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Background/aims: Intravascular volume expansion due to sodium retention is involved in the pathogenesis of obesity-related hypertension. Institution of high fat diet (HFD) feeding leads to an initial state of positive sodium balance due to enhanced tubular reabsorption of sodium, but which tubular sodium transporters are responsible for this remains undefined., Methods: C57/Bl6 mice were fed control or HFD for 3 weeks. Blood pressures were recorded by tail cuff method. Sodium transporter expression and phosphorylation were determined by Western blotting. In vivo activity of NCC was determined using natriuretic responses to hydrochlorothiazide. Expression of NCC mRNA was determined using qPCR., Results: At 3 weeks HFD mice had significant weight gains compared to control mice, but blood pressures were not yet elevated. There were no changes in expression or phosphorylation of the bumetanide-sensitive cotransporter, NKCC2, or in expression of subunits of the amiloride-sensitive ion channel, ENaC. However, there were significant increases in mRNA and protein expression of the thiazide-sensitive co-transporter, NCC, in kidneys from HFD mice. Consistent with this, HFD mice had increased in vivo activity of NCC., Conclusions: Increased expression of NCC promotes the sodium loading response to institution of HFD feeding before onset of hypertension., (© 2015 S. Karger AG, Basel.)

Perturbations of the expression of transporters and drug-metabolizing enzymes (DMEs) by opioids can be the locus of deleterious drug-drug interactions (DDIs). Many transporters and DMEs are regulated by xenobiotic receptors [XRs; e.g., pregnane X receptor (PXR), constitutive androstane receptor (CAR), and Aryl hydrocarbon receptor (AhR)]; however, there is a paucity of information regarding the influence of opioids on XRs. The objective of this study was to determine the influence of oxycodone administration (15 mg/kg intraperitoneally twice daily for 8 days) on liver expression of XRs, transporters, and DMEs in rats. Microarray, quantitative real-time polymerase chain reaction and immunoblotting analyses were used to identify significantly regulated genes. Three XRs (e.g., PXR, CAR, and AhR), 27 transporters (e.g., ABCB1 and SLC22A8), and 19 DMEs (e.g., CYP2B2 and CYP3A1) were regulated (P < 0.05) with fold changes ranging from -46.3 to 17.1. Using MetaCore (computational platform), we identified a unique gene-network of transporters and DMEs assembled around PXR, CAR, and AhR. Therefore, a series of transactivation/translocation assays were conducted to determine whether the observed changes of transporters/DMEs are mediated by direct activation of PXR, CAR, or AhR by oxycodone or its major metabolites (noroxycodone and oxymorphone). Neither oxycodone nor its metabolites activated PXR, CAR, or AhR. Taken together, these findings identify a signature hepatic gene-network associated with repeated oxycodone administration in rats and demonstrate that oxycodone alters the expression of many transporters and DMEs (without direct activation of PXR, CAR, and AhR), which could lead to undesirable DDIs after coadministration of substrates of these transporters/DMEs with oxycodone.

ATP-sensitive K(+) (K(ATP)) channel subunits SUR2A and SUR2B in the rat brain were investigated by RT-PCR assay, western blot analysis, in situ hybridization histochemistry, and immunohistochemical staining. The results show that the mRNA and protein of SUR2A and SUR2B are expressed in whole rat brain extracts and selected regions. SUR2 mRNA is widely expressed in many neurons and glial cells as revealed by in situ hybridization histochemistry. Immunohistochemical staining shows SUR2A to be widely expressed in neurons of the brain, especially in the large pyramidal neurons and their main dendrites in the neocortex and in the Purkinje cells of the cerebellar cortex. In contrast to SUR2A, SUR2B is potently expressed in small cells in the corpus callosum and cerebellar white matter, but is also weakly expressed in some neurons. Double immunostaining shows SUR2B to be localized in astrocytes and oligodendrocytes, while SUR2A is only localized in oligodendrocytes. These results suggest that SUR2A might be mainly a regulatory subunit of the K(ATP) channel in most neurons and part of oligodendrocytes, while SUR2B might be mainly a regulatory subunit of the K(ATP) channel in astrocytes, oligodendrocytes, and some neurons., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

The sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channel is a nonselective cation channel that is regulated by intracellular calcium and adenosine triphosphate. The channel is not constitutively expressed, but is transcriptionally upregulated de novo in all cells of the neurovascular unit, in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). The channel is linked to microvascular dysfunction that manifests as edema formation and delayed secondary hemorrhage. Also implicated in oncotic cell swelling and oncotic (necrotic) cell death, the channel is a major molecular mechanism of 'accidental necrotic cell death' in the CNS. In animal models of SCI, pharmacological inhibition of Sur1 by glibenclamide, as well as gene suppression of Abcc8, prevents delayed capillary fragmentation and tissue necrosis. In models of stroke and TBI, glibenclamide ameliorates edema, secondary hemorrhage, and tissue damage. In a model of SAH, glibenclamide attenuates the inflammatory response due to extravasated blood. Clinical trials of an intravenous formulation of glibenclamide in TBI and stroke underscore the importance of recent advances in understanding the role of the Sur1-regulated NC(Ca-ATP) channel in acute ischemic, traumatic, and inflammatory injury to the CNS.

Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid, are endogenously produced epoxides that act as substrates for the soluble epoxide hydrolase (sEH). Recent studies indicate that EETs increase the tension of rat pulmonary arteries (PAs), and inhibition of sEH augments hypoxic pulmonary vasoconstriction. However, the mechanisms underlying the proconstrictive effects of sEH inhibitors in pulmonary artery smooth muscle cells (PASMCs) are unclear. In the present study, we used a sEH inhibitor, 12-(3-hexylureido) dodec-8-enoic acid (8-HUDE), to examine the ionic mechanisms underlying the constriction of PAs. 8-HUDE increased the tension of rat PAs to 145% baseline in a manner which was effectively eliminated by 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K(+) (K(ATP)) channels. Whole cell currents of HEK cells transfected with Kir6.1 or SUR2B were activated by K(ATP) channel opener pinacidil, inhibited by K(ATP) channel inhibitor glibenclamide or inhibited by 8-HUDE in a concentration-dependent manner with an IC50 value of 40 uM. In addition, 8-HUDE inhibited the expression of Kir6.1 and SUR2B at both mRNA and protein level in rat PASMCs. These observations suggest that 8-HUDE exerts acute effects on K(ATP) channel activity as well as subacute effects through decreased channel expression, and these effects are, at least in part, via the Kir6.1/SUR2B channel., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The sulfonylurea receptor 2A (SUR2A) is an ATP-binding cassette (ABC) protein that forms the regulatory subunit of ATP-sensitive potassium (K(ATP)) channels in the heart. ATP binding and hydrolysis at the SUR2A nucleotide binding domains (NBDs) control gating of K(ATP) channels, and mutations in the NBDs that affect ATP hydrolysis and cellular trafficking cause cardiovascular disorders. To date, there is limited information on the SUR2A NBDs and the effects of disease-causing mutations on their structure and interactions. Structural and biophysical studies of NBDs, especially from eukaryotic ABC proteins like SUR2A, have been hindered by low solubility of the isolated domains. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the precise definition of the domain boundaries. Putative boundaries of SUR2A NBD1 were identified by structure-based sequence alignments and subsequently tested by exploring the solubility of SUR2A NBD1 constructs with different N and C termini. We have determined boundaries of SUR2A NBD1 that allow for soluble heterologous expression of the protein, producing a folded domain with ATP binding activity. Surprisingly, our alignment and screening data indicate that SUR2A NBD1 contains two putative, previously unidentified, regulatory elements: a large insert within the β-sheet subdomain and a C-terminal extension. Our approach, which combines the use of structure-based sequence alignments and predictions of disordered regions combined with biochemical and biophysical studies, may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins., (© 2011 American Chemical Society)

