Dewi Kenneth Rowlands

Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility

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.

Orally Active Peptidic Bradykinin B1 Receptor Antagonists Engineered from a Cyclotide Scaffold for Inflammatory Pain Treatment

Chronic pain is a universal health issue associated with numerous medical conditions, for example after severe burns or following major surgery. Compelling evidence suggests that bradykinin (BK) antagonists could be useful in treating chronic pain and inflammatory pain. Bradykinin and its homolog kallidin (lysyl-BK or KD), which are collectively known as kinins, participate in many pathophysiological insults. They are short-lived peptide mediators and the most potent endogenous pain inducers. Kinins are released during tissue injury or noxious stimulation and modulate pain through the activation of both the B1 and the B2 receptor, which are two G-protein-coupled receptors; the carboxypeptidase metabolites of kinins, des-Arg-BK and des-Arg-KD, activate the B1 receptor. [2b, 4] The B1 receptor stimulates the chronic phase of the inflammatory pain response, while the B2 receptor stimulates the acute phase owing to their differences in ligand dissociation, receptor desensitization, downregulation as well as internalization. Emerging evidence also suggests that the B1 receptor mediates various chronic pain responses through the activation of phospholipase C, thereby leading to the production of diacylglycerol and inositol triphosphate, which further activate protein kinase C and Ca mobilization. Numerous BK-antagonist peptides have been discovered from natural sources and structure–activity studies. Kinestatin isolated from frog skin and helokinestatin from lizard venom are examples of natural BK-antagonist peptides. Structure–activity studies have shown that removing the Cterminal Arg residue and concurrently replacing the penultimate residue Phe to Leu of bradykinin to des-Arg-[Leu]bradykinin or kallidin to des-Arg-[Leu]-kallidin (DALK) changes a bradykinin B2 receptor agonist to a B1 receptor antagonist (Figure 1). To increase potency and in vivo stability, several laboratories also developed BK antagonists

Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive tract

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.

Fat redistribution and adipocyte transformation in uninephrectomized rats.

Dyslipidemia complicates renal function leading to disturbances of major homeostatic organs in the body. Here we examined the effect of chronic renal dysfunction induced by uninephrectomy on fat redistribution and lipid peroxidation in rats treated with an angiotensin-converting enzyme (ACE) inhibitor (lisinopril) for up to 10 months. Uninephrectomized rats developed fat redistribution and hypercholesterolemia typical of chronic renal failure when compared with sham-operated rats or lisinopril-treated uninephrectomized rats. The weight of the peri-renal fat was significantly less in the untreated compared to the lisinopril-treated uninephrectomized rats or those rats with a sham operation. We also found that there was a shift of heat-protecting unilocular adipocytes to heat-producing multilocular fat cells in the untreated uninephrectomized rats. Similarly in these rats we found a shift of subcutaneous and visceral fat to ectopic fat with excessive lipid accumulation and lipofuscin pigmentation. Lisinopril treatment prevented fat redistribution or transformation and lipid peroxidation. This study shows that ACE inhibition may prevent the fat anomalies associated with chronic renal dysfunction.

Differential expression and localization of CFTR and ENaC in mouse endometrium during pre-implantation

Interaction between the cystic fibrosis transmembrane conductance regulator (CFTR), a CAMP‐activated Cl− channel, and epithelial Na+ channel (ENaC) has been proposed as the major mechanism regulating uterine fluid absorption and secretion. Differential expression of these ion channels may give rise to dynamic changes in the fluid environment affecting various reproductive events in the female reproductive tract. This study investigated the expression and localization of CFTR and ENaC during the pre‐implantation period. Semi‐quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry were used to study the expression and localization of CFTR and ENaC in uteri collected from mature superovulated female mice. RT‐PCR showed maximal ENaC and CFTR expression on day 3 after mating. Maximal immunoreactivity was also observed for both ENaC and CFTR on day 3 after mating. However, ENaC was immunolocalized to the apical membrane of both luminal and glandular epithelia, while CFTR was predominantly found in the stromal cells rather than the epithelial cells. Differential expression and localization of CFTR and ENaC provide a molecular mechanism by which maximal fluid absorption can be achieved immediately prior to implantation, to ensure the immobilization of the blastocyst necessary for implantation.

