Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na(+) and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm(2) for 2 h increased ET-1 mRNA content by ∼2.3-fold. Absence of perfusate Ca(2+), chelation of intracellular Ca(2+), or inhibition of Ca(2+) signaling (calmodulin, Ca(2+)/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca(2+) entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca(2+) was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na(+) channels or the mitochondrial Na(+)/Ca(2+) exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca(2+)-dependent signaling pathways that stimulate ET-1 synthesis.

Objective-To analyze the quantity and distribution of intramuscular nerves within the striated urogenital sphincter and test the hypothesis that decreased nerve density is associated with decreased striated sphincter muscle and cadaver age. Methods-Thirteen cadaveric urethras (mean age 47 years, range 15-78 years) were selected for study. A sagittal histologic section was stained with S100 stain to identify intramuscular nerves. The number of times that a nerve was seen within the striated urogenital sphincter (nerve number) was counted. The number of axons within each nerve fascicle was also counted. Regression analysis of nerve density against muscle cell number and age was performed. Results-Remarkable variation was found in the quantity of intramuscular nerves in the striated urogenital sphincter of the 13 urethras studied. The number of nerves ranged from 72 to 543, a sevenfold variation (mean 247.1 ± standard deviation 123.2), and the range of number of axons was 431 to 3523 (2201 ± 1152.6). The larger nerve fascicles were seen predominantly in the distal (13.1 ± 5.7 axons per nerve) compared with the proximal part of the striated urogenital sphincter (1.2 ± 2). Reduced nerve density throughout the striated urogenital sphincter correlated with fewer muscle cells (P = .02). Nerve density also decreased with advancing age (P = .004). Conclusion-Remarkable variation in the quantity of intramuscular nerves was found. Women with sparse intramuscular nerves had fewer striated muscle cells. Intramuscular nerve density declined with age.

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of Na and water transport. CD ET-1 production is stimulated by extracellular fluid volume expansion and tubule fluid flow, suggesting a mechanism coupling CD Na delivery and ET-1 synthesis. A mouse cortical CD cell line, mpkCCDc14, was subjected to static or flow conditions for 2 h at 2 dyn/cm(2), followed by determination of ET-1 mRNA content. Flow with 300 mosmol/l NaCl increased ET-1 mRNA to 65% above that observed under static conditions. Increasing perfusate osmolarity to 450 mosmol/l with NaCl or Na acetate increased ET-1 mRNA to ∼184% compared with no flow, which was not observed when osmolarity was increased using mannitol or urea. Reducing Na concentration to 150 mosmol/l while maintaining total osmolarity at 300 mosmol/l with urea or mannitol decreased the flow response. Inhibition of epithelial Na channel (ENaC) with amiloride or benzamil abolished the flow response, suggesting involvement of ENaC in flow-regulated ET-1 synthesis. Aldosterone almost doubled the flow response. Since Ca(2+) enhances CD ET-1 production, the involvement of plasma membrane and mitochondrial Na/Ca(2+) exchangers (NCX) was assessed. SEA0400 and KB-R7943, plasma membrane NCX inhibitors, did not affect the flow response. However, CGP37157, a mitochondrial NCX inhibitor, abolished the response. In summary, the current study indicates that increased Na delivery, leading to ENaC-mediated Na entry and mitochondrial NCX activity, is involved in flow-stimulated CD ET-1 synthesis. This constitutes the first report of either ENaC or mitochondrial NCX regulation of an autocrine factor in any biologic system.

Aims Endothelin-1 (ET-1) is an autocrine inhibitor of collecting duct (CD) Na+ and water reabsorption. CD ET-1 production is increased by a high salt diet and is important in promoting a natriuretic response. The mechanisms by which a high salt diet enhances CD ET-1 are being uncovered. In particular, elevated tubule fluid flow, as occurs in salt loading, enhances CD ET-1 synthesis. Tubule fluid solute content and interstitial osmolality can also be altered by a high salt diet, however their effect on CD ET-1 alone, or in combination with flow, is poorly understood. Main methods ET-1 mRNA production by a mouse inner medullary CD cell line (mIMCD3) in response to changing flow and/or osmolality was assessed. Key findings Flow or hyperosmolality (using NaCl, mannitol or urea) individually caused an ~2-fold increase in ET-1 mRNA, while flow and hyperosmolality together increased ET-1 mRNA by ~14 fold. The hyperosmolality effect alone and the synergistic effect of flow + hyperosmolality was inhibited by chelation of intracellular Ca2+, however were not altered by blockade of downstream Ca2+-signaling pathways (calcineurin or NFATc), inhibition of cellular Ca2+ entry channels (purinergic receptors or polycystin-2), or blockade of the epithelial Na+ channel. Inhibition of NFAT5 with rottlerin or NFAT5 siRNA greatly reduced the stimulatory effect of osmolality alone and osmolality + flow on mIMCD3 ET-1 mRNA levels. Significance Both flow and osmolality individually and synergistically stimulate mIMCD3 ET-1 mRNA content. These findings may be relevant to explaining high salt diet induction of CD ET-1 production.

Remarkable variation in the quantity of intramuscular nerves was found. Women with sparse intramuscular nerves had fewer striated muscle cells. Intramuscular nerve density declined with age.