To test the possibility that a fast-onset promoting agent repaglinide may initiate prandial insulin secretion through the mechanism of cephalic-phase insulin release, we explored the expression and distribution character of sulfonylurea receptors in rat taste buds. Twenty male Wistar rats aged 10 weeks old were killed after general anesthesia. The circumvallate papillae, fungiform papillae and pancreas tissues were separately collected. Immunohistochemical staining was used to detect the expression and distribution of sulfonylurea receptor 1 (SUR1) or sulfonylurea receptor 2 (SUR2) in rat taste buds. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression of SUR1 or SUR2 mRNA. The pancreatic tissues from the same rat were used as positive control. This is the first study to report that SUR1 is uniquely expressed in the taste buds of fungiform papillae of each rat tongue, while the expression of SUR1 or SUR2 was not detected in the taste buds of circumvallate papillae. SUR1 is selectively expressed in rat taste buds, and its distribution pattern may be functionally relevant, suggesting that the rapid insulin secretion-promoting effect of repaglinide may be exerted through the cephalic-phase secretion pathway mediated by taste buds., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background and Purpose: Spontaneous electrical activity, termed slow waves, drives rhythmic, propulsive contractions in the smooth muscle of the oviduct (myosalpinx). Myosalpinx contractions cause egg transport through the oviduct. Agents that disrupt slow wave pacemaker activity will therefore disrupt myosalpinx contractions and egg transport. Caffeine is commonly used as a ryanodine receptor agonist and has been previously associated with delayed conception. Here we assessed the effects of caffeine on pacemaker activity in the murine myosalpinx., Experimental Approach: The effects of caffeine on electrical pacemaker activity were studied using intracellular microelectrode and isometric force measurements on intact oviduct muscle preparations. Responses to caffeine were compared with responses caused by 3-isobutyl-1-methylxanthine (IBMX) and forskolin., Key Results: Caffeine caused hyperpolarization of membrane potential and inhibited slow wave generation and myosalpinx contractions. The effects of caffeine could be mimicked by the K(ATP) channel agonist pinacidil and antagonized by the K(ATP) channel antagonist glibenclamide. Caffeine is known to inhibit cyclic nucleotide phosphodiesterases (PDEs), leading to an increase in cytosolic cAMP and stimulation of downstream cAMP-dependent mechanisms. The effects of caffeine were mimicked by the PDE inhibitor, IBMX, and the adenylyl cyclase activator forskolin. These effects were also reversed by glibenclamide., Conclusions and Implications: These results suggest that caffeine activates K(ATP) channels in oviduct myosalpinx. Since caffeine abolishes slow waves and associated contractions of the myosalpinx, it would have a negative effect on egg transport through the oviduct and may contribute to the documented delayed conception in women consuming caffeinated beverages., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background: Potassium channels play critical roles in the regulation of cell membrane potential, which is central to the excitability of myometrium. The ATP-sensitive potassium (KATP) channel is one of the most abundant potassium channels in myometrium. The objectives of this study were to investigate the protein expression of KATP channel in human myometrium and determine the levels of KATP channel in lower and upper segmental myometrium before and after onset of labour., Methods: Both lower segmental (LS) and upper segmental (US) myometrial biopsies were collected at cesarean section from pregnant women not-in-labour (TNL) or in-labour (TL) at term. Protein expression level and cellular localization of four KATP channel subunits in US and LS myometrium were determined by Western blot analysis and immunohistochemistry, respectively. The contractile activity of myometrial strip was measured under isometric conditions., Results: Four KATP channel subunits, namely Kir6.1, Kir6.2, SUR1 and SUR2B were identified in pregnant myometrium. While found in vascular myocytes, these subunits appear to be preferentially expressed in myometrial myocytes. Diazoxide, a KATP channel opener, inhibited the spontaneous contractility of pregnant myometrium, suggesting that the KATP channels are functional in human pregnant myometrium. Diazoxide was less potent in TL strips than that in TNL strips. Interestingly, expression of SUR1 was greater in TL than TNL tissues, although no differences were found for SUR2B in these two tissues. For both lower and upper segmental myometrium, Kir6.1 and Kir6.2 were less in TL compared with TNL tissues., Conclusions: Functional KATP channels are expressed in human pregnant myometrium. Down-regulation of Kir6.1 and Kir6.2 expression in myometrium may contribute to the enhanced uterine contractility associated with the onset of labour.

Our study determines alterations in the vasoconstrictor response elicited by electric field stimulation (EFS) in mesenteric arteries from cirrhotic rats treated with CCl(4), and how calcitonin gene-related peptide (CGRP) participates in this response. Vasoconstriction induced by EFS was analysed in the absence and presence of the CGRP receptor antagonist CGRP(8-37) in arterial segments from control and cirrhotic rats. The vasodilator response to exogenous CGRP was tested in both groups of rats, and the interference of the guanylate cyclase inhibitor ODQ or the K(ATP) channel blocker glibenclamide was analysed only in segments from cirrhotic rats. The vasodilator response to the K(ATP) channel opener pinacidil and to 8-bromo-cyclic GMP was tested. The K(ATP) currents were recorded using the patch-clamp technique. Expression of receptor activity-modifying protein 1 (RAMP1), calcitonin receptor-like receptor, Kir 6.1 and sulfonylurea receptor 2B (SUR2B) was also analysed. Release of CGRP and cGMP was measured. The EFS-elicited vasoconstriction was less in segments from cirrhotic rats. The presence of CGRP(8-37) increased the EFS-induced response only in segments from cirrhotic rats. The CGRP-induced vasodilatation was greater in segments from cirrhotic rats, and was inhibited by ODQ or glibenclamide. Both pinacidil and 8-bromo-cyclic GMP induced a stronger vasodilator response in segments from cirrhotic rats. Pinacidil induced greater K(ATP) currents in cirrhotic myocytes. Expression of RAMP1, calcitonin receptor-like receptor, Kir 6.1 and SUR2B was not modified by liver cirrhosis. Liver cirrhosis increased CGRP release, but did not modify cGMP formation. The decreased vasoconstrictor response to EFS in cirrhosis is mediated by increased vasodilator response to CGRP, as well as increased K(ATP) channel gating. This effect of CGRP may play a role in the splanchnic vasodilatation present in liver cirrhosis.

Background: ATP-sensitive K(+) (K(ATP)) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The K(ATP) channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express K(ATP) channels., Methods: Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for K(ATP) channel subunits was determined using real-time RT-PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional K(ATP) channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used., Results: Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate K(ATP) channels, increased whole cell K(+) current. On the other hand, K(+) current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of K(ATP) channels., Conclusions: In conclusion, mammalian oocytes express K(ATP) channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception.