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms.

Nitric oxide (NO), which is produced from l‐arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two‐cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two‐cell embryos/mouse collected after fertilization in vivo (P < 0.01), but the rate of blastocyst formation from two‐cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS‐deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P < 0.001). While all three types of NOS do not seem to play a significant role in pre‐ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.

Systematic Error of Cardiac Output Measured by Bolus Thermodilution With a Pulmonary Artery Catheter Compared With That Measured by an Aortic Flow Probe in a Pig Model

OBJECTIVE The precision of thermodilution cardiac output measurement using a pulmonary artery catheter can be divided into random and systematic errors. This study determined the systematic component. DESIGN Comparative validation against a transonic flow probe on the aortic root. SETTING Animal research laboratory. PARTICIPANTS Eight anesthetized pigs, weight 27 to 32 kg. INTERVENTIONS Thermodilution measurements were compared to those from an aortic flow probe. One (or two) catheters were tested in each pig, with multiple paired readings recorded as cardiac output was varied pharmacologically by esmolol, epinephrine, or dopamine. Linear regression lines were drawn for each pig as well as the slope used to quantify systematic error. Regression analysis of data from each pig was used to assess trending. Bland-Altman analysis also was performed. MEASUREMENTS AND MAIN RESULTS Systematic error derived from slope data was ± 26%. Trending was reliable in 8 out of 10 catheter placements (p value>0.95). Bland- Altman analysis (n = 77 basal anesthesia and 165 pharmacologic intervention data pairs) provided percentage errors of ± 23% and ± 34-39%, respectively. This percentage error increase was due to variations in calibration and the slopes of regression lines for each catheter tested as the lines diverged as cardiac output increased, widening the spread of data. CONCLUSIONS Thermodilution does trend cardiac output reliably in most cases. However, the systematic error is large and has a significant effect on the percentage error as cardiac output increases. The precision error of ± 20% currently used for thermodilution measurement may be set too low and is dependent on the clinical setting.

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.

Regulation of prostacyclin and prostaglandin E2 receptor mediated responses in adult rat dorsal root ganglion cells, in vitro

Primary cultures of adult rat dorsal root ganglia (DRG) were prepared to examine the properties of prostacyclin (IP) receptors and prostaglandin E2 (EP) receptors in sensory neurones. IP receptor agonists, cicaprost and iloprost, stimulated adenylyl cyclase activity with EC50 values of 22 and 28 nM, respectively. Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were 7 fold less potent than cicaprost and iloprost, with PGE2 displaying a lower maximal response. Adenylyl cyclase activation by iloprost, PGE1 and PGE2, but not by forskolin, was highly dependent on DRG cell density. Although the potency of iloprost and PGE2 for stimulating adenylyl cyclase was unchanged, their maximal responses were significantly increased at low cell density. Both IP and EP2/4 receptors could be down‐regulated by agonist pretreatment, however the presence of cyclo‐oxygenase (COX) inhibitors did not prevent this apparent down‐regulation of IP and EP2/4 receptors at high DRG cell densities. Stimulation of adenylyl cyclase by the neuropeptide calcitonin gene‐related peptide was also decreased at high DRG cell density, whereas the responses to β‐adrenoceptor agonists were increased at high DRG cell density. Addition of nerve growth factor (NGF), or the addition of anti‐neurotrophin antibodies during the 5‐day culture of DRG cells, had no effect on IP receptor‐mediated responses. These results indicate that Gs‐coupled receptors involved in nociception are regulated in a variable manner in adult rat sensory neurones, and that this cell density‐dependent regulation may be agonist‐independent for IP and EP2/4 receptors.

CFTR is required for cellular entry and internalization of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular Gram‐negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Δ508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR−/−) mice was significantly less compared with that in the wild‐type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune‐co‐localization and co‐immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion‐channel function. These findings, for the first time, demonstrate that CFTR functions as a cell‐surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections.