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PubMed2006

In vitro storage of unfertilized ova of endangered Caspian brown trout (Salmo trutta caspius) in artificial media.

In order to study the effects of storage media and time of storage on the viability of unfertilized eggs of endangered Caspian brown trout (Salmo trutta caspius), the ova of this fish was stored in coelomic fluid and Cortland artificial media at 2-3 degrees C for 120 h. In this research, Cortland artificial medium was buffered with 20 mM of three different buffers: Hepes (C(8)H(18)N(2)O(4)S), Tris-HCl (C(4)H(11)NO(3)-HCl) and sodium salt Hepes (C(8)H(17)N(2)O(4)SNa). The pH of these media were adjusted according to natural pH of coelomic fluid. The eggs that stored in these media fertilized at times 0 h (eggs fertilized prior to storage), 48, 72 and 120 h of post-stripping, using fresh and pooled sperm obtained from four to six males. According to the results of present study, time of storage showed a significant (p<0.05) main effect on eyeing, hatching and eyed eggs mortality rates. Eyeing and hatching rates significantly (p<0.05) decreased from 97.4+/-2.1% and 95.1+/-4.4% at time 0 (eggs fertilized prior to storage) to 77.9+/-3% and 65.5+/-5% after 120 h of storage. Within a similar period of time, eyed eggs mortality significantly (p<0.05) increased from 2.4+/-2.4% to 17.2+/-3.9%. No significant (p>0.05) main effect was found among media buffered with Tris-HCl (82.8+/-3.2%, 73.4+/-5.4%, 12.1+/-4.5%), Hepes (88.2+/-3.4%, 80.7+/-5.5%, 9.3+/-3.4%), sodium salt Hepes (77.8+/-3.8%, 69.3+/-5.7%, 12.2+/-3.9%) and coelomic fluid (84.8+/-3.8%, 77.7+/-5.1%, 8.9+/-2.7%) for eyeing, hatching and eyed eggs mortality rates. There was a negative correlation (r=-0.895, p<0.001) between eyed eggs mortality and hatching rates. In conclusion, unfertilized eggs of endangered Caspian brown trout can be successfully stored for 48 h without significant loss of fertility. But, storage for 120 h results in the falling of hatching rate. In addition, no significant difference was found between viability rates of ova stored in coelomic fluid and artificial media, 120 h post-storage. It reveals that artificial media could be substituted for coelomic fluid as storage medium at least for 120 h in Caspian brown trout.

Animals↗
PubMed2005

Stretch-dependent modulation of [Na+]i, [Ca2+]i, and pHi in rabbit myocardium--a mechanism for the slow force response.

OBJECTIVE: Rabbit ventricular myocardium is characterized by a biphasic response to stretch with an initial, rapid increase in force followed by a delayed, slow increase in force (slow force response, SFR). The initial phase is attributed to increased myofilament Ca(2+) sensitivity, but the mechanisms of the delayed phase are only incompletely understood. We tested whether stretch-dependent stimulation of Na(+)/H(+) exchange (NHE1) and consecutive changes in pH(i) and/or [Na(+)](i) may underlie the SFR. METHODS: Isometric contractions of rabbit ventricular muscles were recorded in bicarbonate-containing Tyrode's (Tyrode) or bicarbonate-free HEPES-buffered solution (HEPES). Muscles were loaded with the Ca(2+) indicator aequorin, the pH indicator BCECF, or the Na(+) indicator SBFI and rapidly stretched from 88% (L(88)) to 98% (L(98)) of optimal length. The resulting immediate and slow increases in twitch force (1st phase and SFR) as well as changes in [Ca(2+)](i), [Na(+)](i), or pH(i) were quantified before and after inhibition of NHE1 by HOE 642 (3 microM) or reverse-mode Na(+)/Ca(2+) exchange (NCX) by KB-R 7943 (5 microM). RESULTS: In both Tyrode (n=21) and HEPES (n=22), developed force increased to approximately 160% during the 1st phase followed by a further increase to approximately 205% during the SFR. The SFR was accompanied by a 21% increase of the aequorin light transient (n=4; normalized to the 1st phase) and a approximately 3 mM increase in [Na(+)](i) (n=4-7). The SFR was also associated with an increase in pH(i). However, this increase was delayed and was significant only after the SFR had reached its maximum. The delayed pH(i) increase was larger in HEPES than in Tyrode. HOE 642 and/or KB-R 7943 reduced the SFR by approximately 30-40%. In addition, HOE 642 diminished the stretch-mediated elevation of [Na(+)](i) by 72% and the delayed alkalinization. CONCLUSIONS: The data are consistent with the hypothesis that SFR results from increases in [Ca(2+)](i) secondary to altered flux via NCX in part resulting from increases in [Na(+)](i) mediated by NHE1.

