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Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl⁻ and HCO₃⁻ transport. Although >95% of all CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. Here we report that CFTR is detected in both human and mouse sperm. CFTR inhibitor or antibody significantly reduces the sperm capacitation, and the associated HCO₃⁻-dependent events, including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower compared with that of the wild-type. These results suggest that CFTR in sperm may be involved in the transport of HCO₃⁻ important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD.

Cystic fibrosis is caused by mutations in the gene encoding an ion-transport protein, the cystic-fibrosis transmembrane conductance regulator (CFTR). Defective secretion of anions is the primary cause of many of the clinical manifestations of cystic fibrosis, including pancreatic insufficiency. We aimed to identify a molecular mechanism from which a new method to circumvent defective pancreatic secretion could be derived. Multiple-human-tissue RT-PCR and semiquantitative RT-PCR analyses were used to examine gene expression. An antisense technique was used in conjunction with radioimmunoassay, Fura-2 spectrofluorometry, immunohisto-chemistry, and the short-circuit current technique (Ussing chamber) for elucidation of gene function and its application in rescuing defective pancreatic secretion. We cloned a newly identified gene, NYD-SP27, which has structural similarity to an isoform of phospholipase C. NYD-SP27 was expressed endogenously in human pancreatic-duct cells and upregulated in cystic fibrosis. Suppression of NYD-SP27, by transfection of its antisense into human cystic-fibrosis pancreatic-duct cells, resulted in augmentation of phospholipase-C-coupled calcium-ion release and protein kinase C activity, improvement in the amount of mutated CFTR reaching the plasma membrane, and restoration of cAMP-activated pancreatic anion secretion. NYD-SP27 exerts an inhibitory effect on phospholipase-C-coupled processes that depend on calcium ions and protein kinase C, including CFTR trafficking and function. Its upregulation in pancreatic-duct cells may reveal a previously unsuspected defect in cystic fibrosis contributing to pancreatic insufficiency, and thus represents a new target for pharmacological intervention in cystic fibrosis.

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA A receptor. This effect of myricetin was not blocked by the GABA A receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca 2+ /calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca 2+ imaging studies found that myricetin could increase Ca 2+ current and intracellular Ca 2+ concentration, respectively, via T- and L-type Ca 2+ channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABA A receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABA A receptor.

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA(A) receptor. This effect of myricetin was not blocked by the GABA(A) receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca(2+)/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca(2+) imaging studies found that myricetin could increase Ca(2+) current and intracellular Ca(2+) concentration, respectively, via T- and L-type Ca(2+) channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABA(A) receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABA(A) receptor.

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABAA receptor. This effect of myricetin was not blocked by the GABAA receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca2+/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca2+ imaging studies found that myricetin could increase Ca2+ current and intracellular Ca2+ concentration, respectively, via T- and L-type Ca2+ channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABAA receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABAA receptor.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl− and HCO3− transport. Although >95% of all CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. Here we report that CFTR is detected in both human and mouse sperm. CFTR inhibitor or antibody significantly reduces the sperm capacitation, and the associated HCO3−-dependent events, including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower compared with that of the wild-type. These results suggest that CFTR in sperm may be involved in the transport of HCO3− important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD.

Dysmenorrhoea, defined as cramping pain in the lower abdomen occurring during menstruation, is known to affect up to 90% of women of childbearing age to varying degrees. The underlying causes of this condition are believed to be due to a number of factors, but are mainly attributed to increased myometrial activity, increased prostaglandin production and hormonal influences. Although there are pharmaceutical treatments available, they mainly concentrate on symptomatic relief, with the main treatment being the use of non-steroidal anti-inflammatory drugs (NSAIDs) to directly relieve the pain. Other treatments include the use of the combined oral contraceptives which are believed to influence myometrial contractility via regulation of hormonal activity. However due to the gastric and contraceptive side effects of these treatments respectively, other alternative treatments are becoming increasingly popular. One such treatment is the use of Bak foong pills (BFP), a traditional Chinese medicine used in China for the treatment of various gynaecological disorders including primary dysmenorrhoea. The aims of the current project were therefore to highlight the major beneficial effects of BFP and attempt to elucidate its major mechanisms of action in treating dysmenorrhoea. The study demonstrated that BFP's anti-dysmenorrhoeal properties were due to a combination of hormonal, myometrial relaxant and analgesic effects. Treatment of mice with BFP caused an estrogen-like effects as demonstrated with increased cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression. Furthermore, serum estrogen and progesterone levels were also elevated in BFP treated rats. BFP was also able to significantly reduce myometrial contractions, indicating that BFP's anti-dysmenorrhoeal effect may be aided by reduced contractility of the myometrium following treatment. The uterine relaxation caused by BFP was not dependant on increases in nitric oxide or cAMP, but appeared to affect calcium mobilization. Investigation of the analgesic effect of BFP, assessed using a visceral pain model in mice showed that following sub-chronic (72 hour) treatment with BFP, there was a significant reduction in pain response, demonstrating that BFP had direct analgesic effect. (Abstract shortened by UMI.)