Electrodermal level asymmetry in a unilateral brain-damaged patient

To compare the effectiveness of various sperm preparation methods, we examined sperm concentration, percentage of progressive motility, recovery rate, and various motion characteristics in 32 semen samples after Percoll and IxaPrep preparations. Except for sperm concentration, which was much higher after IxaPrep preparation, no statistical differences existed between these two methods in terms of motile sperm concentration (MSC), recovery rate, mean curvilinear velocity (VCL), mean straight-line velocity (VSL) and mean amplitude of lateral head displacement (ALH). Among 22 samples that were allocated in a 37 degrees C, 5% CO2 incubator, the mean MSC after 3 hours was significantly higher following IxaPrep preparation than following Percoll preparation (63.2 x 10(6)/mL vs. 42.8 x 10(6)/mL, p < .03). VCL and VSL were much higher after IxaPrep preparation than after Percoll preparation, both at 3 hours (p < .01) and at 24 hours (p < .03). ALH was also higher after IxaPrep preparation than after Percoll preparation, and it was statistically significant after 3 hours of incubation (p < .03). We conclude that the IxaPrep gradient may be adopted as the preferred method to the Percoll gradient in preparing spermatozoa for assisted reproduction because of the spermatozoa's persistent and better motion activities after incubation.     

Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate

Cryopreservation causes extensive damage to spermatozoa, thereby impairing their fertilizing ability. The purpose of this study was to determine if the direct addition of pentoxifylline to the seminal plasma before cryopreservation improved sperm motility and acrosome reaction. Semen specimens from 15 healthy volunteers were divided into two aliquots. One aliquot was treated by adding 5 mM pentoxifylline directly to the seminal plasma (treatment group) and the other aliquot received no treatment (control group). Both aliquots were then cryopreserved by using the liquid nitrogen freezing method. The percentage of motile spermatozoa and various motion characteristics were then evaluated by performing computer-assisted semen analysis. The sperm viability was determined with a supra-vital dye, Hoechst-33258, and the acrosome reaction (spontaneous and calcium ionophore-induced) was monitored using fluorescein isothiocyanate-conjugated peanut lectin (FITC-PNA) binding assays. Pentoxifylline treatment significantly increased the sperm motility, the amplitude of lateral head displacement, the hyperactivation status, and the frequency of spontaneous acrosome reactions before freezing (P < 0.05). After post-thaw, no difference in motion characteristics (except percentage motility) between treated and control groups were observed. Acrosome loss due to the freeze-thaw process was less in the pentoxifylline-treated group (P = 0.0003). In addition, the percentage of cryopreserved acrosome-intact spermatozoa that underwent further acrosome reactions in response to calcium-ionophore challenge was significantly higher in the treated group (P = 0.03). Pentoxifylline treatment before freezing improved the acrosome reaction to ionophore challenge in cryopreserved spermatozoa. Treatment with pentoxifylline appears to minimize sperm damage during the freeze-thaw process and may improve fertilization rates with assisted reproductive procedures such as intrauterine insemination or in-vitro fertilization.     

Diagnostic accuracy of computer-assisted sperm motion analysis

One retrospective and two prospective studies were conducted among 218 couples treated with in-vitro fertilization (IVF) to establish the reproducibility and diagnostic accuracy of computer-assisted sperm analysis (CASA) with swim-up spermatozoa for the prediction of the fertilization rate of oocytes in vitro. Based on the results of a preliminary retrospective analysis in 49 patients, the 'curvilinear velocity' (VCL) was chosen as the most distinctive motion parameter of sperm function and the median was used to represent the entire sperm population. The number of inseminated motile spermatozoa was then adjusted to median VCL during two subsequent prospective studies with clinical IVF. Whereas in the first prospective study (90 couples) the threshold values of VCL with regard to the number of spermatozoa inseminated were based on the results of the preliminary retrospective study (49 couples), in the second prospective study (79 couples) the settings were based on the results of the first prospective study. The reproducibility of CASA was tested by analysing the motion characteristics of spermatozoa at different intervals after termination of swim-up, by repeated analysis of the same video-recording of the incubated spermatozoa by different observers, and by the repeated video-recording of the freshly prepared sperm samples and analysis of both video-recordings by the same observer. Under these conditions the frequency of disagreement between two measurements varied between 2.0 and 8.2%. In both prospective studies the sensitivity of CASA for the prediction of fertilization was high (74.0%), whereas the specificity was low (40.0%). In contrast to successful fertilization, unsuccessful fertilization of oocytes in vitro could not be predicted reliably with CASA. However, the pregnancy rate per cycle among patients with predicted low fertilization rates was significantly lower (5.3%) than in couples with high predicted fertilization rates (24.3%, P < 0.001). Therefore, CASA of washed spermatozoa may still help to identify couples who would benefit more from intracytoplasmic sperm injection (ICSI) than from IVF. A definite threshold level could not be identified for any of the motion parameters to distinguish the motion characteristics of fertilizing and non-fertilizing spermatozoa. Using various algorithms for hyperactivated motility, the percentage of hyperactivated spermatozoa was significantly higher among the successfully fertilizing patients than among the nonfertilizing group. However, the absolute number of hyperactivated spermatozoa added to the oocytes was higher in non-fertilizing couples. Therefore, the lack of fertilization in some patients may be caused by a generalized defect in sperm function rather than by insufficient hyperactivation.     

The paradoxical effects of pentoxifylline on the binding of spermatozoa to the human zona pellucida

In the presence of pentoxifylline, human spermatozoa are induced to increase certain motion characteristics; however, the role of this drug in fertilization remains equivocal. In this study, the influence of pentoxifylline on one aspect of fertilization, that is sperm-zona binding, has been examined. Results from a fluorescence label competitive zona binding (CZB) test showed that spermatozoa exposed to a pentoxifylline challenge of between 0.1 and 5 mM, which was curtailed after 1 h by washing, had a decreased (P < 0.01) ability to bind to intact zona compared with control spermatozoa. The washing procedures also removed (decrease P < 0.01 compared with peak values) some of the enhanced motion characteristics induced by pentoxifylline. These results were in contrast with those obtained using experimental conditions that maintained an increased curvilinear velocity (VCL) and lateral head displacement (ALH) (increase P < 0.001 above baseline controls) in the continued presence of pentoxifylline. Using a hemizona binding (HZB) assay, 3 mM pentoxifylline increased (P < 0.001) sperm-zona binding almost 20% above zona binding with unexposed control spermatozoa. It was concluded that, in the presence of pentoxifylline, there is increased sperm binding to the zona pellucida; however, if the drug is removed by washing, the sperm binding to the zona is decreased in concert with the removal of the enhanced motion characteristics. The application of zona solubilization by acidic conditions in a microchamber enabled the precise determination of sperm numbers in both of the sperm-zona binding assays, and the results demonstrated that a wide variation in sperm numbers was observed in each test, with 63-580 spermatozoa bound in the CZB assay and 56-1340 spermatozoa bound on a hemizona.     

Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation

OBJECTIVE: To analyze the effect of sodium nitroprusside, a nitric oxide releaser, on sperm motion and lipid peroxidation-induced membrane damage in cryopreserved human sperm. DESIGN: Post-thaw, cryopreserved, human sperm samples were washed and divided into three aliquots. Each aliquot was incubated with either 0, 50, or 100 nM sodium nitroprusside. INTERVENTIONS: Samples were analyzed for lipid peroxidation (measured by malonaldehyde-thiobarbituric acid reactivity) at 3 hours post-thaw. MAIN OUTCOME MEASURES: Percent viability and motion parameters were assessed at 0, 10, and 30 minutes and 2, 3, 5, and 6 hours post-thaw. RESULTS: All results represent a mean +/- SEM, n = 10. Lipid peroxidation in samples incubated with 50 nM sodium nitroprusside (15.1 +/- 2.1 nM malonaldehyde/10(8) sperm) or 100 nM sodium nitroprusside (13.2 +/- 2.1 nM malonaldehyde/10(8) sperm) was significantly lower than in controls (22.7 +/- 3.1 nM malonaldehyde/10(8) sperm). Percent viability was significantly reduced from 0 minutes (60.6% +/- 3.5%) to 6 hours post-thaw in controls (38.0% +/- 5.1%) but not in 50 nM (46.8% +/- 10.4%) or 100 nM (48.8% +/- 6.5%) sodium nitroprusside-treated samples. Compared with controls (18.3% +/- 3.4%), maintenance of percent motility at 3 hours post-thaw was significantly improved in 50 nM (24.5% +/- 2.9%) and in 100 nM (26.3% +/- 3.2%) sodium nitroprusside-treated samples. Straight line velocity maintenance was significantly improved in 50 nM (37.3 +/- 1.3) and in 100 nM (37.0 +/- 1) sodium nitroprusside-treated samples as compared with controls (30.5 +/- 1.7). Significant improvements in curvilinear velocity maintenance compared with controls (56.3 +/- 2.9) also were observed in 50 nM (65.9 +/- 2.1) and 100 nM (72.1 +/- 4.1) sodium nitroprusside-treated samples. Significant differences in the motion parameters of sodium nitroprusside-treated samples were maintained at 5 and 6 hours post-thaw in comparison to controls. CONCLUSION: These results suggest that sodium nitroprusside is beneficial to the maintenance of post-thaw sperm motion and viability for up to 6 hours and that reduction of lipid peroxidative damage to sperm membranes may be the mechanism for these benefits.     