Animals↗
PubMed1985

Intracellular pH and contraction of isolated rabbit and cat papillary muscle: effect of superfusate buffering.

The influence of external buffering on surface pH (pHs), intracellular pH (pHi) and developed twitch tension was investigated in rabbit and cat papillary muscle. pHs and pHi were measured using single and double-barreled microelectrodes respectively. In 20 mM HEPES buffered solution, steady state pHi is close to that in control CO2/HCO-3 (25 mM HCO-3, 5% CO2) solution. pHs and developed tension also do not differ greatly from their control values. Decreasing the HEPES concentration to 5 mM, at constant external pH, lowers pHs considerably. The surface acidosis is associated with a small intracellular acidification; steady state pHi in 5 mM HEPES is always more acid than that in control CO2/HCO-3. A significant decrease in developed tension is also seen in 5 mM HEPES. Alteration of the superfusion velocity influences pHs only slightly. Stimulation of the muscle at high frequency is shown to increase surface acidification, the extent of which is dependent on the buffer concentration. The conclusion from the present experiments is that in papillary muscle external buffering influences intracellular pH and contraction via its effect on pHs.

Acid-Base Equilibrium↗
PubMed1995

pH regulation during ischaemia-reperfusion of isolated rat hearts, and metabolic effects of 2,3-butanedione monoxime.

We investigated changes in pHi during ischaemia-reperfusion of isolated rat hearts using phosphorus nuclear magnetic resonance spectroscopy (31P NMR). Hearts were separated into three groups according to the perfusion buffer: bicarbonate-buffered Krebs solution, HEPES-buffered Krebs solution, or bicarbonate-buffered Krebs solution plus 10(-6) M 5-(N-ethyl-N-isopropyl) amiloride (EIPA). In HEPES buffer and in bicarbonate buffer plus EIPA, pH at the end of 30 min of ischaemia and pH oscillations observed during early reperfusion were lower than in bicarbonate buffer. Thus, the presence of two pH regulation mechanisms (Na(+)-H+ antiport and Na(+)-HCO3- symport) was confirmed in the isolated rat heart, while in HEPES buffer, pH was regulated by Na(+)-H+ antiport, and in bicarbonate buffer plus EIPA, by Na(+)-HCO3- symport. When cardiac contraction was inhibited by 10 mM 2, 3-butanedione 2-monoxime (BDM), we observed, in all cases, a less pronounced decrease in pHi at the end of ischaemia, and in pHi oscillations at the onset of reperfusion. These effects were similar to those observed with 150 x 10(-8) M verapamil and might thus be related to a decrease in intracellular calcium. However, with BDM, a greater reduction in the pH recovery rate was observed only in HEPES buffer, suggesting a possible phosphatase-like effect affecting the Na(+)-H+ exchange. Whatever the buffer used, the protective effect of BDM was reflected by an increase in the rate pressure product, which was not observed with verapamil.

Adenosine Triphosphate↗
PubMed2003

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions.

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.

Animals↗
PubMed2003

Complexation of copper by zwitterionic aminosulfonic (good) buffers.

