole (20 mumol/L), an inhibitor of P-450 AA epoxygenase. Treatment with glutamate (100 mumol/L) for 12 hours increased the expression of P-450 2C11 protein in the microsomal fraction of cultured astrocytes. The response of laser-Doppler cerebral blood flow to administration of glutamate (500 mumol/L) into the subdural space of the rat was significantly attenuated after treatment with miconazole (20 mumol/L for 30 minutes). CONCLUSIONS: These findings suggest a role for a P-450 AA epoxygenase in astrocytes in the coupling between the metabolic activity of neurons and regional blood flow in the brain.

Animals↗

PubMed1996

Inhibition of brain P-450 arachidonic acid epoxygenase decreases baseline cerebral blood flow.

Arachidonic acid (AA) is metabolized by the cytochrome P-450 (P-450) epoxygenase pathway to epoxyeicosatrienoic acids (EETs) in the brain parenchymal tissue and perivascular astrocytes. EETs dilate cerebral microvessels and enhance K+ current in cerebrovascular smooth muscle cells. In the current study, the effect of a subdural administration of miconazole, an inhibitor of P-450 epoxygenase, on microvascular perfusion of rat cerebral cortex was evaluated using laser-Doppler flowmetry (LDF) Baseline cerebral blood flow (CBF) decreased by 29.7 +/- 7.3% (n = 5) after administration of 20 microM miconazole into the subdural space for 30 min. Responses of CBF to sodium nitroprusside and 5-hydroxytryptamine were unaltered by miconazole treatment. Administration of vehicle alone in time-control experiments had no effect on CBF. In other experiments, the effects of miconazole on the metabolism of [14C]AA by cultured rat astrocytes and on nitric oxide synthase activity in homogenates of rat brain were examined. Miconazole inhibited conversion of AA to EETs by cultured astrocytes but had no effect on the conversion of L-arginine to L-citrulline by homogenates of rat brain. These results implicate endogenous P-450 epoxides of AA in the regulation of basal blood flow in cerebral microcirculation.

Animals↗

PubMed1996

Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles.

The present study examined whether preglomerular arterioles of the rat produce 20-hydroxyeicosatetraenoic acid (20-HETE) and whether 20-HETE is vasoactive on these vessels. Raf preglomerular arterioles produced 20-HETE (4.8 +/- 1.0 pmol.min-1.mg-1, n = 7) and, to a lesser extent, 14-, 15-, 11-, and 12-dihydroxyeicosatetraenoic acid, 6-ketoprostaglandin F/alpha and prostaglandin E2 when incubated with [14C]larachidonic acid. The results of immunoblotting and reverse-transcription polymerase chain reaction experiments indicate that these vessels express mRNA and protein for a P-450 4A2 enzyme. With the use of a rat juxtamedullary nephron microvascular preparation perfused in vitro with a cell-free media, addition of 20-HETE (1 nM-1 microM) to the bath reduced the diameter of proximal and distal portions of the efferent arterioles. At a concentration of 1 microM, the diameter of the proximal and distal portions of the afferent arteriole fell by 14 +/- 1 and 16 +/- 3% after 20-HETE. The response to 20-HETE (1 microM) was not altered by blockade of cyclooxygenase, lipoxygenase, and p-450 pathways. Blockade of the large-conductance Ca(2+)-activated K+ channel with tetraethylammonium (1 mM) reduced the diameter of afferent arterioles by 10% and blocked the vasoconstrictor response to 20-HETE (1 microM). These results indicate that 20-HETE is an endogenous constrictor of preglomerular arterioles and suggest a role for the P-450 4A2 enzyme in the regulation of renal vascular tone.

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

Identification of a putative microvascular oxygen sensor.

The vascular response to changes in oxygen levels in the blood and tissue is a highly adaptive physiological response that functions to match tissue oxygen supply to metabolic demand. Defining the cellular mechanisms that can sense physiologically relevant changes in PO2 and adjust vascular diameter are vital to our understanding of this process. A cytochrome P450 (P450) enzyme of the 4A family of omega-hydroxylases was localized in renal microvessels, renal cortex, and a striated muscle microvascular bed (cremaster) of the rat. In the presence of molecular oxygen, this P450 enzyme catalyzes formation of 20-HETE from arachidonic acid (AA). Prior studies have shown that 20-HETE potently contracts renal and cerebral arteries and arterioles. The present study demonstrates that 20-HETE constricts striated muscle arterioles as well. In both intact renal microvessels and enriched renal cortical microsomal enzyme preparations, the formation of 20-HETE was linearly dependent on PO2 between 20 and 140 mm Hg. Homogenates of cremaster tissue produced 20-oxygen HETE when incubated with AA. They also expressed message for P450 4A enzyme, as determined by Southern and Western blots. Administration of 17-octadecynoic acid (17-ODYA), which is a P450 4A inhibitor, attenuated the constriction of third-order cremasteric arterioles in response to elevation of superfusion solution PO2 from approximately equal to 3 to 5 mm Hg to approximately equal to 35 mm Hg. 17-ODYA had no effect on basal vascular tone or response of cremaster arterioles to vasoactive compounds. These results demonstrate the existence of P450 omega-hydroxylase activity and 20-HETE formation in the vasculature and parenchyma of at least two microvascular beds. Our data suggest that a P450 enzyme of the 4A family has the potential to function as an oxygen sensor in mammalian microcirculatory beds and to regulate arteriolar caliber by generating 20-HETE in an oxygen-dependent manner.

Animals↗

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↗

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

Pressurization of isolated renal arteries increases inositol trisphosphate and diacylglycerol.

Inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) concentrations were measured in isolated, cannulated dog renal arteries under control conditions (0 mmHg) and in response to step elevations in transmural pressure. There was a pressure-dependent increase in IP3 at 60 and 120 mmHg, reaching significance at 120 mmHg (P < 0.05) and a significant increase in DAG at both 60 and 120 mmHg measured after maintaining pressure for 15 min. Similarly, IP3 measurements made 90 s after a step increase in transmural pressure also exhibited a pressure-dependent profile, again reaching significance at 120 mmHg. Calculation of active tension demonstrated these renal arteries developed pressure-dependent myogenic tone. To assess the role of the endothelium in this regard, IP3 was measured before and after endothelial removal at 0 and 60 mmHg. Pressure-dependent myogenic tone was still present upon endothelial removal. In the absence of the endothelium, we observed a significant increase in total IP3 at 60 compared with 0 mmHg; furthermore, the increase in IP3 in the absence of the endothelium was significantly greater than that observed when the endothelium was intact. Given that the primary source of IP3 is via the actions of phospholipase C (PLC) on phosphatidylinositol 4,5-bisphosphate, these biochemical data directly demonstrate that elevation of transmural pressure in dog renal arteries activates PLC.

Analysis of Variance↗

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↗