Sigma receptors modulate the excitability of peptidergic nerve terminals in the neurohypophysis by inhibiting voltage-dependent K¤ channels (IK) (Wilke et al. 1999a). The activation of sigma receptors by a variety of ligands reduces current flow through two distinct K¤ channel types, the A-current channel (IA) and the Ca¥-activated K¤ channel (IBK). Current is reduced by the same proportion over the entire accessible voltage range, with no shift in the voltage dependence of activation or inactivation. Furthermore, the residual unblocked currents inactivate with very similar rates, indicating that sigma receptor modulation entails a shutting down of function rather than a modification of gating behaviour (Wilke et al. 1998(Wilke et al. , 1999a. A comparison of the concentration dependence of IA reduction with that of IBK reduction indicated that the sigma receptor ligand PPHT inhibits both of these channels with a very similar EC50 (Wilke et al. 1998); similar results were obtained with haloperidol (Wilke et al. 1999a). Both IA and IK were reduced proportionally by a large number of sigma receptor ligands (including ditolylguanidine, SKF10047, pentazocine, haloperidol, PPHT, U101958, and apomorphine), suggesting that in the rat the same receptor is coupled to two types of K¤ channels. In Dµ, D× and DÚ dopamine receptor-deficient mice, sigma receptor ligands reduced IK as effectively as in wild-type mice, indicating that the responses are not mediated by dopamine receptor subtypes known to interact with some sigma receptor ligands (Wilke et al. 1999a 1. Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K¤ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K¤ channels in peptidergic nerve terminals. 2. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. 3. The sigma receptor ligands pentazocine and SKF10047 modulated K¤ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPâS), a G_protein activator (GTPãS) or a non-hydrolysable ATP analogue (AMPPcP). 4. Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. 5. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor prot...

oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time-and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47 phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47 phox and tyrosine phosphorylation of p47 phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid. superoxide; Rac1/2; hydrogen peroxide; CYP4A; reactive oxygen species PRODUCTS OF CYTOCHROME P-450 (CYP) -hydroxylases (including CYP4 isoforms) mediate key physiological functions including autoregulation of blood flow, tubuloglomerular feedback, Na ϩ reabsorption in the kidney, and relaxation of pulmonary arterioles (29,38). Our studies have focused on the role of CYP4 and its arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), in pulmonary vascular function and biology. 20-HETE is the CYP -hydroxylation product of AA cleaved from membrane phospholipid sources. Enzymes of the CYP4A, -4B, and -4F families catalyze the -hydroxylation of fatty acids, and several isoforms in these families produce 20-HETE when incubated with AA. For example, rat CYP4A1, -4A2, and -4A3 catalyze AA -and -1-hydroxylations with the highest catalytic efficiency accruing to CYP4A1 (35). Although CYP4A2 and CYP4A3 exhibit an additional arachidonate 11,12-epoxidation activity, CYP4A1 operates solely as an -hydroxylase. Most investigators suggest that CYP4 isoforms constitute the major source of 20-HETE synthesis in extrahepatic tissues, including the lung (35, 38). Accordingly, we have investigated the effects of CYP4 product, 20-HETE, in our studies of this system in pulmonary vascular biology.We have identified a unique role f...

The cocaine photoaffinity label 3-iodo-4-azidococaine ([125I]IACoc) binds to the sigma-1 receptor with an affinity that is 2-3 orders of magnitude higher than the parent compound cocaine [Kahoun, J. R., and Ruoho, A. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. In the present study, the binding properties of several cocaine derivatives to the guinea pig liver sigma-1 receptor were determined. The results from assessing the affinity of various derivatives of cocaine which were substituted on the phenyl ring indicated that an important determinant of binding to the guinea pig sigma-1 receptor binding site may be the development of a dipole in the ring in which the pi electron density of the phenyl ring is reduced. This implies that an electron-rich source is present in the sigma-1 receptor binding site, such as the pi system of an aromatic ring or other electron-rich side chains, which interact with the phenyl ring of cocaine. The precise [125I]IACoc derivatization site in the guinea pig sigma-1 receptor was identified using chemical cleavage and purification of the resulting labeled peptides. Cyanogen bromide cleavage of the [125I]IACoc photolabeled sigma-1 receptor followed by radiosequencing identified Asp188, which is located in the putative steroid binding domain-like II (SBDL II) near the carboxyl terminus, as the site of [125I]IACoc insertion. Systematic truncation of the C-terminus indicated the requirement for the last 15 amino acid residues of the receptor for [125I]IACoc photolabeling.

Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membranespanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligands N-allylnormetazocine (SKF10047), ditolylguanidine, and (؎)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (I K ). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition in I K , and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 M. SKF10047 also inhibited I K with a similar EC 50 (14 M). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5 -O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism.Sigma receptors are widely distributed in neuronal and nonneuronal tissue and are distinguished by their ability to bind a broad range of chemically unrelated ligands, including (ϩ)-opiates, neuroleptic drugs, ditolylguanidine (DTG), 1 and phencyclidine-related compounds (1-3). Although the biological activity of ligands suggests that sigma receptors may be involved in behavioral, psychological, and motor functions (1-3), the cellular actions of sigma receptors are poorly understood. Recent studies in melanotrophs (4) and the neurohypophysis (5) showed that sigma receptor ligands inhibit voltage-activated potassium current (I K ), but the signal transduction pathways associated with sigma receptor activation remain unknown.Molecular characterization of sigma receptors has raised intriguing questions about how these receptors generate cellular responses. The high affinity sigma receptor photoprobe iodoazidococaine (IAC) has labeled a 26-kDa protein in rat liver, rat brain, and human placenta (6). Cloning studies have confirmed that both human and rodent sigma receptor cDNAs encode a 25.3-kDa protein (7-10). The protein ...