Copper binding properties were investigated for several popular zwitterionic buffers. The two buffers 4-morpholinoethanesulfonic acid (MES) and 3-N-morpholinopropanesulfonic acid (MOPS) did not bind copper and would be good choices for metal speciation studies within their operational pH range. Conversely, 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO) was observed to weakly bind copper directly (log Kc 2.02). Moreover, strong copper binding was observed for 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (HEPPS), and N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid) (HEPPSO). Log Kc values range from 7.04 to 7.68 and are indicative of strong copper binding ligands. The latter buffer also exhibited weak binding characteristics with a log Kc of 2.05. The strong Cu binding ligands were present in HEPES, HEPPS, and HEPPSO at much lower concentrations than the total buffer concentration. MES, HEPES, MOPSO, and HEPPSO were analyzed by electrospray-ionization quadrapole time-of-flight mass spectroscopy. The most prominent feature of the spectra for each buffer analyzed was the presence of multiple oligomers, indicating a propensity of interaction between buffer molecules. In addition, the presence of several contaminants was identified in the mass spectrum of the HEPES matrix, including a prominent contaminant (at m/z 131) present in levels similar to those obtained from the modeling of the copper titration data. Other contaminants were found in the other matrixes but were not identified as possible copper binding agents.

Journal Article↗
PubMed1996

Fertility of ram spermatozoa pellet-frozen in zwitterion-buffered diluents.

The fertility (positive ultrasound scanning 60-75 days after insemination) of ram spermatozoa frozen in Tris- or zwitterion/glucose/egg yolk/glycerol-based diluents was examined during the natural breeding season (autumn). In Experiment 1 Tris-citrate, high glucose and Hepes-NaOH diluents were used and ewes (n = 266) were inseminated (induced oestrus) with 20 (uterus) or 180 (cervix) million motile spermatozoa. There was no difference in fertility (range: 10.9-71.1%) between diluents, despite the superior post-thawing motility and progressive velocity of spermatozoa in Hepes-NaOH compared with the other diluents (P < 0.001). In Experiment 2, there was no difference in fertility (range: 4.1-50.0%) between Tris-citrate and Hepes-NaOH diluents after insemination (induced oestrus) of ewes (n = 290) with 10 (uterus) or 100 (cervix) million motile spermatozoa, respectively. In Experiment 3 Tris-citrate, Hepes-NaOH and Pipes-NaOH diluents were used and ewes (n = 492) were inseminated (natural oestrus) with 8 or 24 (uterus), or 80 or 240 (cervix) million motile spermatozoa. There were no differences in fertility (range: 35.4-52.9%) between insemination doses or diluents despite the superior motility and acrosome integrity in the zwitterion compared with the Tris-citrate diluents (P < 0.001). In all experiments, the fertility of frozen spermatozoa was higher for intrauterine than cervical insemination (51.9 vs 22.1%, n = 1 048; P < 0.01). It was concluded that post-thawing in vitro quality was superior and the fertility comparable after freeze-thawing spermatozoa in zwitterion compared with Tris-citrate diluents.

Animals↗
PubMedDate not supplied

Comparison of the effect of two different handling media on rabbit zygote developmental ability.

Despite the attention paid to culture media, the relevance of the handling medium at egg recovery/transfer is frequently overlooked. In the present work, we compare the effect of two different handling media (PBS and HEPES-buffered Ham F10, both supplemented with 20% (v/v) FCS), upon in vitro and in vivo developmental ability of in vivo fertilised rabbit zygotes. Zygotes recovered in HEPES-buffered medium (permanence 1 h as maximum) and subsequently cultured in vitro developed more efficiently to the compacted morula (100%) and blastocyst stage (92%) than those recovered in PBS (83% and 76%, respectively, P < 0.05). Zygotes recovered in such media were then further bilaterally transferred to recipient does following a brief in vitro culture period (for 4 hours). At caesarean section (day 28 of pregnancy), significant differences were observed in both the percentage of pregnant uterine horns (PBS: 60% vs. HEPES-buffered Ham F10: 100%) and live birth rates (PBS: 14% vs. HEPES-buffered Ham F10: 34%). Thus when early rabbit zygotes must be handled, even for short incubation periods, the medium is not innocuous.

Animals↗
PubMed1986

Genetic differences among the A/J X C57BL/6J recombinant inbred mouse lines and their degree of association with glucocorticoid-induced cleft palate.

