Type II spiral ganglion afferent neurons drive medial olivocochlear reflex suppression of the cochlear amplifier.

The dynamic adjustment of hearing sensitivity and frequency selectivity is mediated by the medial olivocochlear efferent reflex, which suppresses the gain of the 'cochlear amplifier' in each ear. Such efferent feedback is important for promoting discrimination of sounds in background noise, sound localization and protecting the cochleae from acoustic overstimulation. However, the sensory driver for the olivocochlear reflex is unknown. Here, we resolve this longstanding question using a mouse model null for the gene encoding the type III intermediate filament peripherin (Prph). Prph((-/-)) mice lacked type II spiral ganglion neuron innervation of the outer hair cells, whereas innervation of the inner hair cells by type I spiral ganglion neurons was normal. Compared with Prph((+/+)) controls, both contralateral and ipsilateral olivocochlear efferent-mediated suppression of the cochlear amplifier were absent in Prph((-/-)) mice, demonstrating that outer hair cells and their type II afferents constitute the sensory drive for the olivocochlear efferent reflex

Porcine Bladder Urothelial, Myofibroblast, and Detrusor Muscle Cells: Characterization and ATP Release

ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10–14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically

Vascular microarray profiling in two models of hypertension identifies caveolin-1, Rgs2 and Rgs5 as antihypertensive targets

BACKGROUND: Hypertension is a complex disease with many contributory genetic and environmental factors. We aimed to identify common targets for therapy by gene expression profiling of a resistance artery taken from animals representing two different models of hypertension. We studied gene expression and morphology of a saphenous artery branch in normotensive WKY rats, spontaneously hypertensive rats (SHR) and adrenocorticotropic hormone (ACTH)-induced hypertensive rats. RESULTS: Differential remodeling of arteries occurred in SHR and ACTH-treated rats, involving changes in both smooth muscle and endothelium. Increased expression of smooth muscle cell growth promoters and decreased expression of growth suppressors confirmed smooth muscle cell proliferation in SHR but not in ACTH. Differential gene expression between arteries from the two hypertensive models extended to the renin-angiotensin system, MAP kinase pathways, mitochondrial activity, lipid metabolism, extracellular matrix and calcium handling. In contrast, arteries from both hypertensive models exhibited significant increases in caveolin-1 expression and decreases in the regulators of G-protein signalling, Rgs2 and Rgs5. Increased protein expression of caveolin-1 and increased incidence of caveolae was found in both smooth muscle and endothelial cells of arteries from both hypertensive models. CONCLUSION: We conclude that the majority of differences in gene expression found in the saphenous artery taken from rats with two different forms of hypertension reflect distinctive morphological and physiological alterations. However, changes in common to caveolin-1 expression and G protein signalling, through attenuation of Rgs2 and Rgs5, may contribute to hypertension through augmentation of vasoconstrictor pathways and provide potential targets for common drug development

Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature

<p>Abstract</p> <p>Background</p> <p>HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance.</p> <p>Results</p> <p>Wild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27°C and this correlated with maximal ³H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30°C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca²⁺-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37°C, but not at 30°C.</p> <p>Conclusion</p> <p>Our results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.</p

Diet-Induced Obesity Impairs Endothelium-Derived Hyperpolarization via Altered Potassium Channel Signaling Mechanisms

BACKGROUND: The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study. METHODOLOGY/PRINCIPAL FINDINGS: Membrane potential, vessel diameter and luminal pressure were recorded in 4(th) order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16-20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SK(Ca)/IK(Ca)) inhibition; with such activity being impaired in obesity. SK(Ca)-IK(Ca) activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IK(Ca)-mediated EDH contribution was increased in obesity, and associated with altered IK(Ca) distribution and elevated expression. In contrast, the SK(Ca)-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (K(ir)) and Na(+)/K(+)-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential K(ir) expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density. CONCLUSION/SIGNIFICANCE: In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IK(Ca) and Na(+)/K(+)-ATPase, and decreased K(ir) underlie changes in the EDH mechanism

Factors, fiction and endothelium-derived hyperpolarizing factor

1. The principal mediators of vascular tone are neural, endothelial and physical stimuli that result in the initiation of dilator and constrictor responses to facilitate the control of blood pressure. Two primary vasodilatory stimuli produced by the endothelium are nitric oxide (NO) and prostaglandins. An additional endothelium-dependent vasodilatory mechanism is characterized as the hyperpolarization-mediated relaxation that remains after the inhibition of the synthesis of NO and prostaglandins. This mechanism is due to the action of a so-called endothelium-derived hyperpolarizing factor (EDHF) and is dependent on either the release of diffusible factor(s) and/or to a direct contact-mediated mechanism. 2. Most evidence supports the concept that 'EDHF' activity is dependent on contact-mediated mechanisms. This involves the transfer of an endothelium-derived electrical current, as an endothelium-derived hyperpolarization (EDH), through direct heterocellular coupling of endothelial cells and smooth muscle cells via myoendothelial gap junctions (MEGJ). However, there is a lack of consensus with regard to the nature and mechanism of action of EDHF/EDH (EDH(F)), which has been shown to vary within and between vascular beds, as well as among species, strains, sex and during development, ageing and disease. 3. In addition to actual heterogeneity in EDH(F), further heterogeneity has resulted from the less-than-optimal design, analysis and interpretation of data in some key papers in the EDHF literature; with such views being perpetuated in the subsequent literature. 4. The focus of the present brief review is to examine what factors are proposed as EDH(F) and highlight the correlative structural and functional studies from our laboratory that demonstrate an integral role for MEGJ in the conduction of EDH, which account for the heterogeneity in EDH(F), while incorporating the reported diffusible mechanisms in the regulation of this activity. Furthermore, in addition to the reported heterogeneity in the nature and mechanism of action of EDH(F), the contribution of experimental design and technique to this heterogeneity will be examined

Incidence of Myoendothelial Gap Junctions in the Proximal and Distal Mesenteric Arteries of the Rat Is Suggestive of a Role in Endothelium-Derived Hyperpolarizing Factor - Mediated Responses

Although the chemical nature of endothelium-derived hyperpolarizing factor (EDHF) remains elusive, electrophysiological evidence exists for electrical communication between smooth muscle cells and endothelial cells suggesting that electrotonic propagation of hyperpolarization may explain the failure to identify a single chemical factor as EDHF. Anatomical evidence for myoendothelial gap junctions, or the sites of electrical coupling, is, however, rare. In the present study, serial-section electron microscopy and reconstruction techniques have been used to examine the incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat where EDHF responses have been reported to vary. Myoendothelial gap junctions were found to be very small in the mesenteric arteries, the majority being <100 nm in diameter. In addition, they were significantly more common in the distal compared with the proximal regions of this arterial bed. Pentalaminar gap junctions between adjacent endothelial cells were much larger and were common in both proximal and distal mesenteric arteries. These latter junctions were frequently found near the myoendothelial gap junctions. These results provide the first evidence for the presence of sites for electrical communication between endothelial cells and smooth muscle cells in the mesenteric vascular bed. Furthermore, the relative incidence of these sites suggests that there may be a relationship between the activity of EDHF and the presence of myoendothelial gap junctions

Specialised symapthetic neuroeffector associations in immature rat iris arterioles

Sympathetic nerve-mediated vasoconstriction in iris arterioles of mature rats occurs via the activation of α(1B)-adrenoceptors alone, while in immature rat iris arterioles, vasoconstriction occurs via activation of both α1- and α2-adrenoceptors. In ma

Specialised sympathetic neuroeffector associations in immature rat iris arterioles

Sympathetic nerve-mediated vasoconstriction in iris arterioles of mature rats occurs via the activation of α(1B)-adrenoceptors alone, while in immature rat iris arterioles, vasoconstriction occurs via activation of both α(1)- and α(2)-adrenoceptors. In mature rats the vast majority of sympathetic varicosities form close neuroeffector junctions. Serial section electron microscopy of 14 d iris arterioles has been used to determine whether restriction in physiological receptor types with age may result from the establishment of these close neuroeffector junctions. Ninety varicosities which lay within 4 μm of arteriolar smooth muscle were followed for their entire length. Varicosities rarely contained dense cored vesicles even after treatment with 5-hydroxydopamine. 47% of varicosities formed close associations with muscle cells and 88% formed close associations with muscle cells or melanocytes. Varicosities in bundles were as likely as single varicosities to form close associations with vascular smooth muscle cells, although the distribution of synaptic vesicles in single varicosities did not show the asymmetric accumulation towards the smooth muscle cells seen in the varicosities in bundles which were frequently clustered together. We conclude that restriction of physiological receptor types during development does not appear to correlate with the establishment of close neuroeffector junctions, although changes in presynaptic structures may contribute to the refinement of postsynaptic responses