Combination of magnesium ions and vitamin C alleviates synovitis and osteophyte formation in osteoarthritis of mice.

INTRODUCTION: We previously demonstrated that magnesium ions (Mg2+) was a novel therapeutic alternative for osteoarthritis (OA) through promoting the hypoxia inducible factor-1α (HIF-1α)-mediated cartilage matrix synthesis. However, oxidative stress can inhibit the expression of HIF-1α, amplify the inflammation that potentially impairs the therapeutic efficacy of Mg2+ in OA. Vitamin (VC), a potent antioxidant, may enhance the efficacy of Mg2+ in OA treatment. This study aims to investigate the efficacy of combination of Mg2+ and VC on alleviating joint destruction and pain in OA. MATERIAL AND METHODS: Anterior cruciate ligament transection with partial medial meniscectomy induced mice OA model were randomly received intra-articular injection of either saline, MgCl2 (0.5 mol/L), VC (3 mg/ml) or MgCl2 (0.5 mol/L) plus VC (3 mg/ml) at week 2 post-operation, twice weekly, for 2 weeks. Joint pain and pathological changes were assessed by gait analysis, histology, western blotting and micro-CT. RESULTS: Mg2+ and VC showed additive effects to significantly alleviate the joint destruction and pain. The efficacy of this combined therapy could sustain for 3 months after the last injection. We demonstrated that VC enhanced the promotive effect of Mg2+ on HIF-1α expression in cartilage. Additionally, combination of Mg2+ and VC markedly promoted the M2 polarization of macrophages in synovium. Furthermore, combination of Mg2+ and VC inhibited osteophyte formation and expressions of pain-related neuropeptides. CONCLUSIONS: Intra-articular administration of Mg2+ and VC additively alleviates joint destruction and pain in OA. Our current formulation may be a cost-effective alternative treatment for OA.

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model.

Magnesium (Mg)-based biometal attracts clinical applications due to its biodegradability and beneficial biological effects on tissue regeneration, especially in orthopaedics, yet the underlying anabolic mechanisms in relevant clinical disorders are lacking. The present study investigated the effect of magnesium (Mg) and vitamin C (VC) supplementation for preventing steroid-associated osteonecrosis (SAON) in a rat experimental model. In SAON rats, 50 mg/kg Mg, or 100 mg/kg VC, or combination, or water control was orally supplemented daily for 2 or 6 weeks respectively. Osteonecrosis was evaluated by histology. Serum Mg, VC, and bone turnover markers were measured. Microfil-perfused samples prepared for angiography and trabecular architecture were evaluated by micro-CT. Primary bone marrow cells were isolated from each group to evaluate their potentials in osteoblastogenesis and osteoclastogenesis. The mechanisms were tested in vitro. Histological evaluation showed SAON lesions in steroid treated groups. Mg and VC supplementation synergistically reduced the apoptosis of osteocytes and osteoclast number, and increased osteoblast surface. VC supplementation significantly increased the bone formation marker PINP, and the combination significantly decreased the bone resorption marker CTX. TNFα expression and oxidative injury were decreased in bone marrow in Mg/VC/combination group. Mg significantly increased the blood perfusion in proximal tibia and decreased the leakage particles in distal tibia 2 weeks after SAON induction. VC significantly elevated the osteoblast differentiation potential of marrow cells and improved the trabecular architecture. The combination supplementation significantly inhibited osteoclast differentiation potential of marrow cells. In vitro study showed promoting osteoblast differentiation effect of VC, and anti-inflammation and promoting angiogenesis effect of Mg with underlying mechanisms. Mg and VC supplementation could synergistically alleviate SAON in rats, indicating great translational potentials of metallic minerals for preventing SAON.

An alternative splicing variant of mineralocorticoid receptor discovered in preeclampsia tissues and its effect on endothelial dysfunction.

The pathophysiology of preeclampsia (PE) remains unclear. PE spiral artery remodeling dysfunction and PE offspring cardiovascular future development has been a worldwide concern. We collected placental and umbilical artery samples from nor-motensive and PE pregnancies. Mineralocorticoid receptor (MR) and its alternative splicing variant (ASV) expression and their biological effects on PE were examined. An MR ASV was found to be highly expressed in all PE samples and slightly expressed in about half of the normotensive samples (umbilical artery, ~57.58%; placenta, ~36.84%). The MR ASV expression was positively associated with blood pressure in both groups. The MR ASV protein changed the aldosterone-induced expression pattern of MR target genes related to ion exchanges and cell signaling pathways. The MR ASV can also impair the proliferation, migration, and tube formation ability of endothelial cells. These findings indicate that MR ASV in PE placenta plays a pathogenic role in PE pathophysiology, especially in endothelial dysfunction, and the existence of the MR ASV in PE umbilical artery provides a new direction in the study of PE offspring with increased risk of cardiovascular diseases.