Optimization of the Hamilton-Thorn computerized sperm motility analysis system for use with rat spermatozoa in toxicological studies

To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) approximately 130 microns/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL approximately 50 microns/sec and mean VSL approximately 30 microns/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A soft-ware change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.     

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Human spermatozoal motility, viability and lipid peroxidation (LPO) have been assessed in Ringer-Tyrode supplemented with different concentrations of ascorbic acid (AA) ranging from 50 to 4000 microM. Ascorbic acid in concentrations below 1000 microM protects spermatozoa from free radical damage as evidenced from improvement in their motility and viability. Concomitantly, there is also witnessed depletion of malondialdehyde generation (an end product of LPO) following AA treatment. Ascorbic acid at 1000 microM concentration and above, however, is not protective, as evidenced by abrupt fall in sperm motility and viability and concomitant increase in LPO.     

Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa

The epididymal compounds taurine, hypotaurine and inositol, and the antioxidants carnosine and ascorbic acid, were added to Tris-based diluents containing varying concentrations of glycerol, and their effect on the post-thaw motility characteristics and fertility of ram spermatozoa was examined. Overall, the post-thaw motility characteristics of spermatozoa were better when semen was frozen in the presence rather than in the absence of glycerol. Only taurine protected spermatozoa during cryopreservation; the presence of 25 mM or 50 mM taurine significantly improved the post-thaw percentage of motile spermatozoa but this had no effect on fertility after cervical or laparoscopic insemination of ewes. Increasing the concentration of taurine to more than 100 mM significantly reduced the percentage of motile spermatozoa, compared with the lower concentrations of the amino acid. The presence of more than 50 mM carnosine or ascorbic acid significantly reduced all motility characteristics compared with the control diluent. Given that hypotaurine, carnosine, or ascorbic acid did not improve post-thaw motility, the cryoprotective effect of taurine may be attributable to its osmoregulation rather than to its antioxidant properties.     

Dissociation of the effects of forskolin and dibutyryl cAMP on force of contraction and phospholamban phosphorylation in human heart failure

Forskolin and dibutyryl cyclic adenosine monophosphate (cAMP) stimulate force of contraction independent of beta-adrenoceptor stimulation. We studied their effects on force of contraction and phosphorylation of regulatory proteins in isolated electrically driven trabeculae carneae from failing human ventricles. The phosphorylation state of the regulatory protein phospholamban was studied because its phosphorylation usually faithfully follows contractility. For comparison, the phosphorylation state of the inhibitory subunit of troponin was studied. The phosphorylation state was inferred from in vitro phosphorylation of homogenates with cAMP-dependent protein kinase in the presence of radioactive gamma[32P]ATP Proteins were separated by electrophoresis, and radioactivity in the proteins of interest was quantified. The maximal positive inotropic effects occurred at 30 microM forskolin and were attenuated in comparison with the maximal effects to dibutyryl cAMP (1 mM). Both forskolin and dibutyryl cAMP enhanced phospholamban phosphorylation. However, phospholamban phosphorylation in intact trabeculae treated with 30 microM forskolin and 1 mM dibutyryl cAMP was comparable. It is suggested that phospholamban phosphorylation can be dissociated from inotropy at least in isolated trabeculae from failing human hearts.     

