Heather A. Drummond

The mammalian sodium channel BNC1 is required for normal touch sensation

Of the vertebrate senses, touch is the least understood at the molecular level. The ion channels that form the core of the mechanosensory complex and confer touch sensitivity remain unknown. However, the similarity of the brain sodium channel 1 (BNC1) to nematode proteins involved in mechanotransduction indicated that it might be a part of such a mechanosensor. Here we show that disrupting the mouse BNC1 gene markedly reduces the sensitivity of a specific component of mechanosensation: low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin these mechanoreceptors are excited by hair movement. Consistent with this function, we found BNC1 in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. Although BNC1 has been proposed to have a role in pH sensing, the acid-evoked current in cultured sensory neurons and the response of acid-stimulated nociceptors were normal in BNC1 null mice. These data identify the BNC1 channel as essential for the normal detection of light touch and indicate that BNC1 may be a central component of a mechanosensory complex.

A Molecular Component of the Arterial Baroreceptor Mechanotransducer

Baroreceptor nerve endings detect acute fluctuations in arterial pressure. We tested the hypothesis that members of the DEG/ENaC family of cation channels, which are responsible for touch sensation in Caenorhabditis elegans, may be components of the baroreceptor mechanosensor. We found the gamma subunit of ENaC localized to the site of mechanotransduction in baroreceptor nerve terminals innervating the aortic arch and carotid sinus. A functional role for DEG/ENaC members was suggested by blockade of baroreceptor nerve activity and baroreflex control of blood pressure by an amiloride analog that inhibits DEG/ENaC channels. These data suggest that ENaC subunits may be components of the baroreceptor mechanotransducer and pave the way to a better definition of mechanisms responsible for blood pressure regulation and hypertension.

Protective Effect of Carbon Monoxide–Releasing Compounds in Ischemia-Induced Acute Renal Failure

Heme oxygenase (HO) induction has been demonstrated to be beneficial in limiting the extent of cellular damage after ischemia-induced acute renal failure (ARF). Because increased HO activity is associated with the production of carbon monoxide (CO) as well as the potent antioxidant bilirubin, it is unclear which of the two is of greater importance in the protective effects of HO induction. The purpose of this study was to determine the protective role of CO alone in ischemia-induced ARF. Bilateral clamping of the renal pedicle for 40 min was associated with a ninefold increase in the levels of plasma creatinine 24 h after reperfusion as compared with normal plasma creatinine levels; however, administration of CO donor compounds tricarbonyldichlororuthenium(II) dimer, ([Ru(CO)(3)Cl(2)](2), 10 mg/kg) or tricarbonylchloro(glycinato)ruthenium(II) ([Ru(CO)(3)Cl(glycinate)], (CORM-3) 1 h before the onset of ischemia significantly decreased the levels of plasma creatinine 24 h after reperfusion as compared with vehicle-treated mice. Surprising, treatment with the CO donors was associated with an increase in HO activity 24 h after ischemia. For determining whether the protective effects of the CO donors were due to CO or HO-1 induction, experiments were performed in which HO was inhibited before administration of the CO donors. Pretreatment with the HO inhibitor had no effect on the level of plasma creatinine 24 h after reperfusion after treatment with the CO donor compounds. These results suggest that CO itself may be protective and limit renal damage in ischemia induced ARF.

Degenerin/Epithelial Na+ Channel Proteins: Components of a Vascular Mechanosensor

Mechanosensitive ion channels are thought to mediate stretch-induced contraction in vascular smooth muscle cells (VSMCs); however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although recent reports suggest degenerin/epithelial Na+ channel (DEG/ENaC) proteins may be mechanosensors in sensory neurons, their role as mechanosensors in vascular tissue has not been examined. We first tested whether DEG/ENaC subunits are expressed in cerebral blood vessels and VSMCs and then examined their role as mechanosensors in mediating the myogenic response in intact blood vessels. Using RT-PCR, we found ENaC transcripts expressed in rat cerebral arteries and freshly dissociated rat cerebral VSMCs. We also detected ENaC expression in isolated blood vessels and VSMCs by immunoblotting and immunolocalization. Moreover, inhibition of ENaC with amiloride (1 &mgr;mol/L) and benzamil (30 nmol/L, 1 &mgr;mol), an amiloride analog, blocked myogenic constriction in isolated rat cerebral arteries. These data suggest that DEG/ENaC proteins are required for vessel responses to pressure and are consistent with the evolutionary conservation of mechanosensory function of DEG/ENaC proteins.