Hydrocortisone sodium phosphate was injected intramuscularly into A/J, C57BL/6J and recombinant inbred lines from these two parental lines to study the genetics of steroid-induced cleft palate in a situation of identical maternal and fetal genotypes. The strains were typed for H-2 (the major histocompatibility locus), beta-glucuronidase and beta 2-microglobulin, which served as markers on chromosomes 17, 5 and 2, respectively. Hepatic glucocorticoid binding capacity had been previously measured in Hepes buffer and Hepes buffer plus dithiothreitol (DTT). The level of glucocorticoid binding in Hepes buffer and in Hepes plus DTT, as well as their ratio, was compared to the incidence of steroid-induced cleft palate in the recombinant inbred lines. A correlation was found between the response of glucocorticoid binding to DTT (expressed as a ratio of binding in the presence of DTT to binding without DTT) and hydrocortisone-induced cleft palate. When analyzing the effect of the three chromosomal markers on hydrocortisone-induced cleft palate, the b alleles of beta 2-microglobulin and of beta-glucuronidase were associated with a higher incidence. Genetic analyses of the differences between these two inbred strains of mice in the incidence of steroid-induced cleft palate show it not to be monogenic.

Alleles↗
PubMed2000

Colorimetric indicators of microbial contamination in corneal preservation medium.

PURPOSE: To compare acid-base and oxidation-reduction indicators and to investigate the effect of buffer and temperature on the colorimetric detection of microbial growth in corneal preservation media. METHODS: Corneal preservation media containing gentamicin, without or with HEPES buffer, were prepared with either phenol red or AlamarBlue indicators (AccuMed International, Westlake, OH, U.S.A.). Both media were inoculated with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, Serratia marcescens, or Candida albicans and then incubated at 4 degrees C, 22 degrees C, or 35 degrees C. The pH or percent reduction were determined hourly for eight hours, then daily for one week. RESULTS: The length of time before a confirmed change in pH or reduction occurred varied by microorganism, storage temperature, and buffering capacity. At 4 degrees C, none of the microorganisms caused a detectable pH change in buffered medium within one day after inoculation, although two bacterial species reduced AlamarBlue within four hours. At 22 degrees C and 35 degrees C, all bacteria except P. aeruginosa produced a pH shift within a few hours, and all tested bacterial species reduced AlamarBlue. For bacteria producing detectable pH changes, HEPES-buffered medium took longer to change than medium without HEPES. C. albicans was not detectable in HEPES-buffered medium at any temperature by phenol red and was only detectable by AlamarBlue after 2-3 days at 22 degrees C and 35 degrees C. CONCLUSION: Acidic shifts in refrigerated corneal preservation medium do not occur during contamination by several microorganisms. AlamarBlue, a redox indicator, is more sensitive than phenol red in detecting some bacteria. C. albicans is not reliably detected by pH or redox indicators.

Bacteria↗
PubMed1996

Prevention of ischemia-reperfusion injury with a synthetic metalloprotein superoxide dismutase mimic, SC52608.

Superoxide dismutase (SOD) scavenges free superoxide radicals generated during reperfusion of ischemic tissue and decreases cellular injury. A synthetic manganese-based metalloprotein superoxide dismutase mimic, SC52608 (Monsanto Co.), was tested in the isolated rabbit rectus femoris muscle flap to determine its effects on ischemia-reperfusion injury. The results of our experiments analyzing 38 isolated rectus femoris muscles in 19 New Zealand White rabbits show that administration of SC52608 at the onset of 4 hours of warm ischemia and before reperfusion significantly increases the survival of the muscle from 20.0 +/- 4.9 percent (control, HEPES) to 81.5 +/- 4.6 percent (SC52608) (p < 0.001). It preserved functional contraction in 8 of 10 muscles; only 1 of 12 control muscles (control, HEPES) had contractions (p = 0.0015). SC52608 decreased the neutrophil density from 4.63 +/- 0.6 x 10(4) cells/mm2 in the control (HEPES) muscle to 2.71 +/- 0.6 x 10(4) cell/mm2 in muscles perfused with SC52608 (p = 0.03). The level of malonyldialdehyde decreased from 6.12 +/- 0.26 nmol/gm (control, HEPES) to 4.64 +/- 0.41 nmol/gm (SC52608) (p = 0.0028). Postoperative weights of the muscles showed no statistical difference (p = 0.14) between the controls (16.0 +/- 0.9 gm) and the SC52608 (18.1 +/- 0.7 gm). Our investigation shows that direct intraarterial infusion of a synthetic superoxide dismutase mimic at the onset of ischemia and prior to reperfusion can reduce reperfusion injury in skeletal muscle.