ATP-sensitive potassium (K(ATP)) channels may be linked to mechanisms of pain after nerve injury, but remain under-investigated in primary afferents so far. We therefore characterized these channels in dorsal root ganglion (DRG) neurons, and tested whether they contribute to hyperalgesia after spinal nerve ligation (SNL). We compared K(ATP) channel properties between DRG somata classified by diameter into small or large, and by injury status into neurons from rats that either did or did not become hyperalgesic after SNL, or neurons from control animals. In cell-attached patches, we recorded basal K(ATP) channel opening in all neuronal subpopulations. However, higher open probabilities and longer open times were observed in large compared to small neurons. Following SNL, this channel activity was suppressed only in large neurons from hyperalgesic rats, but not from animals that did not develop hyperalgesia. In contrast, no alterations of channel activity developed in small neurons after axotomy. On the other hand, cell-free recordings showed similar ATP sensitivity, inward rectification and unitary conductance (70-80 pS) between neurons classified by size or injury status. Likewise, pharmacological sensitivity to the K(ATP) channel opener diazoxide, and to the selective blockers glibenclamide and tolbutamide, did not differ between groups. In large neurons, selective inhibition of whole-cell ATP-sensitive potassium channel current (I(K(ATP))) by glibenclamide depolarized resting membrane potential (RMP). The contribution of this current to RMP was also attenuated after painful axotomy. Using specific antibodies, we identified SUR1, SUR2, and Kir6.2 but not Kir6.1 subunits in DRGs. These findings indicate that functional K(ATP) channels are present in normal DRG neurons, wherein they regulate RMP. Alterations of these channels may be involved in the pathogenesis of neuropathic pain following peripheral nerve injury. Their biophysical and pharmacological properties are preserved even after axotomy, suggesting that K(ATP) channels in primary afferents remain available for therapeutic targeting against established neuropathic pain.

Activation of the large-conductance Ca(2+)-activated K(+) channel (BK) in the cardiac inner mitochondrial membrane has been suggested to protect the heart against ischemic injury. However, these findings are limited by the low selectivity profile and potency of the BK channel activator (NS1619) used. In the present study, we address the cardioprotective role of BK channels using a novel, potent, selective, and chemically unrelated BK channel activator, NS11021. Using electrophysiological recordings of heterologously expressed channels, NS11021 was found to activate BK alpha + beta1 channel complexes, while producing no effect on cardiac K(ATP) channels. The cardioprotective effects of NS11021-induced BK channel activation were studied in isolated, perfused rat hearts subjected to 35 min of global ischemia followed by 120 min of reperfusion. 3 microM NS11021 applied prior to ischemia or at the onset of reperfusion significantly reduced the infarct size [control: 44.6 +/- 2.0%; NS11021: 11.4 +/- 2.0%; NS11021 at reperfusion: 19.8 +/- 3.3% (p < 0.001 for both treatments compared to control)] and promoted recovery of myocardial performance. Co-administration of the BK-channel inhibitor paxilline (3 microM) antagonized the protective effect. These findings suggest that tissue damage induced by ischemia and reperfusion can be reduced by activation of cardiac BK channels.

Background and Purpose: Ischemia/hypoxia induces de novo expression of the sulfonylurea receptor 1-regulated NC(Ca-ATP) channel. In rodent models of ischemic stroke, early postevent administration of the sulfonylurea, glibenclamide, is highly effective in reducing edema, mortality, and lesion volume, and in patients with diabetes presenting with ischemic stroke, pre-event plus postevent use of sulfonylureas is associated with better neurological outcome. However, the therapeutic window for treatment with glibenclamide has not been studied., Methods: We examined the effect of low-dose (nonhypoglycemogenic) glibenclamide in 3 rat models of ischemic stroke, all involving proximal middle cerebral artery occlusion (MCAo): a thromboembolic model, a permanent suture occlusion model, and a temporary suture occlusion model with reperfusion (105 minutes occlusion, 2-day reperfusion). Treatment was started at various times up to 6 hours post-MCAo. Lesion volumes were measured 48 hours post-MCAo using 2,3,5-triphenyltetrazolium chloride., Results: Glibenclamide reduced total lesion volume by 53% in the thromboembolic MCAo model at 6 hours, reduced corrected cortical lesion volume by 51% in the permanent MCAo model at 4 hours, and reduced corrected cortical lesion volume by 41% in the temporary MCAo model at 5.75 hours (P<0.05 for all 3). Analysis of pooled data from the permanent MCAo and temporary MCAo series indicated a sigmoidal relationship between hemispheric swelling and corrected cortical lesion volume with the half-maximum cortical lesion volume being observed with 10% hemispheric swelling., Conclusions: Low-dose glibenclamide has a strong beneficial effect on lesion volume and has a highly favorable therapeutic window in several models of ischemic stroke.

ATP-dependent potassium (K(ATP)) channels are the target of multiple vasoactive factors and drugs. Changes in the functional role of ATP-dependent (K(ATP)) potassium channels in hypertension are controversial. The aim of the present study was to analyze the possible changes of ATP-sensitive potassium channels (K(ATP)) expression and function during hypertension. For this purpose, we used endothelium-denuded aorta segments from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) to analyze the 1) expression of K(ATP) subunits Kir6.1, Kir6.2 and SUR2B by immunohistochemistry and Western blot, 2) the K(ATP) currents recorded in the whole cell configuration of the patch-clamp technique and 3) the vasodilator response to the K(ATP) channel openers, pinacidil and cromakalim. Kir6.1 and SUR2B were expressed in the medial layer of the aorta from WKY rats and SHR rats, while Kir6.2 was not detected in aorta from either strain. Kir6.1 and SUR2B expression were decreased in hypertension. However, the vasodilator responses of pinacidil and cromakalim were similar in WKY rats and SHR rats. Moreover, pinacidil induced increase in K+ currents was also similar in WKY rats and SHR rats and also similarly inhibited by glybenclamide. Our data demonstrate for the first time direct evidence of decreased aortic Kir6.1/SUR2B subunit expression in hypertension, but preserved functional responses to K(ATP) channel openers.

The obese Zucker rat reportedly has increased activity of the intrarenal renin-angiotensin-aldosterone system, which conceptually could contribute to elevated salt sensitivity and blood pressure (BP). Our aim was to determine whether there was increased angiotensin II type 1 receptor (AT(1)R)-mediated upregulation of expression or activity of the bumetanide-sensitive Na-K-2Cl cotransporter, the thiazide-sensitive Na-Cl cotransporter (NCC), and/or the epithelial sodium channel (ENaC) in obese vs. lean Zucker rats. Male obese and lean Zucker rats (10-wk old) were fed either 1) control chow (1% NaCl) or 2) chow with candesartan (CAN), an AT(1)R antagonist (25 mg/kg.diet) for 14 wk (n = 8/treatment/body type). BP measured by radiotelemetry, was markedly reduced by CAN ( approximately 20-25 mmHg) in both lean and obese rats with no body-type differences. Obese rats had significantly greater net natriuretic response to single injections of hydrochlorothiazide and benzamil, suggesting increased activity of NCC and ENaC, respectively; however, only the response to benzamil was reduced by CAN. CAN led to a significant reduction in whole kidney levels of NCC and gamma-ENaC (70-kDa band) in both lean and obese rats. However, it significantly increased alpha-ENaC and Na-K-2Cl cotransporter levels, and these increases were greater in obese rats. These studies suggest that relatively increased ENaC, but not NCC activity, in obese rats is due to enhanced AT(1)R activity. CAN attenuated the reduction of several renal transporters in the obese rat kidney. Finally, differences in intrarenal AT(1)R activity do not seem directly responsible for BP differences between lean and obese rats.

Background and Purpose: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5'-triphosphate-sensitive K(+) (K(ATP)) channel opening may cause migraine by dilatating intracranial arteries, including the middle meningeal artery (MMA). We studied the K(ATP) channel expression profile in rat MMA and examined the potential inhibitory effects of the K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA, using the three K(ATP) channel openers levcromakalim, pinacidil and P-1075., Experimental Approach: mRNA and protein expression of K(ATP) channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K(ATP) channel drugs on rat MMA were studied in the genuine closed cranial window model and in myograph baths, respectively., Key Results: Expression studies indicate that inwardly rectifying K(+) (Kir)6.1/sulphonylurea receptor (SUR)2B is the major K(ATP) channel complex in rat MMA. PNU-37883A (0.5 mg kg(-1)) significantly inhibited the in vivo dilatory effect of levcromakalim (0.025 mg kg(-1)), pinacidil (0.38 mg kg(-1)) and P-1075 (0.016 mg kg(-1)) in rat MMA. In vitro PNU-37883A significantly inhibited the dilatory responses of the three K(ATP) channel openers in rat MMA at 10(-7) and 3 x 10(-7) M., Conclusions and Implications: We suggest that Kir6.1/SUR2B is the major functional K(ATP) channel complex in the rat MMA. Furthermore, we demonstrate the potent in vivo and in vitro blocking potentials of PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA.

1. Because chronic activation of the renal sympathetic nervous system promotes sodium and water retention, it is conceivable that long-term exposure of the kidney to the sympathetic neurotransmitter noradrenaline upregulates the expression of key renal epithelial transport systems. 2. To test this hypothesis, we used immunoblotting of renal cortical and medullary tissue to investigate the abundance of major transport systems expressed along the renal tubule in response to long-term (15 days) infusions of noradrenaline (600 ng/min) in rats. 3. Mean arterial blood pressure and heart rate were significantly elevated in rats receiving chronic infusions of noradrenaline (128 +/- 10 mmHg and 492 +/- 16 b.p.m., respectively) compared with animals treated with saline only (89 +/- 3 mmHg and 376 +/- 14 b.p.m., respectively). 4. Chronic infusions of noradrenaline also increased the protein abundance of the cortical Na(+)/H(+) exchanger isoform 3 (NHE-3; 2.5-fold; P = 0.0142), the cortical sodium-bicarbonate cotransporter NBC-1 (2.5-fold; P = 0.0067), the bumetanide-sensitive sodium-potassium-chloride cotransporter BSC-1/NKCC2 in the inner stripe of outer medulla (threefold; P = 0.0020) and aquaporin-2 in the inner medulla (twofold; P = 0.0039). 5. In contrast, noradrenaline did not significantly affect expression of the thiazide-sensitive Na(+)-Cl(-) cotransporter in the cortex, Na(+)/K(+)-ATPase-alpha(1) in the cortex and inner stripe of the outer or inner medulla, the inwardly rectifying K(+) channel (ROMK-1) in the inner stripe of the outer medulla or aquaporin-1 in the cortex or inner medulla. Noradrenaline did significantly, but modestly (less than twofold), increase aquaporin-1 in the inner stripe of the outer medulla. 6. We conclude that noradrenaline-induced increases in the expression of NHE-3, NBC-1, BSC-1 and aquaporin-2 are likely to play an important role in the regulation of salt and water transport by noradrenaline in the kidney and may explain, at least in part, the altered renal sodium and water handling associated with overactivation of the sympathetic system.

Background: Opening of cardiac ATP-sensitive potassium channels (K(ATP) channels) is a well-characterized protective mechanism against ischemia and reperfusion injury. Evidence exists for an involvement of both sarcolemmal and mitochondrial K(ATP) channels in such protection. Classically, cardiac sarcolemmal K(ATP) channels are thought to be composed of Kir6.2 (inward-rectifier potassium channel 6.2) and SUR2A (sulfonylurea receptor type 2A) subunits; however, the evidence is strong that SUR1 (sulfonylurea receptor type 1) subunits are also expressed in the heart and that they may have a functional role. The aim of this study, therefore, was to examine the role of SUR1 in myocardial infarction., Methods and Results: We subjected mice lacking SUR1 subunits to in vivo myocardial ischemia/reperfusion injury. Interestingly, the SUR1-null mice were markedly protected against the ischemic insult, displaying a reduced infarct size and preservation of left ventricular function, which suggests a role for this K(ATP) channel subunit in cardiovascular function during conditions of stress., Conclusions: SUR1 subunits have a high sensitivity toward many sulfonylureas and certain K(ATP) channel-opening drugs. Their potential role during ischemic events should therefore be considered both in the interpretation of experimental data with pharmacological agents and in the clinical arena when the cardiovascular outcome of patients treated with antidiabetic sulfonylureas is being considered.

To understand the possible functions and subcellular localizations of sulfonylurea receptors (SURs) in cardiac muscle, polyclonal anti-SUR2A and anti-SUR2B antisera were raised. Immunoblots revealed both SUR2A and SUR2B expression in mitochondrial fractions of rat heart and other cellular fractions such as microsomes and cell membranes. Immunostaining detected ubiquitous expression of both SUR2A and SUR2B in rat heart in the atria, ventricles, interatrial and interventricular septa, and smooth muscles and endothelia of the coronary arteries. Electron microscopy revealed SUR2A immunoreactivity in the cell membrane, endoplasmic reticulum (ER), and mitochondria. SUR2B immunoreactivity was mainly localized in the mitochondria as well as in the ER and cell membrane. Thus, SUR2A and SUR2B are not only the regulatory subunits of sarcolemmal K(ATP) channels but may also function as regulatory subunits in mitochondrial K(ATP) channels and play important roles in cardioprotection.

ATP-sensitive potassium (KATP) channels play a central role in glucose-stimulated insulin secretion (GSIS) by pancreatic beta-cells. Activity of these channels is determined by their open probability (Po) and the number of channels present in a cell. Glucose is known to reduce Po, but whether it also affects the channel density is unknown. Using INS-1 model beta-cell line, we show that the expression of K(ATP) channel subunits, Kir6.2 and SUR1, is high at low glucose, but declines sharply when the ambient glucose concentration exceeds 5mM. In response to glucose deprivation, channel synthesis increases rapidly by up-regulating translation of existing mRNAs. The effects of glucose deprivation could be mimicked by pharmacological activation of 5'-AMP-activated protein kinase with 5-aminoimidazole-4-carboxamide ribonucleotide and metformin. Pancreatic beta-cells which have lost their ability for GSIS do not show such changes implicating a possible (patho-)physiological link between glucose-regulated KATP channel expression and the capacity for normal GSIS.

The present study was done to determine whether endogenous nitric oxide (NO) plays a role in the regulation of sodium transporters in the kidney. Male Sprague-Dawley rats were treated with NG-nitro-L-arginine methyl ester (L-NAME, 100 mg/L drinking water) for 4 weeks. Control rats were supplied with tap water without drugs. Expression of Na, K-ATPase, type 3 Na/H exchanger (NHE3), Na/K/2Cl cotransporter (BSC1), and thiazide-sensitive Na/Cl cotransporter (TSC) proteins was determined in the kidney by Western blot analysis. Catalytic activity of Na,K-ATPase was also determined. The treatment with L-NAME significantly and steadily increased the systemic blood pressure. Total and fractional excretion of urinary sodium decreased significantly, while creatinine clearance remained unaltered. Neither plasma renin activity nor aldosterone concentration was significantly altered. The alpha1 subunit expression and the catalytic activity of Na, K-ATPase were increased in the kidney. The expression of NHE3, BSC1 and TSC was also increased significantly. These results suggest that endogenously-derived NO exerts a tonic inhibitory effect on the expression of sodium transporters, including Na, K-ATPase, NHE3, BSC1, and TSC, in the kidney.

1. ATP-sensitive K(+) channels (K(ATP) channels) are tetradimeric complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). The SURs SUR2A (cardiac) and SUR2B (smooth muscle) differ only in the last 42 amino acids. In SUR2B, the mutation Y1206S, located at intracellular loop 8, increases the affinity for glibenclamide (GBC) about 10-fold. Here, we examined whether the mutation Y1206S in SUR2A had effects similar to those in SUR2B.2. GBC bound to SUR2A with K(D)=20 nM; the mutation increased affinity approximately 5 x. 3. In cells, coexpression of SUR2A with Kir6.2 increased the affinity for GBC approximately 3 x; with the mutant, the increase was 9 x. 4. The mutation did not affect the affinity of SUR2A for openers; coexpression with Kir6.2 reduced opener affinity of wild-type and mutant SUR2A by about 2 x. 5. The negative allosteric interaction between the opener, P1075, and GBC at wild-type and mutant SUR2A was markedly affected by the presence of MgATP and by coexpression with Kir6.2. 6. In inside-out patches, GBC inhibited the wild-type Kir6.2/SUR2A and 2B channels with IC(50) values of 27 nM; the mutation shifted the IC(50) values to approximately 1 nM. 7. The data show that the mutation Y1206S increased the affinity of SUR2A for GBC and modulated the effects of coexpression. Overall, the changes were similar to those observed with SUR2B(Y1206S), suggesting that the differences in the last 42 carboxy-terminal amino acids of SUR2A and 2B are of limited influence on the binding of GBC and P1075 to the SUR2 isoforms.

ATP-sensitive K+ channels, termed K(ATP) channels, provide a link between cellular metabolism and membrane electrical activity in a variety of tissues. Channel isoforms have been identified and are targets for compounds that both stimulate and inhibit their activity resulting in membrane hyperpolarization and depolarization, respectively. Examples include relaxation of vascular smooth muscle and stimulation of insulin secretion. This article reviews the cloning, molecular biology, and structure of K(ATP) channels, with particular focus on the SUR1/K(IR)6.2 neuroendocrine channels that are important for the regulation of insulin secretion. We integrate the extensive pharmacologic structure-activity-relationship data on these channels, which defines a bipartite drug binding pocket in the SUR (sulfonylurea receptor), with recent structure-function studies that identify domains of SUR and K(IR)6.2, the channel pore, which are critical for channel assembly, for gating, and for the ligand-receptor interactions that modulate channel activity. The atomic structure of a sulfonylurea in a protein pocket is used to develop insight into the recognition of these compounds. A homology model of K(ATP) channels, based on VC-MsbA, another member of the ABC protein family, is described and used to position amino acids important for the action of channel openers and blockers within the core of SUR. The model has a central chamber which could serve as a multifaceted binding pocket.

Herein, we provide the first evidence on the capsaicin (CPS) receptor vanilloid receptor type-1 (VR1) by rat thymocytes, and its involvement in CPS-induced apoptosis. VR1 mRNA was identified by quantitative RT-PCR in CD5(+) thymocytes. By immunofluorescence and flow cytometry, we found that a substantial portion of CD5+ thymocytes, namely CD4+ and double negative (DN) cell subsets, express VR1 that was present on plasma membrane on discrete spots. By Western blot, VR1 protein was identified as a single band of 95 kDa. We also described that CPS could trigger two distinct pathways of thymocyte death, namely apoptosis and necrosis depending on the dose of CPS exposure. CPS-induced apoptosis involved intracellular free calcium (Ca2+) influx, phosphatidylserine exposure, mitochondrial permeability transmembrane pore (PTP) opening and mitochondrial transmembrane potential (Delta Psi m) dissipation leading to cytochrome c release, activation of caspase-9 and -3 and oligonucleosomal DNA fragmentation. VR1 was functionally implicated in these events as they were completely abrogated by the VR1 antagonist, capsazepine (CPZ). Finally, we demonstrated that VR1 expression on distinct thymocytes was associated with the selective ability of CPS to trigger DNA fragmentation in VR1+ CD4+ and DN thymocytes. Overall, our results suggest that the expression of VR1 on thymocytes may function as a sensor of harmful stimuli present in the thymic environment.

Capsaicin, a pungent ingredient of chili pepper, activates vanilloid receptor subtype 1 (VR1), which is a nonselective cation channel with high Ca(2+) permeability. Although VR1 and its splice variant are highly expressed in sensory neurons, they are expressed in neuronal cells in brain and peripheral non-neuronal cells. In this study, we investigated whether VR1 is expressed in PC12 cells, rat pheochromocytoma. Capsaicin at concentrations above 100 microM induced an increase in intracellular free Ca(2+) concentrations by influx from extracellular spaces, and the effect was blocked by capsazepine, a selective antagonist of VR1. VR1 transcript and protein were detected by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Immunocytochemical analysis revealed that VR1 protein was expressed in the cytosol and the plasma membrane of PC12 cells, and treatment with the antisense oligonucleotide for VR1 decreased the expression. VR1 in PC12 cells showed different characters from that in sensory neurons; capsaicin concentration-dependency and heat- and nerve growth factor-sensitivities. These results suggested that VR1 was functionally expressed in PC12 cells. The usefulness of PC12 cell line for studying functions and/or expression of VR1 is discussed.

We investigated the neurotoxic effects of capsaicin (CAP) on pain sensitivity and on the expression of capsaicin receptor, the vanilloid receptor (VR1), in rats. High-dose application of CAP has been known to degenerate a large fraction of the sensory neurons. Although the neurotoxic effects of CAP are well documented, the effects of CAP on the vanilloid receptor (VR1) are not yet known. In this paper, we investigated the effects of high-dose application of CAP on the expression of VR1 in rats. Thermal and mechanical pain sensitivity was reduced when neonatal rats were treated with a high dose of CAP. This reduction of pain sensitivity was significantly decreased after initiating carrageenan-induced inflammation. The expression of VR1 in dorsal root ganglia (DRG) isolated from the CAP-treated rats was reduced compared to that from the vehicle-treated rats. Therefore, we can conclude that the neurotoxic effect of CAP is related to the decrease of VR1 expression.

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.

Little is known about the central projection patterns of trigeminal afferent neurons expressing the vanilloid receptor TRPV1 and their coexpression of neuromodulatory peptides. To address these issues, we examined the distribution of TRPV1-positive neurons in the trigeminal ganglion (TG) and trigeminal sensory nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc) in the rat via light and electron microscopy. In addition, we studied the colocalization of TRPV1-positive neurons with substance P (SP) and calcitonin gene-related peptide (CGRP) via confocal microscopy. In TG, only small and medium-sized neurons were immunopositive for TRPV1. The staining for TRPV1 was found in axon collaterals in the dorsal parts of Vp, Vo, and Vi and in terminals and fibers throughout lamina I and the outer zone of lamina II (IIo) of Vc. With electron microscopy, TRPV1-positive fibers in the ascending and descending trigeminal tracts were found to be unmyelinated. Almost all TRPV1-positive terminals in Vc contained numerous large dense-core vesicles and formed synaptic contacts with single small dendrites. Multiple immunofluorescence revealed a high degree of colocalization of TRPV1 with SP and CGRP in TG neurons as well as in fibers and terminals confined to laminae I and IIo of Vc. These results suggest that the central projections of unmyelinated (C) afferents sensitive to noxious heat and capsaicin are organized differently between Vc and the rostral trigeminal nuclei and that Vc may play a role in the development of hyperalgesia.

Capsaicin, the pungent ingredient in red pepper, has been used since ancient times as a spice, despite the burning sensation associated with its intake. More than 50 years ago, Nikolaus Jancso discovered that capsaicin can selectively stimulate nociceptive primary afferent neurons. The ensuing research established that the neuropharmacological properties of capsaicin are due to its activation of the transient receptor potential ion channel of the vanilloid type 1 (TRPV1). Expressed by primary afferent neurons innervating the gut and other organs, TRPV1 is gated not only by vanilloids such as capsaicin, but also by noxious heat, acidosis and intracellular lipid mediators such as anandamide and lipoxygenase products. Importantly, TRPV1 can be sensitized by acidosis and activation of various pro-algesic pathways. Upregulation of TRPV1 in inflammatory bowel disease and the beneficial effect of TRPV1 downregulation in functional dyspepsia and irritable bladder make this polymodal nociceptor an attractive target of novel therapies for chronic abdominal pain.