Role of Reactive Oxygen Species in Endothelin-Induced Hypertension

Abstract—Recent reports have indicated that endothelin-induced vasoconstriction in isolated aortic vascular rings may be mediated by the production of superoxide anion. The purpose of this study was to determine the role of superoxide anion in mediating the chronic renal and hypertensive actions of endothelin. Endothelin-1 (5 pmol/kg per minute) was chronically infused into the jugular vein by use of mini-osmotic pump for 9 days in male Sprague-Dawley rats and in rats treated with the superoxide anion scavenger tempol (30 mg/kg per day). Mean arterial pressure in the endothelin-1–treated rats was 141±3 mm Hg, compared with 125±2 mm Hg in control rats. Endothelin-1 increased renal vascular resistance (15.3±2.5 versus 10±1.3 mm Hg/mL per minute) and decreased renal plasma flow (6.5±0.9 versus 8.7±0.7 mL/min) in control rats. Endothelin-1 also significantly increased TBARS in the kidney and urinary 8-isoprostaglandin F2&agr; excretion. The increase in arterial pressure in response to endothelin-1 was completely abolished by tempol (127±4 versus 127±4 mm Hg). Tempol also markedly attenuated the renal plasma flow and renal vascular resistance response to endothelin-1. Tempol also significantly decreased the level of 8-isoprostaglandin F2&agr; in the endothelin-1–treated rats. Tempol had no effect on arterial pressure or renal hemodynamics in control rats. These data indicate that formation of reactive oxygen species may play an important role in mediating hypertension induced by chronic elevations in endothelin.

Localization of β and γ subunits of ENaC in sensory nerve endings in the rat foot pad

Abstract The molecular mechanisms underlying mechanoelectrical transduction and the receptors that detect light touch remain uncertain. Studies in Caenorhabditis elegans suggest that members of the DEG/ENaC cation channel family may be mechanoreceptors. Therefore, we tested the hypothesis that subunits of the mammalian epithelial Na+ channel (ENaC) family are expressed in touch receptors in rat hairless skin. We detected βENaC and γENaC, but not αENaC transcripts in cervical and lumbar dorsal root ganglia (DRG). Using immunofluorescence, we found βENaC and γENaC expressed in medium to large lumbar DRG neurons. Moreover, we detected these two subunits in Merkel cell–neurite complexes, Meissner-like corpuscles, and small lamellated corpuscles, specialized mechanosensory structures of the skin. Within these structures, βENaC and γENaC were localized in the nerve fibers believed to contain the sensors responsive to mechanical stress. Thus β and γENaC subunits are good candidates as components of the molecular sensor that detects touch.

A New Trick for an Old Dogma: ENaC Proteins as Mechanotransducers in Vascular Smooth Muscle

Myogenic constriction is a vasoconstriction of blood vessels to increases in perfusion pressure. In renal preglomerular vasculature, it is an established mechanism of renal blood flow autoregulation. Recently, myogenic constriction has been identified as an important protective mechanism, preventing the transmission of systemic pressure to the fragile glomerular vasculature. Although the signal transduction pathways mediating vasoconstriction are well known, how the increases in pressure trigger vasoconstriction is unclear. The response is initiated by pressure-induced stretch of the vessel wall and thus is dependent on mechanical signaling. The identity of the sensor detecting VSMC stretch is unknown. Previous studies have considered the role of extracellular matrix-integrin interactions, ion conduction units (channels and/or transporters), and the cytoskeleton as pressure detectors. Whether, and how, these structures fit together in VSMCs is poorly understood. However, a model of mechanotransduction in the nematode Caenorhadbditis elegans (C. elegans) has been established that ties together extracellular matrix, ion channels, and cytoskeletal proteins into a large mechanosensing complex. In the C. elegans mechanotransducer model, a family of evolutionarily conserved proteins, referred to as the DEG/ENaC/ASIC family, form the ion-conducting pore of the mechanotransducer. Members of this protein family are expressed in VSMC where they may participate in pressure detection. This review will address how the C. elegans mechanotransducer model can be used to model pressure detection in mammalian VSMCs and provide a new perspective to pressure detection in VSMCs.

Sensing Tension Epithelial Sodium Channel/Acid-Sensing Ion Channel Proteins in Cardiovascular Homeostasis

The epithelial sodium (Na+) channel (ENaC) plays a critical role in blood pressure regulation by controlling renal salt and water reabsorption. Channel overactivity can lead to severe hypertension and underactivity to salt wasting and hypotension.1 In addition to their role in salt/water homeostasis, recent studies suggest that ENaC proteins, and their relatives, the acid-sensing ion channel (ASIC) proteins, may play more ubiquitous roles in cardiovascular regulation than considered previously. Recent evidence suggests that ENaC/ASIC proteins may act as mechanosensors and chemosensors in the cardiovascular system. ENaC/ASIC proteins are expressed in mechanosensing and chemosensing tissues, such as vascular smooth muscle cells (VSMCs), carotid body glomus cells, and sensory neurons innervating arterial baroreceptors, heart, and skeletal muscle. Disruption of the ENaC/ASIC channels alters myogenic constriction, arterial chemoreceptor and baroreceptor responses, and acid-induced responses in heart and skeletal muscle. This brief review summarizes the evidence supporting a role for ENaC and ASIC proteins in diverse systems of cardiovascular mechanosensing and chemosensing. Together, these studies suggest that ENaC/ASIC proteins contribute to cardiovascular homeostasis by mediating neural and local regulatory mechanisms. ENaC and ASIC proteins are members of a protein family termed the degenerin (DEG)/ENaC/ASIC family. Members of this family are expressed in a wide range of species (nematode Caenorhabditis elegans , Drosophila , and mammals) and participate in diverse biological functions, including neurodegeneration, acid sensation, taste, learning and memory, proprioception, Na+/water transport, and mechanosensation. All of the members of the DEG/ENaC/ASIC family share a highly conserved structure: intracellular N and C termini and 2 membrane-spanning domains separated by a large extracellular domain. Most DEG/ENaC/ASIC proteins form amiloride sensitive, nonvoltage, gated cation channels.1,2 ### C elegans DEGs Members were first identified in the nematode, where a chemically induced mutation caused a subset of neurons to swell and lyse. This phenotype led to the first …

20-Hydroxyeicosatetraenoic Acid (20-HETE) Stimulates Migration of Vascular Smooth Muscle Cells

Aim: We tested the hypothesis that 20-HETE production contributes to platelet derived growth factor (PDGF)-BB stimulated migration of VSMC in a cell culture model. Methods: Studies were performed with A10 cells which are a rat vascular smooth muscle derived cell line. Migration was determined using a Boyden chamber chemotactic assay. Results: Pre-treatment of cells with two doses of 20-HETE (100 and 500 nM) significantly increased PDGF-BB stimulated VSMC migration by 34-58% of control; whereas, prior incubation of cells with inhibitors of 20-HETE production, 17-ODYA (1-25 M) or HET0016 (100 nM), significantly decreased PDGF-BB stimulated migration by 40-90%. 20-HETE mediated increase in PDGF-BB migration was completely prevented by the 20-HETE antagonist, WIT-002. In order to determine what second messenger pathways are involved in the 20-HETE mediated stimulation of VSM migration, experiments were performed with specific inhibitors of tyrosine kinase (tyrphostin 25, 10 µM), mitogen-activated extracellular signal-regulated kinase (MEK, PD98059, 20 µM and U0126, 10 µM), protein kinase C (Myr-PKC, 50 µM), and phosphoinositide 3-kinases (PI3Ks) (wortmannin, 50 nM). Blockade of MEK and PI3K all abolished the increase in 20-HETE mediated migration. Conclusion: 20-HETE stimulates PDGF-mediated VSM migration acting through pathways that involve MEK and PI3K.

Impaired pressure-induced constriction in mouse middle cerebral arteries of ASIC2 knockout mice.

Recent studies from our laboratory demonstrated the importance of mechanosensitive epithelial Na(+) channel (ENaC) proteins in pressure-induced constriction in renal and cerebral arteries. ENaC proteins are closely related to acid-sensing ion channel 2 (ASIC2), a protein known to be required for normal mechanotransduction in certain sensory neurons. However, the role of the ASIC2 protein in pressure-induced constriction has never been addressed. The goal of the current study was to investigate the role of ASIC2 proteins in pressure-induced, or myogenic, constriction in the mouse middle cerebral arteries (MCAs) from ASIC2 wild-type (+/+), heterozygous (+/-), and null (-/-) mice. Constrictor responses to KCl (20-80 mM) and phenylephrine (10(-7)-10(-4) M) were not different among groups. However, vasoconstrictor responses to increases in intraluminal pressure (15-90 mmHg) were impaired in MCAs from ASIC2(-/-) and (+/-) mice. At 60 and 90 mmHg, MCAs from ASIC2(+/+) mice generated 13.7 +/- 2.1% and 15.8 +/- 2.0% tone and ASIC2(-/-) mice generated 7.4 +/- 2.8% and 12.5 +/- 2.4% tone, respectively. Surprisingly, MCAs from ASIC2(+/-) mice generated 1.2 +/- 2.2% and 3.9 +/- 1.8% tone at 60 and 90 mmHg. The reason underlying the total loss of myogenic tone in the ASIC2(+/-) is not clear, although the loss of mechanosensitive beta- and gamma-ENaC proteins may be a contributing factor. These results demonstrate that normal ASIC2 expression is required for normal pressure-induced constriction in the MCA. Furthermore, ASIC2 may be involved in establishing the basal level of myogenic tone.