Animals↗
PubMed1999

Functional assessment of rat aorta after cold storage in different media.

Cold storage is frequently used to store isolated blood vessels for a limited period of time. However preservation of vascular smooth muscle and endothelial functions is time and medium-dependent. The present study was designed to compare the reactivity of rat aorta before and after cold storage for 24 and 48 h in one of four different solutions consisting of Hepes-buffered Krebs solution, Belzer solution, Krebs solution, and Eurocollins solution. Smooth muscle and endothelial functions of the rat aorta were assessed using in vitro isometric tension measurement. The results obtained for vessels preserved for 24 and 48 h were compared with those for vessels studied immediately after harvesting. Sensitivity and maximum contraction to KCl and norepinephrine were not altered in rat aorta preserved up to 48 h in Hepes-Krebs and Belzer solutions. In contrast, the amplitude of contraction elicited by KCl was significantly reduced by 50% and 77% in aorta stored for 24 and 48 h in Krebs solution and by 77% and 96% in those stored in Eurocollins solutions. Similarly, the maximal contraction elicited by norepinephrine was significantly reduced by 60% and 45% in arteries stored for 24 and 48 h in Krebs solution and by 34% and 86% in those stored in Eurocollins solution. In contrast, cold storage in the different media did not alter the relaxations elicited by sodium nitroprusside and forskolin. The endothelium-dependent relaxations in response to acetylcholine were not statistically modified after preservation up to 48 h in Hepes-Krebs solution. In contrast, the maximal relaxations to acetylcholine were significantly decreased after storage for 24 and 48 h in Belzer, Krebs and Eurocollins solutions. These results suggest that among the four media studied, Hepes-Krebs solution is the most suitable medium for the storage of blood vessels under hypothermic conditions.

Animals↗
PubMed1989

Improved procedures for pineal gland fixation for electron microscopy.

The common procedures used for preparing some organs and tissues for electron microscopy, in which a fixative with the buffer portion adjusted to near-isotonicity to plasma is perfused in vivo, causes intolerable shrinkage of rat pineal cells. The present study was undertaken to optimize the parameters involved in the fixation of the pineal gland. The buffer and its concentration and the aldehyde or aldehydes used were among the variables investigated. The buffers tried were phosphate, cacodylate, PIPES, and HEPES. Decreasing the buffer concentration prevented shrinkage with all four buffers. The optimum concentrations were 0.05 M phosphate, 0.07 M cacodylate, 0.05 M or 0.057 M PIPES, and 0.1 M HEPES. PIPES and HEPES were clearly superior in retaining cytoplasmic density when compared with phosphate or cacodylate. The use of lithium PIPES and HEPES instead of the sodium equivalents enhanced membrane detail. A small volume of more concentrated aldehyde fixative perfused ahead of the main perfusate (a strong prewash) definitely helped prevent shrinkage. Using a mixture of aldehydes consisting of glutaraldehyde, formaldehyde, and acrolein reduced the tendency for shrinkage when compared with glutaraldehyde only. Some of the shrinkage space artefacts could be easily misinterpreted as normal features. Since the pineal gland commonly contains degenerating structures, a dependable fixation procedure is particularly needed. Also, accurate preservation is essential in the evaluation of physiological changes.

Animals↗
PubMed1977

Cell envelope of Neisseria gonorrhoeae: relationship between autolysis in buffer and the hydrolysis of peptidoglycan.

Neisseria gonorrhoeae readily underwent autolysis when suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at alkaline pH values. Autolysis was inhibited by the addition of Mg2+ or other divalent cations. Autolysis was also suppressed at acid pH (pH 6.0). Suspension of cells in buffer was accompanied by the hydrolysis of peptidoglycan. The rate of peptidoglycan hydrolysis in HEPES buffer was maximal at pH 8.5 and was similar in the presence or absence of Mg2+. Therefore, divalent cation stabilization against autolysis is not mediated by inhibition of peptidoglycan hydrolysis. Peptidoglycan hydrolysis occurred in HEPES buffer (pH 6.0), but at a rate that was 50% of the maximum. Incubation of cells with chloramphenicol or rifampin before suspension in HEPES buffer (pH 8.5) partially prevented autolysis; under these conditions, peptidoglycan hydrolysis still occurred, but at a reduced rate. Old and new peptidoglycans were hydrolyzed at similar rates. Peptidoglycan hydrolysis results in solubilization of both the peptide and glycan moieties.