Hybrid fracture fixation systems developed for orthopaedic applications: A general review.

Orthopaedic implants are applied daily in our orthopaedic clinics for treatment of musculoskeletal injuries, especially for bone fracture fixation. To realise the multiple functions of orthopaedic implants, hybrid system that contains several different materials or parts have also been designed for application, such as prosthesis for total hip arthroplasty. Fixation of osteoporotic fracture is challenging as the current metal implants made of stainless steel or titanium that are rather rigid and bioinert, which are not favourable for enhancing fracture healing and subsequent remodelling. Magnesium (Mg) and its alloys are reported to possess good biocompatibility, biodegradability and osteopromotive effects during its in vivo degradation and now tested as a new generation of degradable metallic biomaterials. Several recent clinical studies reported the Mg-based screws for bone fixation, although the history of testing Mg as fixation implant was documented more than 100 years ago. Truthfully, Mg has its limitations as fixation implant, especially when applied at load-bearing sites because of rather rapid degradation. Currently developed Mg-based implants have only been designed for application at less or non-loading-bearing skeletal site(s). Therefore, after years research and development, the authors propose an innovative hybrid fixation system with parts composed of Mg and titanium or stainless steel to maximise the biological benefits of Mg; titanium or stainless steel in this hybrid system can provide enough mechanical support for fractures at load-bearing site(s) while Mg promotes the fracture healing through novel mechanisms during its degradation, especially in patients with osteoporosis and other metabolic disorders that are unfavourable conditions for fracture healing. This hybrid fixation strategy is designed to effectively enhance the osteoporotic fracture healing and may potentially also reduce the refracture rate. The translational potential of this article: This article systemically reviewed the combination utility of different metallic implants in orthopaedic applications. It will do great contribution to the further development of internal orthopaedic implants for fracture fixation. Meanwhile, it also introduced a titanium-magnesium hybrid fixation system as an alternative fixation strategy, especially for osteoporotic patients.

CCR6 is required for ligand-induced CatSper activation in human sperm.

CatSper channel has been considered the principal sperm Ca2+ channel responsible for the cytosolic Ca2+ elevation required for various sperm functions necessary for fertilization [1-4]. However, the mechanism underlying the activation of CatSper channel by various physiological ligands remain incompletely understood. We have recently demonstrated the expression of C-C chemokine receptor 6 (CCR6) in sperm and Ca2+ influx upon binding of human ß-defensin 1 (DEFB1) to CCR6, which is important for sperm motility [5]. In the present study, we have demonstrated that CCR6 receptor and CatSper channel are both required for the Ca2+ entry/current induced by physiological ligands DEFB1, chemokine (C-C motif) ligand 20 (CCL20) and progesterone in human sperm. CCR6 is co-localized and interacts with CatSper in human sperm. Ca2+ influx mediated by CCR6 and CatSper is required for essential sperm functions, including motility, hyperactivation and acrosome reaction, which are impaired in infertile sperm showing reduced levels of CCR6 and CatSper. The present finding suggests a critical role of CCR6 receptor in mediating ligand-induced, CatSper-dependent Ca2+ influx required for various sperm functions and thus male fertility.

XCI-escaping gene KDM5C contributes to ovarian development via downregulating miR-320a.

Mechanisms underlying female gonadal dysgenesis remain unclarified and relatively unstudied. Whether X-chromosome inactivation (XCI)-escaping genes and microRNAs (miRNAs) contribute to this condition is currently unknown. We compared 45,X Turner Syndrome women with 46,XX normal women, and investigated differentially expressed miRNAs in Turner Syndrome through plasma miRNA sequencing. We found that miR-320a was consistently upregulated not only in 45,X plasma and peripheral blood mononuclear cells (PBMCs), but also in 45,X fetal gonadal tissues. The levels of miR-320a in PBMCs from 45,X, 46,XX, 46,XY, and 47,XXY human subjects were inversely related to the expression levels of XCI-escaping gene KDM5C in PBMCs. In vitro models indicated that KDM5C suppressed miR-320a transcription by directly binding to the promoter of miR-320a to prevent histone methylation. In addition, we demonstrated that KITLG, an essential gene for ovarian development and primordial germ cell survival, was a direct target of miR-320a and that it was downregulated in 45,X fetal gonadal tissues. In conclusion, we demonstrated that downregulation of miR-320a by the XCI-escaping gene KDM5C contributed to ovarian development by targeting KITLG.