Clinical biomechanic correlates of cervical dysfunction: Part 4. Altered regional motor behavior

The in vitro effect of zinc on superoxide anion (O2-) generation and on experimentally induced lipid peroxidation (LPO) in spermatozoa of infertile men was investigated. Washed spermatozoa pre-incubated for 30 min at 37 degrees C in the presence of 1 or 3 mmol l-1 zinc, released less superoxide anions (P < 0.03 and P < 0.02, respectively; n = 9) than the untreated spermatozoa. Similar results were obtained using activated polymorphonuclear leukocytes (1 x 10(6) cells ml-1) in the presence of 1 or 3 mmol l-1 Zn (P < 0.001 and P < 0.0002, respectively; n = 9). The in vitro evidence of the inhibitory effect of zinc on O2- generation by human spermatozoa and leukocytes indicates that zinc may act in vivo as a scavenger of excessive O2- production by defective spermatozoa and/or leukocytes in semen after ejaculation. A significant stimulatory effect of Zn (3 mmol l-1) on iron-induced lipid peroxidation, measured by the formation of thiobarbituric acid reactive substances (TBARS), was detected in the spermatozoa of 16 normo- and 17 asthenozoospermic males (P < 0.0001 and P < 0.001, respectively). In 11 samples with sperm concentration 20.3 +/- 2.1 x 10(6) ml-1, exhibiting initial TBARS concentration two times higher than in normo- and asthenozoospermic samples (40.5 +/- 2.4 vs. 17.1 +/- 1.1 and 28.5 +/- 4.1 nmoles TBARS 10(-8) spermatozoa), no effect of zinc on the LPO rate was found. The observed inhibitory effect of zinc on superoxide anion regardless of the initial O2- level and stimulatory effect of zinc depending on the initial LPO rate in human spermatozoa suggests that this metal ion participates in the oxidative changes occurring after ejaculation and thus may modulate the properties of germ cells.     

Synthesis and antibacterial evaluation of novel water-soluble organic peroxides

Various compounds have been used in the attempt to improve sperm motility, including pentoxifylline (PF), a methylxanthine derivative. It has been postulated that PF, being a phosphodiesterase inhibitor, increases sperm kinematic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. However, it has not been clarified whether the biological effects of PF on sperm motility correlate with its ability to increase intracellular cAMP levels. To examine this relationship, the kinematic parameters, hyperactivation, and intracellular cAMP content were evaluated in motile spermatozoa, obtained by discontinuous Percoll gradient and swim-up from 21 normozoospermic semen samples, incubated without and with PF for 0, 1, 2, and 4 h. PF increased beat cross frequency after 1 and 2 h of incubation, curvilinear velocity and lateral head displacement (ALH) after 4 h, and hyperactivation after 1, 2, and 4 h, and decreased linearity (LIN) after 1 h of incubation. The intracellular cAMP content of spermatozoa incubated with PF increased at all time-points examined. Both intracellular cAMP content and increase in hyperactivation in response to PF decreased with the length of incubation. In the absence of PF, cAMP content was unchanged and was correlated significantly only with ALH and the percentage of spermatozoa with hyperactivated motility. Following incubation with PF, cAMP content correlated with hyperactivation and all sperm kinematic parameters, with the exception of LIN and straightness. These findings suggest that the beneficial effects of PF on sperm kinematic parameters and hyperactivation are related to its ability to increase intracellular cAMP content.     

A direct role for arrestins in desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes

A heterologous bovine in vitro fertilization (IVF) system was used to study the functional competence of scimitar-horned oryx spermatozoa after cryopreservation. Four sperm-freezing methods were compared after dilution of ejaculates from six oryx with an equine semen extender: 1) dry ice, 2) dry shipper one-step, 3) dry shipper two-step, and 4) liquid nitrogen vapor. Post-thaw sperm motility, longevity, and acrosomal status were assessed and zona pellucida penetration, fertilization, and embryo cleavage were evaluated after coincubation of thawed oryx spermatozoa with in vitro-matured domestic cow oocytes. Sperm motility index (SMI) decreased (p < 0.05) over a 6-h period, but a high percentage (>/= 65%) of spermatozoa contained intact acrosomes in all treatments. Despite differences in sperm motility among methods, oocyte penetration, fertilization, and embryo cleavage did not differ (p >/= 0.05). However, cleavage success was < 50% across all treatments. There were positive correlations (p < 0.05; r = 0.81-0.97) between sample SMI at 3 and 6 h and fertilization, penetration, and cleavage, but no correlations (p >/= 0.05) between SMI at 0 or 1 h and IVF success. This study demonstrates that compatible heterologous gamete interaction allows thorough assessment of post-thaw sperm function in an endangered antelope. Scimitar-horned oryx spermatozoa appear relatively tolerant of varied cryopreservation methods, and preserved samples exhibit adequate post-thaw function to warrant use for assisted reproduction.     

Metabolism of platelet-activating factor in rat epididymal spermatozoa

A role for platelet-activating factor (PAF) in sperm function has been proposed. In the present investigation the metabolism of PAF was examined in sperm and epididymal tissue. The major regulatory enzymes for the synthesis of PAF via the de novo pathway have been established in spermatozoa; these include acetyltransferase and cholinephosphotransferase specific for PAF biosynthesis. De novo acetyltransferase activity for PAF biosynthesis in spermatozoa was significantly higher than that of the acetyltransferase of the remodeling pathway. A metabolic pathway was described for the catabolism of PAF in sperm, involving PAF-acetylhydrolase (-AH), lysophospholipase D, and a phosphohydrolase. An isozyme of PAF-AH similar to that reported in sera was also demonstrated in epididymal fluid and tissue. This isozyme was distinctly different from that found in the spermatozoa. The partial inactivation of PAF-AH by the vaginal pH, and/or its detachment from sperm during migration to the site of fertilization, may allow increased motility and migration to the site of fertilization. It is suggested that a decapacitation factor previously described may be related to PAF-AH.     

Progesterone induces Ca++-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm

Progesterone (P) has been reported to modulate numerous sperm functions through the binding of P to plasma membrane. One of the effects is an increase in sperm hyperactivation, which is known to be cAMP-dependent. To evaluate the effect of P on cAMP levels, human spermatozoa were incubated 2 h with increasing P concentrations. P significantly induced cAMP increase in a dose-dependent manner, reaching a 3-fold increase at 100 micromol/L (P < 0.01). During the study of the kinetics of P effect, two cAMP peaks were observed: one occurring after a 30-min incubation, with a 1.5-fold increase (P < 0.05), and the second one after a 120-min incubation, with a 2.5-fold increase (P < 0.01). These effects of P on cAMP levels correlated with significant rises in the percentage of hyperactivated spermatozoa, occurring at the same times as those of cAMP. To evaluate the Ca++-dependence of these P effects, the experiments were performed in the presence of and in the absence of Ca++ in the incubation medium. The effects of P at the 30th min and the 120th min were completely abolished in the absence of Ca++. Moreover, calcium ionophore A23187, after a 30-min incubation, induced an increase in cAMP levels identical to that obtained with P. The effect of P was partially reproduced by gamma-amino-butiric acid (GABA) and inhibited by GABA antagonist picrotoxin. It was also inhibited by tyrosine kinase inhibitor genistein but not by RU486. Based on these findings, we conclude that P induces Ca++-dependent cAMP increase in human sperm, that this effect is likely caused by the influx of Ca++ (previously reported), and that the effect partially involves GABA(A)-like receptors.     

In vitro capacitating effect of gamma-aminobutyric acid in ram spermatozoa

We have evaluated the capacitating effect of gamma-aminobutyric acid (GABA) in ram spermatozoa in vitro, in a chemically defined medium, by means of the chlortetracycline (CTC) binding assay. Semen from adult Australian Merino rams was collected in an artificial vagina; spermatozoa were washed once in modified Biggers, Whitten, and Wittingham medium (m-BWW), without BSA or serum, and incubated in m-BWW alone or in m-BWW containing GABA, GABA agonists, or antagonists for 2 h at 38.5 degrees C under 5% CO2 in air. Samples were taken for assessment of CTC binding pattern or were further incubated for 15 min in the presence of 5 microM calcium ionophore A23187. Acrosomal exocytosis was evaluated by Pisum sativum agglutinin binding. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of CTC forms II and III, corresponding to mid-capacitated and capacitated spermatozoa, respectively. The effect was marginally significant at 1 microM and maximal at 20 microM. The action of 20 microM GABA was mimicked by the GABAB-receptor agonist, muscimol, but not by the GABAA-receptor agonist, baclofen, and completely blocked by the GABAA-receptor antagonists, bicuculline and picrotoxin, which lacked effect per se. In a separate set of experiments, incubation of spermatozoa with GABA at a concentration of 1 microM, which was insufficient to stimulate sperm capacitation, together with the neuroactive steroid allopregnanolone (1 microM) provoked a capacitating effect similar to that achieved by 20 microM GABA alone. These results show that GABA has a capacitating action on ram spermatozoa through a GABAA receptor-mediated mechanism.     

Movements of sodium and potassium into ejaculated boar spermatozoa suspended in seminal plasma and a biological salt solution

Uptake of 22Na and 42K into ejaculated boar spermatozoa was measured in vitro. Cells were suspended either in seminal plasma or in a biological salt solution of essentially the same composition as boar seminal plasma. Samples were incubated at 30 degrees C. Correction was made for extracellular space in the centrifuged sperm pellet. This was determined as 22Na-space, which was less (P less than 0.001) than [14C] carboxyinulin space. Large differences were observed among individual ejaculates. The half-time for potassium uptake into the spermatozoa averaged 11.5 min, which is much faster than that for leukocytes or erythrocytes. When the spermatozoa were suspended in the biological salt solution, the initial rate of 42K uptake was significantly decreased. This may be due to disturbances of the protein components of the sperm membrane. The uptake of 22Na into the spermatozoa was slow. Sodium and potassium transport appeared not to be coupled in the 3/2 ratio which has been reported for erythrocyte membranes. The average concentration of sodium was 108 mM in seminal plasma and 26 mM in the spermatozoa (112 mmol/kg water and 38 mmol/kg water, respectively). The corresponding figures for potassium were 26 mM and 51 mM (27 mmol/kg water and 74 mmol/kg water). The random error for a single determination for the various methods used varied between 2.4 and 13.3% of the mean.     

Cyclic AMP-dependent protein kinase enhances hippocampal dentate granule cell GABAA receptor currents

1. The effects of activation of endogenous adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), intracellular application of PKA and inhibition of endogenous PKA by protein kinase inhibitory peptide (PKIP) on hippocampal dentate granule cell gamma-aminobuturic acid A (GABAA) receptor (GABAR) currents were characterized. 2. GABAR currents evoked by repeated application of GABA (30 or 100 microM) were enhanced by application of 1 mM norepinephrine (52 +/- 26%; mean +/- SE; n = 11) and of 500 mM 8-bromo cAMP (15 +/- 2%, n = 7). 3. GABA concentration response curves were obtained from six dentate granule cells before and after application of 500 microM 8-bromo cAMP. The maximal current was increased significantly by 89 +/- 36%, but the mean EC50 was not significantly changed (68 +/- 42 microM vs. 25 +/- 10 microM). 4. The GABA concentration response relationship was studied in a group of 7 granule cells recorded with pipettes containing PKIP and 2 mM ATP and compared with another group of 12 cells recorded with 2 mM ATP in the pipette. When currents were recorded with intracellular PKIP, the mean EC50 for GABA was no different (43 +/- 9 microM vs. 45 +/- 16 microM); however, the maximal current obtained was smaller, (961 +/- 102 pA vs. 658 +/- 104 pA). 5. Concentration response data were obtained from four granule cells using recording pipettes containing the cPKA and an ATP regeneration system and compared with seven cells recorded with the ATP regeneration system. With cPKA, the maximal GABAR current was significantly larger (1,224 +/- 132 pA vs. 718 +/- 56 pA), but the EC50 for GABA was not significantly altered (21 +/- 2.0 microM vs. 79 +/- 25 microM).     

