Yiu-Wa Chung

Regulation of anion secretion by cyclo-oxygenase and prostanoids in cultured epididymal epithelia from the rat

1 The role of cyclo‐oxygenase (COX) in the regulation of anion secretion (measured as short‐ circuit current, Isc) in cultured epididymal epithelia from immature rats was investigated. 2 COX inhibitors attenuated the increase of anion secretion caused by bradykinin (LBK) but had no effect on that caused by PGE2, suggesting that prostaglandin synthesis mediates the secretory response of the tissues to LBK. 3 The apparent IC50 values for indomethacin, piroxicam and L‐745,337 in inhibiting the LBK‐induced Isc were 0·14, 1·34 and 15·7 μM, respectively. This order of potency: indomethacin > piroxicam > L‐745,337 >> DFU suggests the involvement of the COX‐1 isozyme in the mediation of the secretory response to LBK. 4 Among the COX products (prostaglandins, thromboxane and prostacyclins) tested, only PGE2 and, to a much lesser extent, PGF2α stimulated anion secretion by cultured rat epididymal epithelia. 5 The effect of PGE2 was mimicked by 11‐deoxyl PGE1, a specific prostaglandin E (EP)2/4 receptor agonist, but not by sulprostone, a specific EP1/3 receptor agonist, indicating that cyclic AMP‐coupled EP2/4 receptors are involved in the LBK‐stimulated anion secretion. 6 A reverse transcriptase‐polymerase chain reaction study detected the expression of COX‐1 and COX‐2 mRNA in intact rat epididymis and in cultured epididymal epithelia. The expression of COX‐1 mRNA was reduced by LBK by 44 %. 7 Immunohistochemical studies demonstrated the presence of COX‐1 immunoreactivity in the basal cells of the intact rat epididymis. By comparision, COX‐2 immunoreactivity was detected in the apical pole of the principal cells. 8 The role of COX in the formation of the epididymal microenvironment and the implication of long term administration of non‐steroidal anti‐inflammatory drugs (NSAIDs) on male fertility are discussed.

Stimulation of anion secretion by β‐adrenoceptors in the mouse endometrial epithelium

1 Regulation of anion secretion by adrenoceptors in primary culture of mouse endometrial epithelium was investigated using the short circuit current (ISC) technique. 2 Adrenaline stimulated a sustained increase in the ISC in a concentration‐dependent manner. The adrenaline‐induced ISC could be inhibited by pretreatment with diphenylamine 2,2‐dicarboxylic acid (DPC) or replacement of external Cl− and HCO3−, but not by amiloride or replacement of Na+ in apical solution. 3 The concentration‐dependent responses of the adrenaline‐induced ISC to the CF channel blockers glibenclamide and DPC were examined and exhibited IC50 values of 380 and 960 μm, respectively. 4 The effect of various adrenoceptor agonists on the ISC was examined. The order of potency appeared to be isoprenaline > adrenaline > noradrenaline, while no response was elicited by the α‐adrenoceptor agonist methoxamine, indicating a predominant involvement of β‐adrenoceptors. 5 The β‐adrenoceptor antagonist propranolol was found to be much more effective than the α‐adrenoceptor antagonist phentolamine in inhibiting the ISC responses induced by all adrenoceptor agonists examined. 6 The effect of adrenaline on the ISC was mimicked by an adenylate cyclase activator, forskolin, but suppressed by the adenylate cyclase inhibitor MDL 12,330A, indicating the involvement of cAMP. 7 Our results demonstrate that anion secretion by the mouse endometrial epithelium is regulated by β‐adrenoceptors and involves a cAMP‐dependent mechanism.

A sperm GPI-anchored protein elicits sperm-cumulus cross-talk leading to the acrosome reaction.

Abstract.The acrosome reaction has long been thought to be induced by the zona pellucida. Here we report the identification and function of a novel human sperm glycosylphosphatidylinositol (GPI)-anchored membrane protein, NYD-SP8. The release of the protein during sperm-egg interaction and its binding to the cumulus, the first layer of egg investment, elicits cross-talk between the gametes and produces calcium dependant release of progesterone, which lead to the acrosome reaction. An in vivo mouse model of NYD-SP8 immunization is also established showing a reduced fertility rate. Thus, contrary to accepted dogma, our study demonstrates for the first time that, prior to reaching the zona pellucida, sperm may release a surface protein that acts on the cumulus cells leading to the acrosome reaction, which may be important for determining the outcome of fertilization.

Androgen dependent expression of AT1 receptor and its regulation of anion secretion in rat epididymis.

Our previous studies have provided solid evidence for the presence of an intrinsic angiotensin‐generating system in the rat epididymis, which plays an important role in the regulation of the anion and thus fluid secretion by the epididymal epithelium. In the present study, the effect of androgen on the expression of AT1receptor and its subsequent regulation of anion secretion by the epididymis were investigated using Western blotting, semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) and in vitro electrophysiological approaches. Results from Western blotting analysis showed that the expression of AT1receptor protein was almost abolished by castration whereas its expression was completely restored to the control level when the castrated rats were hormonally replaced with testosterone. Efferent duct ligation, however, appeared not to affect the expression of AT1receptor protein by the epididymis. Results from RT‐PCR showed that mRNA expression of AT1receptor was consistent with that observed in protein expression. Results from short‐circuit current (ISC) showed that castration almost abolished the angiotensin II‐induced ISC. However, efferent duct ligation did not affect the angiotensin II‐induced ISC, which was completely blocked in the presence of losartan, a specific antagonist of the AT1receptor. These data indicate that the expression of epididymal AT1receptor is predominantly influenced by testicular androgens but not by testicular factors. This androgen‐dependent expression of AT1receptor could have a role in the control of AT1receptor‐mediated anion secretion and thus fluid secretion by the rat epididymis.

Swelling-induced anion and cation conductances in human epididymal cells.

1. Activation of both anion and cation conductances was observed in primary cultured human epididymal cells during osmotic swelling under the patch‐clamp whole‐cell configuration. The swelling‐induced anion conductance was 25.66 +/‐ 4.70 nS and the cation conductance was 7.35 +/‐ 1.40 nS. The permeability ratio of K+ to Cl‐ (PK/PCl) was calculated to be 0.40. Known anion or cation channel blockers could inhibit both conductances simultaneously. 2. When the major permeant ion species in the pipette and bath solution was Cl‐, the mean conductance was found to be 17.06 +/‐ 1.8 nS, significantly smaller than that obtained in the presence of intracellular K+, 25.66 +/‐ 4.70 nS (P < 0.05). No significant current activation was observed when solutions containing only K+ as the permeant ion were used. 3. When the anionic amino acids glutamate and aspartate were used to replace extracellular Cl‐, the permeability ratios were calculated to be PGlut/PCl = 0.20 and PAsp/PCl = 0.17. 4. The cation conductance was found to be non‐selective since its permeability to other cations such as Na+ and choline, an organic compound highly concentrated in epididymal fluid, was similar to that of K+. 5. Regulatory volume decrease (RVD) was observed after initial osmotic swelling; this could be inhibited by either anion or cation channel blockers. 6. The results of this study suggest that both anion and cation conductances are activated during cellular swelling, and indicate the existence of an interdependent relationship between the swelling‐induced cation and anion conductances. Both swelling‐induced cation and anion conductances are involved in the volume regulatory process and may be responsible for transporting amino acids or organic compounds in human epididymal cells.

Expression of sperm Ca2+-activated K+ channels in Xenopus oocytes and their modulation by extracellular ATP

Ionic fluxes across the sperm membrane have been shown to be important in the initiating process of sperm activation and gamete interaction; however, electrophysiological investigation of the ion channels involved has been precluded by the small size of the sperm, especially in mammalian species. In the present study sperm ion channels were expressed in Xenopus oocytes by injection of RNAs of spermatogenic cells isolated from the rat testes. The RNA‐injected oocytes responded to ATP, a factor known to regulate sperm activation, with the activation of an outwardly rectifying whole‐cell current which was dependent on K+ concentrations and inhibitable by K+ channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The ATP‐induced current could be mimicked by a Ca2+ ionophore but suppressed by a Ca2+ chelator applied intracellularly, indicating a Ca2+ dependence of the current. Single‐channel measurements on RNA‐injected oocytes revealed channels of large conductance which could be blocked by CTX and TEA. Co‐injection of germ cell RNAs with the antisense RNA for a mouse gene encoding slowpoke ‘Maxi’ Ca2+‐activated K+ channels resulted in significant reduction of the ATP‐ and ionomycin‐induced current. The expression of the ‘Maxi’ Ca2+‐activated K+ channels in sperm collected from the rat epididymis was also confirmed by Western blot analysis. These results suggest that sperm possess Ca2+‐activated K+ channels which may be involved in the process of sperm activation.

UPREGULATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR EXPRESSION BY OESTROGEN AND BAK FOONG PILL IN MOUSE UTERI

Although cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to be expressed in the female reproductive tract, its functional role in the uterus is not fully understood. The present study investigated a possible physiological role of CFTR by comparing the effects of 17β‐oestradiol and Bak Foong Pill (BFP), an over‐the‐counter Chinese medicine used for centuries for the treatment of various gynaecological disorders, on uterus size and the expression of CFTR in the uterus of ovariectomised mice using RT‐PCR. Treatment of ovariectomised mice with 17β‐oestradiol (0.2mg/kg, p.o.) for 12 days caused a significnat increase in uterine wet weight compared to vehicle. However, treatment with BFP (3g/kg, p.o.) for the same period failed to increase uterine wet weight, indicating a lack of direct oestrogen‐like activity of BFP. Analysis of CFTR mRNA expression in the harvested uteri using RT‐PCR showed that both 17β‐oestradiol and BFP induced an increase in CFTR mRNA expression in mouse uteri compared to levels observed in vehicle‐treated animals. These results suggest that CFTR can be upregulated by oestrogen and BFP, however, the effect exerted by BFP does not seem to be mediated by direct oestrogen‐like activity. Regulation of CFTR expression by both oestrogen and gynaecological medication BFP indicates an important role of CFTR in reproductive functions.

Involvement of intracellular and extracellular Ca2+ in tetramethylpyrazine-induced colonic anion secretion

In the present study we investigated the role of Ca2+ in tetramethylpyrazine (TMP)‐induced anion secretion in the human colonic epithelial cell line, Caco‐2, using the short‐circuit current (ISC) technique in conjunction with intracellular Ca2+ measurements. The results showed that TMP‐induced ISC response was significantly reduced by 58.8% and 38.3% after inhibiting Ca2+ ATPase of endoplasmic reticulum (ER) with thapsigargin and mobilizing ER stored Ca2+ release with ATP, respectively. Conversely, thapsigargin‐ and ATP‐evoked ISC responses were also significantly reduced by pretreatment with TMP by 43.2% and 38.5%, respectively. Conversely, removal of extracellular Ca2+, apical but not basolateral, or the presence of the Ca2+ chelator (EGTA) significantly increased TMP‐induced ISC by 47.1% and 37.8%, respectively. Similar to TMP, thapsigargin‐induced current increase was also enhanced by chelating extracellular Ca2+ or in Ca2+ free solution; however, removal of extracellular Ca2+ did not significantly affect 3‐isobutyl‐1‐methylxanthine (IBMX)‐ and forskolin‐induced transepithelial current. Measurement of the intracellular concentration of free Ca2+ ([Ca2+]i) with fura‐2/AM showed that TMP could induce an increase in [Ca2+]i but pretreatment with TMP significantly reduced thapsigargin‐evoked, but not ATP‐induced, [Ca2+]i increase. These results suggest that the effect of TMP on colonic anion secretion is partly mediated by TMP‐increased [Ca2+]i by acting on a target similar to thapsigargin. The observed inhibitory effect of extracellular Ca2+ on Ca2+‐dependent anion secretion represents a novel mechanism by which Ca2+‐dependent regulation of epithelial electrolyte transport may be fine‐tuned by extracellular Ca2+ in the apical domain.

Local regulation of anion secretion by pituitary adenylate cyclase-activating polypeptide in human colonic T84 cells.

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a novel hypothalamic peptide, which has been shown to exert various functions in a number of tissues, including exocrine and endocrine tissues. The present study investigated the role of local PACAP in the control of anion secretion by the human colonic T84 cell. Both bioactive forms of PACAP‐27 and PACAP‐38 gave rise to a dose‐dependent increase in the short‐circuit current (ISC). However, there was a reversal in the order of potency observed at different concentration ranges for the two bioactive forms. PACAP‐27 was greater than PACAP‐38 when the peptide concentrations were below 10n m; PACAP‐38 was greater than PACAP‐27 in the range of 10–80n m. The effects of both PACAP forms were restricted to the apical aspect of the T84 cell. The ISCresponses to both PACAP‐27 and PACAP‐38 were suppressed respectively by the non‐selective Cl−channel blocker, diphenylamine‐dicarboxylic acid (DPC), by the Ca2+dependent Cl−channel blocker, diisothiocyanatostilbene‐disulfonic acid (DIDS) and by the Ca2+chelator, BAPTA‐AM, indicating the involvement of Ca2+. The expression of PACAP was demonstrated and localized specifically to the perinuclear cytoplasm of the T84 cell using immunocytochemistry, indicating its epithelial origin. Thus, the present data suggest that, in addition to the well‐known cAMP‐dependent pathway, PACAP may play a role in regulating colonic Cl−secretion via a Ca2+‐dependent pathway, perhaps through two distinct PACAP receptor subtypes. Moreover, the regulation of anion secretion by T84 cells may be mediated by locally formed PACAP in an autocrine or paracrine fashion.

Functional expression of sperm angiotensin II type I receptor in Xenopus oocyte: modulation of a sperm Ca2+-activated K+ channel.

In addition to Ca2+ and K+ fluxes, angiotensin II (Ang II) has been shown to influence sperm motility. The present study investigated the involvement of angiotensin II type 1 receptor (AT1) in mediating the modulatory effect of Ang II on a sperm Ca2+-activated K+ channel expressed in Xenopus oocytes injected with RNAs of spermatogenic cells. Ang II at a concentration of 1 microM was found to potentiate the ionomycin-induced current, previously demonstrated to be mediated by a Maxi Ca2+-activated K+ channel. However, at higher concentration, 20 microM, Ang II was found to suppress the ionomycin-induced current. Both potentiating and inhibitory effects of Ang II were blocked by losartan, a specific antagonist of AT1 receptors. Immunohistochemical studies further confirmed the presence of AT1 receptors in spermatogenic cells while expression of AT1 receptor mRNA was demonstrated by RT-PCR. These results suggest that Ang II may influence sperm motility as well as other sperm function by acting on AT1 receptors, and exerting potentiating and inhibitory effects on the Ca2+-activated K+ channels.