Abstract:BackgroundThe vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids.ResultsShort circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by for… Show more
“…Further studies are needed to confirm the relationship between the elevation in AC expression and cAMP levels with a putative increase in CFTR functional activity. Patch‐clamp studies (Gao et al, ; Fisher et al, ; Pondugula et al, ) would be particularly useful to establish such a relationship: For example, a cultured cervical epithelial monolayer could be used to identify an enhanced Cl − conductance by determining the magnitude of a short circuit current (I sc ) and its response to the specific CFTR inhibitor, CFTRinh‐172. In the presence of forskolin, an activator of AC (Chan et al, ), I sc is expected to increase.…”
The consistency of the cervical mucus changes with the reproductive cycle, which we hypothesized involved changing levels of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC), and cyclic adenosine mono-phosphate (cAMP). We therefore measured the abundance of each in the rat cervix under estrogen and progesterone influence to determine if the activity of these components could explain the changes in the consistency of cervical mucus. Ovariectomised adult female rats were treated with three days of either estrogen (1 μg/kg/day) or progesterone (20 mg/kg/day), or three days of estrogen followed by two days of either vehicle or progesterone or estrogen plus progesterone. In some groups, mifepristone (7 mg/kg/day) was concurrently given with progesterone. Animals were then sacrificed, and the cervix was harvested for protein and mRNA expression analyses by Western blot and real-time PCR, respectively. The distribution of proteins was investigated by immunohistochemistry, and levels of cAMP were determined by enzyme-linked immunosorbent assay (ELISA). Cftr mRNA, AC protein, and cAMP levels in cervical homogenates as well as the tissue distribution of CFTR and AC in endocervical epithelia were highest under estrogen influence; the opposite pattern was seen under progesterone influence. Cervical lumen circumference was highest under estrogen and lowest under progesterone. The effects of progesterone were antagonized by mifepristone. Therefore, increased abundance of CFTR, AC, and cAMP under estrogen influence could account for the increased fluid accumulation within the cervical lumen, which would contribute to lower cervical mucus consistency, whereas progesterone reverses this effect at the molecular and organ level.
“…Further studies are needed to confirm the relationship between the elevation in AC expression and cAMP levels with a putative increase in CFTR functional activity. Patch‐clamp studies (Gao et al, ; Fisher et al, ; Pondugula et al, ) would be particularly useful to establish such a relationship: For example, a cultured cervical epithelial monolayer could be used to identify an enhanced Cl − conductance by determining the magnitude of a short circuit current (I sc ) and its response to the specific CFTR inhibitor, CFTRinh‐172. In the presence of forskolin, an activator of AC (Chan et al, ), I sc is expected to increase.…”
The consistency of the cervical mucus changes with the reproductive cycle, which we hypothesized involved changing levels of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC), and cyclic adenosine mono-phosphate (cAMP). We therefore measured the abundance of each in the rat cervix under estrogen and progesterone influence to determine if the activity of these components could explain the changes in the consistency of cervical mucus. Ovariectomised adult female rats were treated with three days of either estrogen (1 μg/kg/day) or progesterone (20 mg/kg/day), or three days of estrogen followed by two days of either vehicle or progesterone or estrogen plus progesterone. In some groups, mifepristone (7 mg/kg/day) was concurrently given with progesterone. Animals were then sacrificed, and the cervix was harvested for protein and mRNA expression analyses by Western blot and real-time PCR, respectively. The distribution of proteins was investigated by immunohistochemistry, and levels of cAMP were determined by enzyme-linked immunosorbent assay (ELISA). Cftr mRNA, AC protein, and cAMP levels in cervical homogenates as well as the tissue distribution of CFTR and AC in endocervical epithelia were highest under estrogen influence; the opposite pattern was seen under progesterone influence. Cervical lumen circumference was highest under estrogen and lowest under progesterone. The effects of progesterone were antagonized by mifepristone. Therefore, increased abundance of CFTR, AC, and cAMP under estrogen influence could account for the increased fluid accumulation within the cervical lumen, which would contribute to lower cervical mucus consistency, whereas progesterone reverses this effect at the molecular and organ level.
“…Patch clamp experiment can be used to investigate this relationship [31][32][33]. In patch-clamp study, the magnitude of a short circuit current (Isc) in the cultured epithelial monolayer that can be obtain from seminal vesicles of animals receiving different treatment (as above) can be measured.…”
“…The low affinity targets were taken to be in metabolic pathways rather than membrane transporters; interference with metabolism was shown to decrease the apical H + flux (e.g., removal of glucose, above). High concentrations of drugs (e.g., >100 μM) that are in equilibrium with a lipophilic chemical form (e.g., DIOA and NPPB) [ 30 , 31 ], are able to integrate into mitochondrial membranes and interfere with ATP production. Fig.…”
BackgroundDisturbance of acid–base balance in the inner ear is known to be associated with hearing loss in a number of conditions including genetic mutations and pharmacologic interventions. Several previous physiologic and immunohistochemical observations lead to proposals of the involvement of acid–base transporters in stria vascularis.ResultsWe directly measured acid flux in vitro from the apical side of isolated stria vascularis from adult C57Bl/6 mice with a novel constant-perfusion pH-selective self-referencing probe. Acid efflux that depended on metabolism and ion transport was observed from the apical side of stria vascularis. The acid flux was decreased to about 40 % of control by removal of the metabolic substrate (glucose-free) and by inhibition of the sodium pump (ouabain). The flux was also decreased a) by inhibition of Na,H-exchangers by amiloride, dimethylamiloride (DMA), S3226 and Hoe694, b) by inhibition of Na,2Cl,K-cotransporter (NKCC1) by bumetanide, and c) by the likely inhibition of HCO3/anion exchange by DIDS. By contrast, the acid flux was increased by inhibition of gastric H,K-ATPase (SCH28080) but was not affected by an inhibitor of vH-ATPase (bafilomycin). K flux from stria vascularis was reduced less than 5 % by SCH28080.ConclusionsThese observations suggest that stria vascularis may be an important site of control of cochlear acid–base balance and demonstrate a functional role of several acid–base transporters in stria vascularis, including basolateral H,K-ATPase and apical Na,H-exchange. Previous suggestions that H secretion is mediated by an apical vH-ATPase and that basolateral H,K-ATPase contributes importantly to K secretion in stria vascularis are not supported. These results advance our understanding of inner ear acid–base balance and provide a stronger basis to interpret the etiology of genetic and pharmacologic cochlear dysfunctions that are influenced by endolymphatic pH.Electronic supplementary materialThe online version of this article (doi:10.1186/s12899-016-0024-1) contains supplementary material, which is available to authorized users.
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