We have recently identified imidazoline I(1)-receptors in the heart. In the present study, we tested regulation of cardiac I(1)-receptors versus alpha(2) -adrenoceptors in response to hypertension and to chronic exposure to agonist. Spontaneously hypertensive rats (SHR, 12-14 weeks old) received moxonidine (10, 60, and 120 microg/kg/h s.c.) for 1 and 4 weeks. Autoradiographic binding of (125)I-paraiodoclonidine (0.5 nM, 1 h, 22 degrees C) and inhibition of binding with epinephrine (10(-10)-10(-5) M) demonstrated the presence of alpha(2)-adrenoceptors in heart atria and ventricles. Immunoblotting and reverse transcription-polymerase chain reaction identified alpha(2A)-alpha(2B)-, and alpha(2C), and -adrenoceptor proteins and mRNA, respectively. However, compared with normotensive controls, cardiac alpha(2) -adrenoceptor kinetic parameters, receptor proteins, and mRNAs were not altered in SHR with or without moxonidine treatment. In contrast, autoradiography showed that up-regulated atrial I(1)-receptors in SHR are dose-dependently normalized by 1 week, with no additional effect after 4 weeks of treatment. Moxonidine (120 microg/kg/h) decreased B(max) in right (40.0 +/- 2.9-7.0 +/- 0.6 fmol/unit area; p < 0.01) and left (27.7 +/- 2.8-7.1 +/- 0.4 fmol/unit area; p < 0.01) atria, and decreased the 85- and 29-kDa imidazoline receptor protein bands, in right atria, to 51.8 +/- 3.0% (p < 0.01) and 82.7 +/- 5.2% (p < 0.03) of vehicle-treated SHR, respectively. Moxonidine-associated percentage of decrease in B(max) only correlated with the 85-kDa protein (R(2) = 0.57; p < 0.006), suggesting that this protein may represent I(2)-receptors. The weak but significant correlation between the two imidazoline receptor proteins (R(2) = 0.28; p < 0.03) implies that they arise from the same gene. In conclusion, the heart possesses I(1)-receptors and alpha(2)-adrenoceptors, but only I(1)-receptors are responsive to hypertension and to chronic in vivo treatment with a selective I(1)-receptor agonist.

The vanilloid receptor-1 (TRPV1), the capsaicin receptor, a transducer of several nociceptive stimuli, is principally expressed by nociceptive fibers that specifically target supraspinal-projecting neurons expressing the NK1 receptor. TRPV1 is also expressed by intraspinal neurons. Using double immunocytochemistry, we show that 14.2% of these TRPV1 dorsal horn cell bodies also express the NK1 receptor suggesting that endogenous vanilloids may directly modulate second-order ascending neurons.

Objectives: Delta(9)-Tetrahydrocannabinol, the active agent of Cannabis sativa, exhibits well-documented antitumor properties, but little is known about the possible effects mediated by endogenous cannabinoids on human tumors. In the present study, we analyzed the effect of arachidonyl ethanolamide (AEA) on cervical carcinoma (CxCa) cell lines., Methods: To assess the sensitivity of CxCa cells to AEA, we selected three cell lines that were exposed to increasing doses of AEA with or without antagonists to receptors to AEA. DNA fragmentation and caspase-7 activity were used as apoptosis markers. The expression of receptors to AEA were analyzed in CxCa cell lines as well as CxCa biopsies., Results: The major finding was that AEA induced apoptosis of CxCa cell lines via aberrantly expressed vanilloid receptor-1, whereas AEA binding to the classical CB1 and CB2 cannabinoid receptors mediated a protective effect. Furthermore, unexpectedly, a strong expression of the three forms of AEA receptors was observed in ex vivo CxCa biopsies., Conclusions: Overall, these data suggest that the specific targeting of VR1 by endogenous cannabinoids or synthetic molecules offers attractive opportunities for the development of novel potent anticancer drugs.

The vanilloid receptor-like 1 protein (VRL-1, also called TRPV2) is a member of the TRPV family of proteins and is a homolog of the capsaicin/vanilloid receptor (VR1, or TRPV1). Although VRL-1 does not bind capsaicin, like VR1 it is activated by noxious heat (>52 degrees C). Unlike VR1, however, VRL-1 is primarily expressed by medium- and large-diameter primary afferents, which suggests that nociceptive processing is but one of the functions to which VRL-1 contributes. To provide information on the diverse spinal circuits that are engaged by these VRL-1-expressing primary afferents, we completed a detailed immunocytochemical map of VRL-1 in rat spinal cord, including light and electron microscopic analysis, and generated a more comprehensive neurochemical characterization of VRL-1-expressing primary afferents. Consistent with previous reports, we found that VRL-1 and VR1 are expressed in different dorsal root ganglion (DRG) cell bodies. Almost all VRL-1-expressing cells labeled for N52 (a marker of myelinated afferents), consistent with VRL-1 expression in Adelta and Abeta fibers. EM analysis of the DRG and dorsal roots confirmed this and revealed two categories of neurons based on the intensity of immunolabeling. The densest VRL-1 immunoreactivity in the spinal cord was found in lamina I, inner lamina II, and laminae III/IV. This is consistent with the expression of VRL-1 by myelinated nociceptors that target laminae I and IIi and in nonnociceptive Abeta fibers that target laminae III/IV. Dorsal rhizotomy reduced, but did not eliminate, the immunostaining in all dorsal horn laminae, which indicates that VRL-1 expression derives from both DRG cells and from neurons intrinsic to the brain or spinal cord. Spinal hemisection reduced immunostaining of the ipsilateral dorsal columns in segments rostral to the lesion and in the dorsal column nuclei, presumably from the loss of ascending Abeta afferents, but there was no change caudal to the lesion. Thus, supraspinal sources of dorsal horn VRL-1 immunoreactivity are likely not significant. Although we never observed VRL-1 immunostaining in cell bodies in the superficial dorsal horn, there was extensive labeling of motoneurons and ventral root efferents-in particular, in an extremely densely labeled population at the lumbosacral junction. Finally, many ependymal cells surrounding the central canal were intensely labeled. These results emphasize that VRL-1, in contrast to VR1, is present in a diverse population of neurons and undoubtedly contributes to numerous functions in addition to nociceptive processing., (Copyright 2004 Wiley-Liss, Inc.)

The vanilloid receptor-1 (TRPV1), expressed by nociceptive fibers, is a transducer of thermal and chemical nociceptive messages. However, endogenous ligands excite TRPV1 receptors localized on central nociceptive terminals and interneurons. Using immunocytochemistry at the ultrastructural level, we show that TRPV1 is also expressed in spinal glial cells characterized as astrocyte by double labeling with glial fibrillary acid protein. Quantification of the labeling shows that the most numerous labeling is neuronal and that 7% of the total TRPV1 labeling is localized in astrocytes. The total absence of staining in TRPV1 knock out mice strongly suggests that true TRPV1 protein is present in astrocytes. The localization of TRPV1-containing astrocytes apposed to nociceptive C-terminals suggests that they may be involved in the control of pain transmission.

Oncostatin M (OSM) is a member of the interleukin-6 family of cytokines, and we have reported previously that the murine OSM receptor beta subunit (OSMR) was expressed in some neurons in the adult trigeminal and dorsal root ganglia (DRGs) and in the perineonatal hypoglossal nucleus. In the present study, we investigated the development of OSMR-positive neurons of DRGs in OSM-deficient mice. In situ hybridization revealed that OSMR-positive neurons in DRGs began to appear at postnatal day 0 (P0) and reached the adult level at P14. In the DRGs of the OSM-deficient mice, vanilloid receptor 1 (VR1)- and P2X3-positive small-sized neurons were significantly decreased. In addition, OSMR-positive neurons decreased, resulting in the reduced number of VR1/P2X3/OSMR-triple positive neurons. OSM-deficient mice displayed significantly reduced noxious responses in models of acute thermal, mechanical, chemical, and visceral pain. Thus, OSM plays an essential role in the development of a subtype of nociceptive neurons in the DRGs.

