The genesis and unique properties of the lymphovascular tumor embolus are because of calpain-regulated proteolysis of E-cadherin.

The genesis and unique properties of the lymphovascular tumor embolus are poorly understood largely because of the absence of an experimental model that specifically reflects this important step of tumor progression. The lymphovascular tumor embolus is a blastocyst-like structure resistant to chemotherapy, efficient at metastasis and overexpressing E-cadherin (E-cad). Conventional dogma has regarded E-cad as a metastasis-suppressor gene involved in epithelial-mesenchymal transition. However, within the lymphovascular embolus, E-cad and its proteolytic processing by calpain and other proteases have a dominant oncogenic rather than suppressive role in metastasis formation and tumor cell survival. Studies using a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated the equivalence of xenograft-generated spheroids with lymphovascular emboli in vivo with both structures demonstrating E-cad overexpression and specific proteolytic processing. Western blot revealed full-length (FL) E-cad (120 kDa) and four fragments: E-cad/NTF1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa) and E-cad/NTF4 (80 kDa). Compared with MARY-X, only E-cad/NTF1 was present in the spheroids. E-cad/NTF1 was produced by calpain, E-cad/NTF2 by γ-secretase and E-cad/NTF3 by a matrix metalloproteinase (MMP). Spheroidgenesis and lymphovascular emboli formation are the direct result of calpain-mediated cleavage of E-cad and the generation of E-cad/NTF1 from membrane-associated E-cad rather than the de novo presence of either E-cad/NTF1 or E-cad/CTF1. E-cad/NTF1 retained the p120ctn-binding site but lost both the ß-catenin and α-binding sites, facilitating its disassembly from traditional cadherin-based adherens junctions and its 360° distribution around the embolus. This calpain-mediated proteolysis of E-cad generates the formation of the lymphovascular embolus and is responsible for its unique properties of increased homotypic adhesion, apoptosis resistance and budding.

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.

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.

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.

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.

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.

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.

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.

Diminished influenza A virus-specific MHC class I-restricted cytotoxic T lymphocyte activity among elderly persons.

Influenza A virus-specific MHC class I-restricted cytotoxic T lymphocyte (CTL) activities among young and elderly adults were compared. Peripheral blood lymphocytes from 10 young adults, (mean age 27 +/- 2.4 years) and elderly persons (mean age 71 +/- 1.6 years) were stimulated with influenza A/Taiwan/1/86 (H1N1) virus for 7 days and assayed for lytic activity against A/Taiwan, A/Shanghai (H3N2), and B/USSR virus-infected autologous target cells. Young adults exhibited significantly higher influenza A cross-reactive CTL activity against A/H1N1 and A/H3N2 target cells when compared to aged persons. This was true at all effector-to-target cell ratios tested. Negligible lysis of B/USSR-infected target cells or nonautologous A/Taiwan-infected cells was observed. The number of leukocytes recovered per milliliter of blood was also significantly higher in young adults than in old donors; however, the percentage of CD45+ (common leukocyte antigen), CD3+ (T cells), CD4+ (T helper), and CD8+ (T cytotoxic/suppressor) as well as the CD4+/CD8+ ratios was similar in both groups. Depletion of cells with monoclonal antibodies indicated that the effector cells were CD8+ T cells. Serum-neutralizing antibody (Nt Ab) titers were similar among young and elderly persons and there was no correlation between Nt Ab and CTL activity. These results demonstrate a reduced influenza virus-specific MHC class I-restricted CTL activity among elderly persons. The deficiency in this cell-mediated immune function may contribute to the morbidity and mortality from influenza virus infections in this population.

Pharmacokinetics of 4-aminopyridine in human volunteers. A preliminary study using a new GLC method for its estimation.

The concentrations of 4-aminopyridine hydrochloride in the blood and urine from volunteers were measured following a bolus injection i.v. of 0.3 mg kg-1. The drug was assayed by means of a new GLC method which is described. The pharmacokinetics of 4-aminopyridine are complicated by an additional increase in plasma concentration during the elimination phase of the drug.

Hepatic and renal disposition of pancuronium and gallamine in patients with extrahepatic cholestasis.

The plasma clearance of pancuronium in patients with extrahepatic cholestasis was 16% lower than in a control group (1.47 +/- 0.11 ml min-1 kg-1 v. 1.76 +/- 0.21 ml min-1 kg-1), but the difference was not significant. A significant increase in the elimination half-life T 1/2 beta of pancuronium (from 141 to 224 min) and a significant increase in the volume of the peripheral compartment (V2) was found in patients with extrahepatic cholestasis when compared with control patients. There was a significantly lower cumulative biliary excretion of pancuronium (0.3 +/- 0.3% v. 10.9 +/- 3.2% in the controls) during the 48-h period of observation. The biotransformation and cumulative urinary excretion patterns of pancuronium revealed no significant differences between the two groups of patients. The increase of T 1/2 beta pancuronium in patients with extrahepatic cholestasis was mainly a consequence of the increase in the volume of distribution. No significant differences in the plasma clearance, T 1/2 beta or in the volume of distribution were observed with gallamine in the patients with extrahepatic cholestasis when compared with the control group. The cumulative urinary excretion of gallamine during 48 h in both groups of patients was approximately 100%. We concluded that in patients with cholestasis and normal glomerular filtration, gallamine is probably more reliable than pancuronium for neuromuscular blockade.

Effect of experimental cholestasis on neuromuscular blocking drugs in cats.

Pancuronium, Org 6368 and gallamine were compared in control cats and in cats with experimental cholestasis. A decrease in the plasma clearance and a prolongation of neuromuscular blockade with Org 6368 and pancuronium were found in the latter; no significant difference was detected in the biotransformation pattern of Org 6368 and pancuronium compared with controls. Inhibition of hepatic uptake of Org 6368 and pancuronium in extrahepatic cholestasis might explain the significant alterations in the pharmacokinetics of the two steroid neuromuscular blocking drugs. The pharmacokinetics of gallamine were normal during cholestasis. The results suggest that, under pathological conditions involving increased plasma concentrations of bile salts, neuromuscular blocking agents that are cleared from the plasma by the liver may have an impaired hepatic uptake and consequently a prolonged duration of action.