Guanine nucleotide-binding inhibitory protein-mediated inhibition of adenylyl cyclase is enhanced in spontaneously hypertensive rat preglomerular arteriolar smooth muscle cells

The purpose of our study was to determine whether Gi-mediated control over adenylyl cyclase in preglomerular arteriolar smooth muscle cells (PGASMC) is enhanced in the spontaneously hypertensive rat (SHR). PGASMC were cultured from preglomerular microvessels isolated from adult SHR (14-15 wk of age) and age-matched WKY rats. Confluent monolayers of cells in third passage were used for the experiments. cAMP released into the media (30 min) as well as cellular levels of cAMP were measured in the presence of a phosphodiesterase inhibitor, 1-isobutyl-3-methyl-xanthine (IBMX; 100 microM) and expressed as pmol/mg protein. Total (released + cellular) cAMP was significantly lower in SHR (14.19 +/- 2.30 pmol/mg protein) as compared with WKY (28.3 +/- 3.04 pmol/mg protein). Correspondingly, the released (4.6 +/- 0.4 pmol/mg protein) as well as cellular (9.78 +/- 2.18 pmol/mg protein) cAMP levels were also significantly lower in SHR when compared with WKY (8.85 +/- 1.26 and 18.86 +/- 2.0 pmol/mg protein, respectively). The steady-state levels of none of the Gi alpha subunits, namely Gi alpha 1, Gi alpha 2 and Gi alpha 3, were higher in the SHR PGASMC. Pertussis toxin treatment (PTX; 100 ng/ml; 24 hr) caused complete ADP-ribosylation of Gi alpha subunits in both WKY and SHR PGASMC. The same treatment of PTX also produced a significant increase in total cAMP in SHR, but not in WKY, such that the total cAMP levels after PTX treatment were not significantly different between the two strains. Interestingly, PTX significantly increased the released (20.26 +/- 0.90 pmol/mg protein) but not the cellular (13.63 +/- 1.63 pmol/mg protein) cAMP in SHR. Forskolin (1 microM) induced similar increases in total cAMP and isoproterenol (1 microM) caused greater increases in total cAMP in SHR cells compared with WKY cells. These data strongly suggest that in SHR PGASMC total adenylyl cyclase activity is not altered. Furthermore, steady-state expressions of Gi alpha-1, Gi alpha-2 and Gi alpha-3 are not increased whereas Gi-mediated inhibition of adenylyl cyclase is augmented in SHR PGASMC.     

Increased urinary excretion of glycerol: metabolic studies on a patient

The ratio of the zinc content of boar spermatozoa obtained from semen cooled to 4 degrees C for 30 min to that of the original room temperature control (20-26 degrees C) was constant at 2-28 +/- 0-16 in 22 samples of fresh whole semen from 12 animals. The same ratio occurred when zinc (0 to 0.6 mM in citrate buffer) was added to semen or washed spermatozoa. The increase is dependent only on the initial sperm zinc content at room temperature.     

Adenylate cyclase of gastric mucosa in patients with chronic renal failure

It has been shown previously that antisecretory response of famotidine is altered in patients with renal failure. To evaluate the underlying mechanism(s) of this clinical observation we obtained biopsy specimens of fundic mucosa from 3 groups of patients with variable renal function (group 1 normal renal function (n = 16); group 2 chronic renal failure (n = 16), CLCR > or = 5 < 90 ml/min; group 3 hemodialysis therapy (n = 16)) (matched for age, sex, and Helicobacter pylori (Hp) status. In the homogenized samples adenylate cyclase (AC) activity was assessed and the influence of uremia on this second messenger system involved in gastric acid secretion was tested. AC activity was measured as the formation of cAMP, which was determined by RIA. The mean basal AC activity was 150 in group 1, 190 in group 2, and 120 pmol cAMP/mg protein/20 min in group 3. There was a dose-dependent stimulation by histamine (1 microM-1 mM). Emax of cAMP formation ranged between 230 and 403 pmol cAMP/mg protein/20 min and EC50 between 5.9 and 20.1 microM histamine, dependent on Hp status. Histamine-stimulated AC activation was reduced to about 50% by 0.1 mM famotidine. The sensitivity of AC to histamine seems to decrease in patients undergoing hemodialysis. Similarly, the colonization with Hp may result in decreased maximal response of the AC system towards histamine.