Buffers↗
PubMed1969

Enhanced growth of T-strain mycoplasmas with N-2-hydroxyethylpiperazone-N'-2-ethanesulfonic acid buffer.

The small size of T-strain mycoplasma colonies obtained on agar medium has been a drawback to the study of these organisms. The incorporation of 0.05 mN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), a new hydrogen ion buffer, into agar medium at pH 6.8 led to the production of colonies at least 60% greater in diameter than those obtained on media without HEPES at pH 6.5 or 6.0. In addition, the colonies often had the "fried-egg" appearance typical of "classical" large-colony-forming mycoplasmas. The addition of HEPES to liquid medium in the presence of 0.1% urea resulted in a slightly higher number of viable organisms and a corresponding increase in ammonia production. Rapid death of the mycoplasmas occurred on continued incubation of the liquid medium cultures even in the presence of HEPES. The larger colony size facilitated the study of hemadsorption and tissue-culture cell adsorption. Preliminary results of such tests in which these phenomena have been demonstrated are presented. The advantages and disadvantages of having larger colonies are discussed, and the terminology of T-strain mycoplasmas is considered in the light of the present findings.

Adsorption↗
PubMed1985

Cl- requirement for saliva secretion in the isolated, perfused rat submandibular gland.

Replacement of perfusate Cl- with Br- reduced by 37% the volume of saliva secreted by isolated, perfused rat submandibular glands exposed to 10(-6) M acetylcholine. Replacement of perfusate Cl- with SO4(2-) reduced fluid secretion by 73%, while replacement of bicarbonate with HEPES or addition of acetazolamide had no effect on salivary flows or volumes. Furosemide (10(-3) M) reduced saliva secretion when added to Cl- -(73%) or Br- -containing (56%) perfusates and also to HEPES- (95%) or acetazolamide-containing (82%) perfusates. Perfusion of the glands with furosemide or SO4- containing perfusates resulted in significantly reduced salivary Cl- concentrations and increased residual anion (Na+-K+-Ca2+-Cl-) concentrations, while perfusion with solutions containing acetazolamide or with HCO3- -free (HEPES) perfusates resulted in opposite changes in salivary anions. Addition of furosemide to HEPES- or acetazolamide-containing perfusates reversed these changes and caused a reduction in salivary Cl- concentrations and an increase in residual anion concentrations. It is concluded that 1) extracellular Cl- is required for saliva secretion in the rat submandibular gland, 2) the required external Cl- enters the salivary cells by a furosemide-sensitive transport system, which is likely to be similar to a Na+-K+-Cl- cotransport system described in other epithelia, 3) HCO3- is not normally required for saliva secretion but can sustain it partially when Cl- transport is inhibited by either furosemide or Cl- replacement, and 4) salivary electrolyte changes induced by Cl- or HCO3- replacement or by furosemide probably represent combined effects on acinar secretion and transductal electrolyte transport.

Animals↗
PubMed1982

A comparison of two buffering systems for lens organ culture.

Bovine lenses were incubated in a pairwise assay for 24 h in TC 199 which was buffered with Hepes or NaHCO3, respectively. In lenses incubated with Hepes, the content of ATP was higher and that of AMP lower than in those incubated with NaHCO3. The specific activity of phosphofructokinase (PFK), was higher in the lenses from the Hepes medium than in lenses incubated with NaHCO3. A heat-labile metazyme of the PFK which is found in the nucleus of unincubated lenses disappeared when the lenses were incubated in the presence of bicarbonate, but remained intact after 22 h incubation with Hepes.

Adenosine Monophosphate↗
PubMed1983

Primary culture of purified Leydig cells isolated from adult rat testes.

Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function.

Age Factors↗