Nicorandil is an adenosine triphosphate sensitive K (K-ATP) channel opener and a nitric oxide donor. K-ATP channels and nitric oxide are important factors in ischemic preconditioning, which in turn suppresses reperfusion arrhythmias. The present study sought to evaluate whether nicorandil suppresses ischemic-induced ventricular arrhythmias and enhances sulfonylurea receptors (SUR 2; subunit of K-ATP channel), endothelial nitric oxide (eNOS), and inducible nitric oxide (iNOS) expression in the left ventricle after myocardial infarction without reperfusion. Thirty male Sprague-Dawley rats at 7 weeks of age were separated into three groups, as follows. Acute myocardial infarction was induced in twenty rats by ligating the left main coronary artery. Ten of these twenty rats were continuously administered nicorandil at 3 mg/kg/day i.p. The other ten rats were left untreated. The ten controls were untreated and sham-operated. After coronary ligation, ventricular arrhythmias were evaluated from stored ECG signals. At 24 hours after treatment, eNOS, iNOS, and SUR2 mRNA levels and eNOS, iNOS expression in the left ventricle were determined by reverse transcription polymerase chain reaction (RT-PCR) and by immunohistochemical staining, respectively. Nicorandil suppressed the total number of ventricular arrhythmias from 1 to 2 hours, the total duration of ventricular tachycardia from 2 to 3 hours, and that of ventricular fibrillation from 1 to 2 and from 4 to 5 hours after coronary ligation. Nicorandil improved the survival rate 24 hours after coronary ligation. Levels of SUR2 mRNA increased only in left ventricles treated with nicorandil, particularly in the non-ischemic myocardium. eNOS mRNA was enhanced 2.2-fold in the area at risk in infarcted controls compared to sham-operated rats. In the non-ischemic area and area at risk of rats treated with nicorandil compared to sham-operated rats, eNOS mRNA was enhanced 3.3- and 2.7-fold, respectively. Staining indicated that the highest concentrations of eNOS occurred in the endothelium and myocardium of the non-ischemic area of rats treated with nicorandil. iNOS mRNA was present in both the area at risk and the non-ischemic area only in infarcted rats, and levels thereof were higher in the area at risk than in the non-ischemic area. However, there was no difference in iNOS mRNA levels between nicorandil-treated rats and controls. iNOS exhibited stronger staining in the area at risk than in the non-ischemic area of both nicorandil-treated and infarcted controls, with no differences between these two groups of rats. The mechanisms of protection against lethal ventricular tachyarrhythmia in nicorandil may increase nitric oxide release by upregulated eNOS expression through the opening of K-ATP channels and/or a K-ATP channels opener itself after acute myocardial infarction.

To study the effects of free fatty acids on insulin secretion and expression of SUR1 gene in rat pancreatic B cells in vitro, and to explore the molecular mechanisms in lipotoxicity inducing insulin secretion dysfunction, pancreatic islet cells were isolated and digested from male SD rats. Purified islets were incubated with either 0.25 mmol/L palmitate or 0.125 mmol/L oleate for 48 h in vitro. Then islets were stimulated with either 5.6 mmol/L or 16.7 mmol/L glucose for 1 h. Insulin release was measured by using radioimmunoassay, and the expression of SUR1 gene mRNA was quantified by reserve transcription-polymerase chain reaction (RT-PCR). The islets exposed to both palmitate and oleate for 48 h showed an increased basal and a decreased glucose-indused insulin release as compared with control islets. Palmitate increased basal insulin secretion by 110% (P< 0.01), decreased glucose stimulated insulin secretion by 43% (P<0.01); while oleate increased basal insulin secretion by 80% (P<0.01) and decreased glucose stimulated insulin secretion by 32 % (P<0.05). RT-PCR showed that oleate significantly suppressed SUR1 gene expression by 64 % (P<0.01) as compared with the control group, while palmitate group manifested a light decrease of 15% (P>0.05) of SUR1 gene expression. Our results suggested that chronic exposure to free fatty acids of pancreatic beta cells inhibited glucose stimulated insulin secretion. Regulation of SUR1 gene expression may be involved in such effects, which may also be one of the molecular mechanisms in lipotoxocity inducing beta cells secretion dysfunction.

Rat sciatic nerve axons express capsaicin, proton and heat sensitivity and respond to stimulation with a Ca2+-dependent and graded calcitonin gene-related peptide (CGRP) release. In this study we demonstrate that similar functions, including capsaicin-induced CGRP release, are to be found in the desheathed sciatic nerve of the mouse. We have morphologically investigated the mechanisms of this axonal release in regions away from the active zones of synapses. Capsaicin receptor 1 (TRPV1) and CGRP immunostaining was performed using electron microscopic visualization. TRPV1 was identified in the axoplasm and inside vesicles--presumably on axonal transport--as well as in considerable quantity in the axonal plasma membrane of unmyelinated nerve fibers. Most of the unmyelinated axons were immunopositive for CGRP and in unstimulated nerves CGRP-containing vesicles almost entirely filled the axoplasm. After capsaicin stimulation (10(-6) M for 5 min), the fibers appeared depleted of CGRP with only few vesicles remaining as well as some residual staining of the axoplasm. In addition a large number of vesicles were fused with the axonal membrane, forming classical exocytotic figures--the omega structures--lined with CGRP immunoreactive product. These results present morphological evidence for the distribution of TRPV1 along unmyelinated axons in peripheral nerve and also provide the first demonstration of vesicular neuropeptide exocytosis along unmyelinated axons in peripheral nerve.

Expression of the heat sensitive cation channels TRPV1 and TRPV2 was investigated by immunofluorescence in rat dorsal root ganglion (DRG) neurons. TRPV1-positive neurons were more frequent and had smaller diameters than TRPV2-positive neurons (35.7% vs 7.3%; 22.3 microm vs 27.6 microm), but size distributions overlapped and significant co-expression was seen in 20.7% of TRPV2-positive neurons (1.7% of all). Expression patterns did not differ between tissue sections typically used in immunocytochemistry and dissociated DRG neurons typically used in electrophysiology. Rectangular temperature pulses revealed two patterns of heat-evoked inward currents in small DRG neurons: low-threshold rapidly activating and high-threshold slowly activating. Slowly activating heat-evoked currents have not been described previously, but correspond to the type I heat responses of primary nociceptive afferents, which have been suggested to be mediated by TRPV2.

Resiniferatoxin (RTX), a potent analog of capsaicin, was evaluated electrophysiologically in dorsal root ganglion (DRG) cells and cell lines ectopically expressing the vanilloid receptor type 1 (VR1) to determine if cell phenotype influenced RTXs neurotoxic properties. Furthermore, capsaicin and heat activation of VR1 were evaluated in these cells to determine if cellular damage was unique to RTX activation of the receptors. RTX application to DRG cells identified as type 1, 2 or 5, cell types known to express VR1, induced large inward currents. RTX did not induce currents in DRG cells that do not express the receptor (type 4 cells). In cell lines ectopically expressing VR1, RTX-induced similar currents. RTX produced no effect in non-transfected cells. After exposure to RTX both DRG cells and transfected cells failed to respond to subsequent applications of the agonist. In addition, whole cell capacitance was reduced up to 70%. The decrease in capacitance was associated with the loss of plasma membrane, as determined by confocal microscopy. Cell phenotype, other than VR1 expression, did not influence the response to RTX. Interestingly, capsaicin and heat activation of vanilloid receptors also decreased cell capacitance, but the loss of membrane was not as great as with RTX and responses to these stimuli were not lost after the initial exposure. The loss of cell membrane required elevated intracellular levels of Ca2+. From these data it was concluded that the loss of cell membrane was dependent on the presence of both VR1 and intracellular Ca2+ accumulation, but not on cell phenotype.

Vanilloid receptors subtype 1 (VR1), a nonselective cation channel responsive to capsaicin, protons, and noxious heat, has been recently identified in not only neural but also non-neural cells. In the present study, we demonstrated the peripheral expression of VR1 in gastric mucosal epithelial cells and investigated the role of the receptor in cellular protection. The rat gastric mucosal epithelial cell line was used. The expression of VR1 was examined by Western blotting and RT-PCR. Cell damage was induced by immersion in 10% ethanol or acid (pH 4.0) for 30 min, and cell viability was determined by MTT assay. Capsaicin or resiniferatoxin was added 30 min before the challenge with ethanol or acid, while capsazepine or ruthenium red (a VR1 antagonist) was added simultaneously with capsaicin. The distinct expression of VR1 protein and mRNA was detected in rat gastric mucosal epithelial cell line as well as in the rat stomach and spinal cord by Western blotting and RT-PCR, respectively. The cDNA sequence of the PCR product was found to be almost identical to that of the authentic VR1 (99.8%) when the product was subcloned and sequenced. On the other hand, the cell damage induced by ethanol or acid was dose-dependently prevented by pretreatment with capsaicin. The protective effect of capsaicin was mimicked by resiniferatoxin and almost totally abolished by co-addition of capsazepine or ruthenium red. These findings suggest that VR1 is expressed peripherally in gastric mucosal epithelial cells and plays a cellular protective role.

Myocardial ischaemia causes the release of metabolites such as bradykinin, which stimulates cardiac sensory receptors to evoke a sympathoexcitatory reflex. However, the molecular identity of the afferent neurons and fibres mediating this reflex response is not clear. In this study, we tested the hypothesis that the cardiogenic sympathoexcitatory reflex is mediated by capsaicin-sensitive afferent fibres. Enhanced immunofluorescence labelling revealed that vanilloid receptor 1 (VR1)-containing afferent nerve fibres were present on the epicardial surface of the rat heart. Resiniferatoxin (RTX), a potent analogue of capsaicin, was used to deplete capsaicin-sensitive afferent fibres in rats. Depletion of these fibres was confirmed by a substantial reduction of VR1 immunoreactivity in the epicardium and dorsal root ganglia. The thermal sensitivity was also diminished in RTX-treated rats. Renal sympathetic nerve activity (RSNA) and blood pressure were recorded in anaesthetized rats during epicardial application of bradykinin or capsaicin. In vehicle-treated rats, epicardial bradykinin (10 microg ml-1) or capsaicin (10 microg ml-1) application produced a significant increase in RSNA and arterial blood pressure. The RSNA and blood pressure responses caused by bradykinin and capsaicin were completely abolished in RTX-treated rats. Furthermore, epicardial application of iodo-RTX, a highly specific antagonist of VR1 receptors, blocked capsaicin- but not bradykinin-induced sympathoexcitatory responses. Thus, these data provide important histological and functional evidence that the heart is innervated by VR1-expressing afferent nerves and these afferent nerves are essential for the cardiogenic sympathoexcitatory reflex during myocardial ischaemia.

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7-1,125.6 microm(2), mean+/-S.D. 407.7+/-219.7 microm(2)), whereas VRL-1-IR neurons were medium-sized to large (range 223.6-1,341.1 microm(2), mean+/-S.D. 584.3+/-253.5 microm(2)). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5-1464.4 microm(2), mean+/-S.D. 554.3+/-207.4 microm(2); VRL-1: range 161.7-1166.2 microm(2), mean+/-S.D. 541.9+/-186.2 microm(2)). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons.

The application of cyclophosphamide to rats was used to induce interstitial cystitis. Behavioural studies indicated a strong pain reaction that developed within 2 h and levelled off thereafter causing a constant pain during the following 18 h. Neurons prepared from L6/S1 dorsal root ganglia innervating the urinary bladder responded to the application of capsaicin or alpha,beta-methylene ATP (alpha,beta-meATP) with an increase of intracellular Ca2+ ([Ca2+]i). The [Ca2+]i responses to capsaicin were identical in the dorsal root ganglion cells of cyclophosphamide- and saline-treated rats, whereas alpha,beta-meATP induced less increase in [Ca2+]i in the cyclophosphamide-treated animals than in their saline-treated counterparts. Hence, alpha,beta-meATP-sensitive P2X3 and/or P2X2/3 receptors of L6/S1 dorsal root ganglion neurons were functionally downregulated during subacute pain caused by experimental cystitis. In contrast, capsaicin-sensitive vanilloid 1 receptors did not react to the same procedure. Thoracal dorsal root ganglia, not innervating the urinary bladder, were also unaltered in their responsiveness to alpha,beta-meATP by cyclophosphamide treatment.

Cannabinoids have been shown to disrupt memory processes and these effects occur primarily through cannabinoid CB1 receptors in the brain. The present study investigates, for the first time, the regional variations and age-related changes in CB1 protein expression in the hippocampus and its neighbouring entorhinal, perirhinal, postrhinal and temporal cortices using Western blotting. In young adult rats, CB1 protein was highly expressed in the hippocampus and within the hippocampus, the greatest density of CB1 protein was located in CA1. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, CB1 protein expression was significantly increased in the aged entorhinal and temporal cortices and was significantly decreased in the aged postrhinal cortex. The present study demonstrates region-specific changes in CB1 protein expression during ageing and further suggests that cannabinoid CB1 receptors may contribute to the aging process.

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB(2) RNA, was employed for the high-level expression of the murine CB(2) (mCB(2)) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate-polyacrylamide gel electrophoresis (urea-SDS-PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40kDa were produced by the construct. The 40kDa product, but not the 26kDa product, was glycosylated as determined by 2-deoxy-D-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [3H]CP55940 ([3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB(2) RNA indicated a K(d) of 0.35+/-0.04nM and a B(max) of 24.4+/-2.7pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB(2) receptors, was observed. The CB(2) receptor-specific antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [3H]CP55940, while the CB(1) receptor-specific antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB(2) RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB(2) receptor. However, the presence of both 26 and 40kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB(2) RNA expression system.

Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a potential outcome of therapeutic application of the drug. Moreover, endogenous cannabinoids are physiological immune regulators. In the present report, we describe alterations in gene transcription that occur after cannabinoid exposure in a mast cell line, RBL2H3. Cannabinoid exposure causes marked changes in the transcript levels for numerous genes, acting both independently of and in concert with immunoreceptor stimulation via Fc epsilon RI. In two mast cell lines, we observed mRNA and protein expression corresponding to both CB1 and CB2 cannabinoid receptor isoforms, contrary to the prevailing view that CB1 is restricted to the CNS. We show that coexpression of the two isoforms is not functionally redundant in mast cells. Analysis of signaling pathways downstream of cannabinoid application reveals that activation of extracellular signal-regulated kinase, AKT, and a selected subset of AKT targets is accomplished by CB2 ligands and nonselective CB1/CB2 agonists in mast cells. CB1 inhibition does not affect AKT or extracellular signal-regulated kinase activation by cannabinoids, indicating that CB2 is the predominant regulatory receptor for these kinases in this cell context. CB1 receptors are, however, functional in these mast cells, since they can contribute to suppression of secretory responses.