Follicular hyperandrogenism downregulates aromatase in luteinized granulosa cells in polycystic ovary syndrome women.

Women with polycystic ovary syndrome (PCOS) undergoing IVF-embryo transfer based-assisted reproductive technology (ART) treatment show variable ovarian responses to exogenous FSH administration. For better understanding and control of PCOS ovarian responses in ART, the present study was carried out to compare the follicular hormones and the expression of granulosa cell genes between PCOS and non-PCOS women during ART treatment as well as their IVF outcomes. Overall, 138 PCOS and 78 non-PCOS women were recruited for the present study. Follicular fluid collected from PCOS women showed high levels of testosterone. The expression of aromatase was found significantly reduced in luteinized granulosa cells from PCOS women. In cultured luteinized granulosa cells isolated from non-PCOS women, their exposure to testosterone at a level that was observed in PCOS follicles could decrease both mRNA and protein levels of aromatase in vitro. The inhibitory effect of testosterone was abolished by androgen receptor antagonist, flutamide. These results suggest that the hyperandrogenic follicular environment may be a key hazardous factor leading to the down-regulation of aromatase in PCOS.

Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate.

In the earlier study, sodium orthovanadate (SOV) has been reported to be a powerful inhibitor of (Na(+), K(+)) adenosine triphosphatase, exhibit widespread actions on the renal and cardiovascular systems, induces smooth muscle contraction by inhibiting the phosphorylation of the protein tyrosine phosphatases. In the current study, we aimed to investigate the cellular mechanisms by which SOV facilitated contractile response of vas deferens smooth muscle and its potential therapeutic advantage. Exogenous application of ATP and NA-caused contraction was strengthened by pretreatment with SOV. This facilitation was inhibited not by bath with the inhibitor of P2 receptor, PPADS, or the inhibitor of α1 receptor, Prazosin, but by bath with the protein tyrosine kinase inhibitor, Genistein. SOV induced a sustained increase in intracellular Ca(2+) of smooth muscle cells, which was abolished by 100 µM Genistein or Ca(2+)-free solution. The facilitation of SOV could also be inhibited by the selective inhibitors of TRP channel, 2-APB and non-selective cation channel, Gd(3+), Ni(+). The in vivo study showed that peritoneal injection of SOV in dystrophic mice (mdx mice) enhanced the contraction of vas deferens smooth muscle stimulated by electrical field stimulation, ATP, noradrenaline, or KCl. The above results suggest that SOV facilitates the concentration of vas deferens smooth muscle through the tyrosine phosphorylation activated the non-selective cation channels, which has potential use in the therapy for muscle dysfunction.

Physiology and pathophysiology of the epithelial barrier of the female reproductive tract: role of ion channels.

The epithelium lining the female reproductive tract forms a selectively permeable barrier that is responsible for creating an optimal luminal fluid microenvironment essential to the success of various reproductive events. The selective permeability of the epithelial barrier to various ions is provided by the gating of epithelial ion channels, which work together with an array of other ion transporters to drive fluid movement across the epithelium. Thus, the luminal fluid is fine-tuned by the selective barrier with tight regulation of the epithelial ion channels. This chapter discusses the role of epithelial ion channels in regulating the epithelial barrier function and thus the fluid volume and ionic composition of the female reproductive tract; physiological factors regulating the ion channels and the importance of the regulation in various reproductive events such as sperm transport and capacitation, embryo development and implantation. Disturbance of the fluid microenvironment due to defects or abnormal regulation of these ion channels and dysregulated epithelial barrier function in a number of pathological conditions, such as ovarian hyperstimulation syndrome, hydrosalpinx and infertility, are also discussed.

Dedifferentiation-reprogrammed mesenchymal stem cells with improved therapeutic potential.

Stem cell transplantation has been shown to improve functional outcome in degenerative and ischemic disorders. However, low in vivo survival and differentiation potential of the transplanted cells limits their overall effectiveness and thus clinical usage. Here we show that, after in vitro induction of neuronal differentiation and dedifferentiation, on withdrawal of extrinsic factors, mesenchymal stem cells (MSCs) derived from bone marrow, which have already committed to neuronal lineage, revert to a primitive cell population (dedifferentiated MSCs) retaining stem cell characteristics but exhibiting a reprogrammed phenotype distinct from their original counterparts. Of therapeutic interest, the dedifferentiated MSCs exhibited enhanced cell survival and higher efficacy in neuronal differentiation compared to unmanipulated MSCs both in vitro and in vivo, with significantly improved cognition function in a neonatal hypoxic-ischemic brain damage rat model. Increased expression of bcl-2 family proteins and microRNA-34a appears to be the important mechanism giving rise to this previously undefined stem cell population that may provide a novel treatment strategy with improved therapeutic efficacy.

