Studies on relationship between Na,K-ATPase activity and sperm capacitation in guinea pig

In order to elucidate the biochemical mechanism of sperm capacitation, the relationship between plasmalemma Na,K-ATPase, capacitation and acrosome reaction was investigated. Plasmalemma of guinea pig spermatozoa was isolated by sucrose gradient centrifugation for the determination of Na,K-ATPase activity. As far as the authors are aware, the Na,K-ATPase activity in plasmalemma of the guinea pig has never been detected. By treating sperm plasmalemma with 0.05% DOC (deoxycholate), enzyme activity could be quantitatively measured. After spermatozoa were incubated in capacitation medium for 8 h, Na,K-ATPase activity was found to be decreased to 35.6% as compared with that before incubation. The spermatozoa incubated for 10.5 h in capacitation medium containing 1 and 5 mumol/L ouabain showed 46.5% and 64.4% acrosome reactions respectively, while the acrosome reaction of the control group was only 27.4%. The above results suggest that the decrease in the Na,K-ATPase activity of guinea pig spermatozoa may be a prerequisite for capacitation. Experimental results demonstrated for the first time that Na,K-ATPase activity exists in the sperm plasmalemma of the guinea pig. It was further found that the decrease of Na,K-ATPase activity of plasmalemma enhances sperm capacitation. It is suggested that sperm capacitation in the guinea pig is possibly induced by the decrease in plasmalemma Na,K-ATPase and, as a consequence, the intracellular Na+ is increased, which would benefit the exchange of Na+out/Ca++in and the onset of acrosome reaction.     

Transfer of diclofenac sodium across excised guinea pig skin on high-frequency pulse iontophoresis. I. Equivalent circuit model

High frequency pulse iontophoresis of diclofenac sodium across excised guinea-pig skin was carried out in vitro. An equivalent circuit model was constructed to simulate the time courses of voltage drop across the donor solution and the skin. Parameter values obtained by the least-squares adaptation of the model to observed data were consistent with expectation and validated the proposed model.     

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.     

Identification of a botulinum neurotoxin A protease inhibitor displaying efficacy in a cellular model

Herein we describe a small-molecule, non-peptidic, inhibitor for botulinum neurotoxin A protease, that displays for the first time efficacy in a cell-based assay.     

Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (I(CCh)) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited I(CCh) dose-dependently with IC(50) of 1.1 0.1 nM (n = 6). GTPgS-induced current, however, was not inhibited by 10 nM 4-DAMP. I(CCh) was not recorded in pertussis-toxin (PTX)-pretreated smooth muscle cells of gastric antrum. I(CCh) values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 3 pA (n = 6) in the control and PTX-treated cells, respectively (P 0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that G(o) protein is important for activation of ACh-activated NSC channels, our results suggest that I(CCh) might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.     

Effects of L-glutamine, glutaminase and glutamine synthetase on CAP threshold of cochlear nerve of guinea pig.

Negative direct current (-DC 300 microA) stimulation was applied to the round window of the guinea pig cochlea to exhaust the pre-synaptic intracellular reserves of the transmitter in hair cells, and then the scala tympani was perfused respectively with L-glutamine, glutamine synthetase and glutaminase. Experimental results showed that the negative DC electrical stimulation applied to the round window elevated the CAP threshold of the cochlear nerve in the basal turn of the cochlea, which recovered over a period of approximately 17-39 min. The perfusion of L-glutamine apparently elevated the CAP threshold. The recovery of the CAP threshold following electrical stimulation, however, was accelerated by the perfusion of 10 mmol/L L-glutamine. The time for recovery only took about 5-6 min. The perfusion of enzyme glutamine synthetase elevated the CAP threshold by 50 dB, while glutaminase had little effect. These results suggest that the effect of L-glutamine on the CAP threshold in the cochlea of the guinea pig appears to be that of a potent depolarizing agent which accelerates the recovery of the CAP threshold during the depletion of the transmitter, and L-glutamine may be the candidate for the afferent excitatory transmitter.     

Refined homology model of monoacylglycerol lipase: toward a selective inhibitor

Monoacylglycerol lipase (MGL) is primarily responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both cannabinoid receptors. Increased tissue 2-AG levels consequent to MGL inhibition are considered therapeutic against pain, inflammation, and neurodegenerative disorders. However, the lack of MGL structural information has hindered the development of MGL-selective inhibitors. Here, we detail a fully refined homology model of MGL which preferentially identifies MGL inhibitors over druglike noninhibitors. We include for the first time insight into the active-site geometry and potential hydrogen-bonding interactions along with molecular dynamics simulations describing the opening and closing of the MGL helical-domain lid. Docked poses of both the natural substrate and known inhibitors are detailed. A comparison of the MGL active-site to that of the other principal endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, demonstrates key differences which provide crucial insight toward the design of selective MGL inhibitors as potential drugs.     

The distribution of beta-adrenergic receptors in guinea pig lungs and their changes in experimental allergic asthma

In this paper, the distribution of pulmonary beta-adrenergic receptors (beta-receptors) in normal and experimental allergic asthmatic guinea pigs was determined using autoradiographic method. The results showed that the distribution of beta-receptors in the lung sections was widespread. There was a significant decrease of beta-receptor density in several tissue structures of asthmatic guinea pig lungs compared with the controls, a 23.73% decrease in beta-receptor binding sites in bronchiolar smooth muscles and a 18.65% decrease in bronchial smooth muscles; a 23.53%, a 14.23% and a 17.16% decrease in bronchiolar epithelium, in bronchial epithelium and in alveolar epithelium respectively. The results indicated that the beta-receptor decrease in smooth muscles in experimental asthmatic guinea pig lungs might be one of the important factors which made the tension of smooth muscles increase. The relationship between the beta-receptor decrease of other sites and bronchial asthma needs to be studied further.     

Measurement of albuterol in guinea pig serum by high performance liquid chromatography with fluorescence detection

A sensitive, simple and reproducible high performance liquid chromatographic method for detecting and quantifying albuterol in guinea pig serum is described. A structurally related compound, bamethan, was used as an internal standard. The method employs ion-pair extraction with di(2-ethylhexyl)phosphate followed by chromatography on a Zorbax SB C18 reversed-phase column. Fluorescence detection was used to identify the compounds of interest. The calibration curve was linear between 1 and 50 ng/mL albuterol hemisulfate salt (0.83 and 41.50 ng/mL albuterol base), and the limit of detection for a 1 mL sample was 1 ng/mL albuterol hemisulfate salt (0.83 ng/mL albuterol base). Serum levels of albuterol were quantified from guinea pigs that had received the drug by continuous subcutaneous infusion at a dose of 0.2 mg/kg/day for 1, 5 or 10 days, or 10 days followed by a 24 h washout period.     

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

Summary In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.     

Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and microdialysis samples using liquid chromatography/tandem mass spectrometry

Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective separation was achieved on an alpha1-acid glycoprotein column within 14 min for all methods. A cetirizine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were detected by multiple reaction monitoring using transitions m/z 389.1 --> 200.9 and 396.1 --> 276.1, respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea pig plasma, the assay was linear over the range 0.25-5000 ng/mL for both S-CZE and R-CZE, with a lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples, the assays were linear over the range 2.5-250 ng/g and 0.25-50 ng/mL, respectively, and the LLOQ values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were < or =7.1% and < or =12.6%, respectively, and the intra- and inter-day accuracy varied by less than +/-8.0% and +/-6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.