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

BACKGROUND: The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. METHOD/PRINCIPAL FINDINGS: Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. CONCLUSIONS: The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Tyrosine phosphorylation modulates store-operated calcium entry in cultured rat epididymal basal cells.

Store-operated calcium entry (SOCE) is essential for many cellular processes. In this study, we investigated modulation of SOCE by tyrosine phosphorylation in rat epididymal basal cells. The intracellular Ca(2+) ([Ca(2+)]i) measurement showed that SOCE occurred in rat epididymal basal cells by pretreating the cells with thapsigargin (Tg), the inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPase. To identify the role of Ca(2+) channels in this response, we examined the effects of transient receptor potential canonical channel blockers 2-aminoethoxydiphenyl borate (2-APB), 1-[ß-[3-(4-methoxyphenyl)pro-poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride(SKF96365), Gd(3+), and non-selective cation channel blocker Ni(2+) respectively on SOCE and found that these blockers could inhibit the Ca(2+) influx to different extent. Furthermore, we studied the regulation of SOCE by tyrosine kinase pathway. The inhibitor of tyrosine kinase genistein remarkably suppressed the SOCE response, whereas sodium orthovanadate, the inhibitor of tyrosine phosphatase, greatly enhanced it. The results suggest that tyrosine kinase pathway plays a significant role in the initiation of SOCE and positively modulates SOCE in epididymal basal cells.

Stimulating effects of dopamine on chloride transport across the rat caudal epididymal epithelium in culture.

The present study investigated the effects of dopamine on chloride transport across cultured rat caudal epididymal epithelium. The results showed that dopamine induced a biphasic short-circuit current (Isc) in a concentration-dependent manner. The dopamine-induced response consisted of an initial rapid spike followed by a sustained phase. The alpha and beta adrenoreceptor inhibitors, phentolamine and propranolol, inhibited the initial spike and the sustained phase, respectively, suggesting a contribution of adrenergic receptors. The response was almost abolished by removing the extracellular Cl-, suggesting that the dopamine-induced short-circuit current is primarily a Cl- current. The response was inhibited by the apical Cl- channel blocker, diphenylamine-dicarboxylic acid, and the Ca2+-activated Cl- channel blocker, disulfonic acid stilbene, indicating that Cl- may pass through two types of Cl- channels on the apical side. Preloading monolayers with the intracellular Ca2+ chelator BAPTA/AM abolished the initial spike and greatly reduced the second phase in the Isc response to dopamine. Pretreating the monolayers with an adenylate cyclase inhibitor, MDL12330A, inhibited all of the second Isc response and part of the initial spike. Also, characteristics of the Cl- currents induced by dopamine were observed in whole-cell patch-clamp recording. The increases of intracellular cAMP and Ca2+ induced by dopamine were also measured. The results suggest that extracellular dopamine activates Ca2+-dependent and cAMP-dependent regulatory pathways, leading to activation of both Ca2+-dependent and cAMP-dependent Cl- conductances in epididymal epithelial cells.

Cellular mechanisms underlying the laxative effect of flavonol naringenin on rat constipation model.

BACKGROUND & AIMS: Symptoms of constipation are extremely common, especially in the elderly. The present study aim to identify an efficacious treatment strategy for constipation by evaluating the secretion-promoting and laxative effect of a herbal compound, naringenin, on intestinal epithelial anion secretion and a rat constipation model, respectively. METHODS/PRINCIPAL FINDINGS: In isolated rat colonic crypts, mucosal addition of naringenin (100 microM) elicited a concentration-dependent and sustained increase in the short-circuit current (I(SC)), which could be inhibited in Cl- free solution or by bumetanide and DPC (diphenylamine-2-carboxylic acid), but not by DIDS (4, 4'- diisothiocyanatostilbene-2, 2'-disulfonic acid). Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced I(SC). In addition, significant inhibition of the naringenin-induced I(SC) by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl- secretion. Naringenin-evoked whole cell current which exhibited a linear I-V relationship and time-and voltage- independent characteristics was inhibited by DPC, indicating that the cAMP activated Cl- conductance most likely CFTR (cystic fibrosis transmembrane conductance regulator) was involved. In rat constipation model, administration of naringenin restored the level of fecal output, water content and mucus secretion compared to loperamide-administrated group. CONCLUSIONS: Taken together, our data suggest that naringenin could stimulate Cl- secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model. Naringenin appears to be a novel alternative treatment strategy for constipation.

Cellular mechanism of adrenalin stimulated chloride secretion via beta-adrenoceptor in T84 cells.

In the present study, the intracellular regulatory pathways involved in the adrenalin-stimulated chloride secretion across T84 cells were investigated. Biphasic characteristics were observed in the Isc response to the basolateral addition of adrenalin (0.25 nM-100 microM). The biphasic response was almost abolished by removing ambient Cl(-). Chloride secretion was found to depend on the activities of basolaterally located Na+-K+-2Cl(-) cotransporters and K+ channels. The alpha-adrenoceptor antagonist phentolamine did not have any effect on either phase of adrenalin-induced Isc, while after pretreatment of the beta-adrenoceptor antagonist propranolol, the adrenalin-induced Isc was substantially abolished, suggesting the biphasic response may be mediated by the beta-adrenoceptor. Under whole cell patch-clamp conditions, T84 cells responded to adrenalin with a rise in inward current. The current, which exhibited a linear I-V relationship and time- and voltage-independent characteristics, was inhibited by the chloride channel blocker DPC and the reverse potential was close to the equilibrium potential for Cl(-) (0 mV), implying that the current was Cl(-) selective. When preloaded with a Ca2+-chelating agent, BAPTA/AM did not affect the Isc response to adrenalin, whereas the Isc was destroyed by pretreating the cells with an adenyl cyclase inhibitor, MDL12330A. These observations were further supported by the intracellular [cAMP] measurement experiment, indicating that adrenalin induced chloride secretion could be mediated by a beta-adrenoceptor only involving cAMP as an intracellular second messenger.

The role of extracellular ATP in the male reproductive tract.

In addition to its well established role as a neurotransmitter, extracellular ATP has been considered as a paracrine/autocrine factor, either released from sperm or epithelial cells, in the male reproductive tract and shown to play a versatile role in modulating various reproductive functions. This review summarizes the signal pathways through which ATP induces anion secretion by the epithelia of the epididymis, as well as its epithelium-dependent modulation of smooth muscle contraction of the vas deferens. Finally, the overall role of ATP in coordinating various reproductive events in the male genital tract is discussed.

Herpes simplex virus VP22 enhances adenovirus-mediated microdystrophin gene transfer to skeletal muscles in dystrophin-deficient (mdx) mice.

One of the obstacles to efficient vector-mediated gene therapy for Duchenne's muscular dystrophy (DMD) is its limited transduction efficiency. The VP22 tegument protein of herpes simplex virus type 1 (HSV-1) is able to cross biological membranes and translocate the VP22 fusion protein from transfected primary cells to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of vector-mediated gene therapy and to investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, the recombinant adenoviruses Ad-VP22, Ad-MICDYS, and Ad-VP22-MICDYS were constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo. About 92 +/- 3.6% of cells were microdystrophin positive 48 hr postinfection with Ad-VP22-MICDYS. The number of centralized nuclei in Ad-VP22-MICDYS-transduced tibialis anterior (TA) muscle was significantly reduced, from 78 +/- 5.2 to 20 +/- 2.5%, by 2 weeks postinjection. By 2 months postinjection, the average number of microdystrophin-positive fibers in TA muscle injected with Ad-VP22-MICDYS was 2.2 times more than that of TA muscle injected with Ad-MICDYS. Ad-VP22-MICDYS led to significant recovery of force-producing capabilities in TA muscle. These results demonstrate that VP22 greatly augmented adenovirus-mediated microdystrophin delivery to C2C12 cells and to the skeletal muscles of dystrophin-deficient (mdx) mice. These results highlight the efficiency of VP22-mediated intercellular protein delivery for the potential therapy of DMD and suggest that VP22 may be a promising tool with which to enhance the efficacy of adenoviral gene transfer for somatic gene therapy of DMD.

Cellular mechanisms of carbachol-stimulated Cl- secretion in rat epididymal epithelium.

Neurotransmitter-controlled Cl- secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca2+ activated Cl- channel blocker disulfonic acid stilbene (DIDS, 300 microM) only inhibited part of the CCH-induced Isc response, while nonselective Cl- channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 microM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca2+-chelating agent BAPTA/AM (50 microM) and an endoplasmic reticulum Ca2+ pump inhibitor, Thapsigagin (Tg, 1 microM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways.