DNA integrity in human spermatozoa: relationships with semen quality.

The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.

Physiological properties of ATP-activated cation channels in rat brain microvascular endothelial cells.

Endothelial cells mediate the actions of a variety of vasoactive substances, including ATP. ATP vasodilatatory actions have been shown to depend on a calcium-dependent release of endothelium-derived relaxing factor(s) (EDRF). ATP induced a vasodilatation of pial penetrating microvessels when applied intraluminally; these relaxations were mediated by the endothelium and followed release of nitric oxide (NO), since they were sensitive to blockade of NO-synthesizing enzymes by NG-nitro-L-arginine (1 mM) and NG-mono-methyl-L-arginine (0.1 mM). We have also investigated the electrophysiological actions of extracellular ATP on rat brain microvascular (RBMEC) and bovine aortic endothelial cells (BAEC) using the patch-clamp technique. While BAEC were hyperpolarized by ATP (10 microM), ATP caused the activation of a depolarizing nonselective cation current in brain endothelial cells. NO production measurements by [3H]citrulline assay and by direct amperometric determination also revealed that after exposure to 1-100 microM ATP, RBMEC released NO. NO release from RBMEC was abolished by removal of external calcium. We conclude that, in the brain, ATP exerts its vasoactive roles by altering the electrophysiological properties of endothelial cells by acting on receptor-operated ion channels, thus providing a mechanism for calcium entry and subsequent release of EDRF.

Anesthetic-dependent pial arteriolar response to ethanol.

Anesthetic agents are often administered in the presence of ethyl alcohol, both in research and in the clinical setting. The authors tested the hypothesis that anesthetic agents may affect cerebrovascular responses to ethanol. A closed cranial window preparation in the rat was used to compare the response of pial arterioles to topically applied ethanol (0.01% to 1% vol/vol) in the presence of alpha-chloralose/urethane (50 and 600 mg/kg, respectively) or halothane (0.5% to 1%) anesthesia. Heart rate, mean arterial blood pressure, and blood gas levels were maintained stable and within the physiological range throughout each experiment. Ethanol induced significant vasoconstriction in alpha-chloralose/urethane-anesthetized animals (multivariate analysis of variance (MANOVA), p = 0.039); conversely, ethanol induced significant vasodilation of the pial arterioles in halothane-anesthetized animals (MANOVA, p = 0.017). These responses were significantly different from one another (MANOVA, p = 0.001). Thus, the choice of anesthetic agent alters the cerebrovascular response to ethanol, and care should be taken to ascertain the influence of anesthesia in both research and clinical settings.

Differential effects of alcohols on intracerebral arterioles. Ethanol alone causes vasoconstriction.

We compared the effect of the acute application of ethanol, methanol, 1-propanol, 1-butanol, urea, and mannitol (1-100 mM) on the basal tone of isolated-cannulated rat intracerebral arterioles to determine if the response of these arterioles to ethanol could be attributed to alteration of membrane fluidity or changes in osmolality. These arterioles spontaneously developed tone to 62.0 +/- 8.4% of passive diameter (44.2 +/- 11.9 vs. 70.9 +/- 14.7 microns). Ethanol caused a dose-dependent reduction in arteriolar diameter starting at 3 mM (p = 0.03), reaching a diameter of 81.4 +/- 3.0% of basal tone at 100 mM. In comparison, all other agents tested caused the arterioles to dilate, with the exception of 1-propanol, which produced inconsistent vessel responses. At 100 mM concentration, methanol, 1-butanol, urea, and mannitol dilated intracerebral arterioles by 116.1 +/- 12.7, 151.5 +/- 12.4, 131.1 +/- 17.0, and 149.8 +/- 6.6%, respectively. Thus, in a concentration range associated with acute intoxication, ethanol causes constriction of isolated intracerebral arterioles. The mechanism of action of ethanol cannot be accounted for solely based upon its physicochemical characteristics of osmolality or lipid solubility, but rather may reflect a more specific action on one or more cellular mechanisms responsible for determining basal intracerebral arteriolar tone. The characterization of the response of intracerebral arterioles to ethanol is important in view of epidemiologic links between ethanol consumption and cerebrovascular disease.

ATP-sensitive K+ channels in rat aorta and brain microvascular endothelial cells.

The endothelium plays an important role in the modulation of vascular tone and blood cell activation. Extensive work has demonstrated that the release of endothelium-derived relaxing factor (EDRF) from the endothelium is evoked by a number of physical and chemical stimuli requiring Ca2+. Because endothelial cells do not express voltage-dependent Ca2+ channels, Ca2+ influxes following receptor activation may be facilitated by cell hyperpolarizations mediated by the activation of K+ conductances. There has been recent interest in the role of ATP-sensitive K+ channels (KATP) suggesting that KATP may play a role in the regulation of blood flow. We have investigated the electrophysiological properties of an ATP-sensitive K+ conductance in whole cell and membrane patches from rat aorta and brain microvascular endothelial cells. Whole cell as well as single-channel currents were increased by either intracellular dialysis of ATP or application of glucose-free/NaCN (2 mM) solutions. Both currents were reversibly blocked by glibenclamide (1-100 microM). The KATP channel opener pinacidil (30 microM) caused activation of an outward current in the presence of physiological intracellular ATP concentrations. In inside-out patches, 10 microM-1 mM ATP invariably caused a dramatic decrease in channel activity. We conclude that both rat aorta and brain microvascular endothelial cells express KATP channels. KATP may play a role in the regulation of endothelial cell resting potential during impaired energy supply and therefore modulate EDRF release and thus cerebral blood flow.

Influence of hyperglycemia on cerebral adenosine production during ischemia and reperfusion.

We hypothesized that systemic hyperglycemia would alter cerebral adenosine concentrations during ischemia and reperfusion. In the present study, we analyzed brain tissue and cerebrospinal fluid (CSF) from hyperglycemic and normoglycemic rats before ischemia, after 15 min of incomplete forebrain ischemia, and during 60 min of reperfusion. Hyperglycemic rats received 3 g/kg of 17% D-glucose intraperitoneally, which increased blood glucose to 357 +/- 23 mg/100 ml compared with 128 +/- 12 mg/100 ml in normoglycemic rats. Brain tissue was sampled by the freeze-blow technique, and CSF was obtained by collecting cortical perfusate from the closed cranial window. Tissue and CSF were analyzed for adenosine and its metabolites inosine and hypoxanthine, and tissue was also analyzed for adenine nucleotides. Hyperglycemia significantly attenuated the increase in brain tissue and CSF adenosine and its metabolites during ischemia while preserving adenine nucleotide concentrates. This attenuation of ischemic adenosine production persisted after 5 min of reperfusion in tissue and throughout 60 min of reperfusion in CSF. Because adenosine, a cerebral vasodilator, can inhibit the release of neuronal excitotoxins as well as affect neutrophil-endothelial interactions, adenosine has been proposed as an endogenous neuroprotector. Thus the attenuation of adenosine and its metabolites may be a factor in the pathogenesis of increased ischemic brain injury associated with systemic hyperglycemia.

A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells.

A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or "weeding." The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin approximately laminin much greater than gelatin greater than poly-D-lysine approximately Matrigel greater than hyaluronic acid approximately plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-D-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7 +/- 2.6% (n = 6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of gamma-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation.

The hydrolysis of extracellular adenine nucleotides by arterial smooth muscle cells. Regulation of adenosine production at the cell surface.

The extracellular reaction sequence ATP----ADP----AMP----adenosine participates in regulating the time course of cellular response during crisis or signaling events, such as thrombus formation or neurotransmission. We have investigated the whole time course of hydrolysis of ATP to adenosine by recirculating adenine nucleotide substrates over smooth muscle cells attached to polystyrene beads. Kinetic parameters were estimated for each reaction by fitting observed time courses to models of the pathway. In spite of the inhibition of 5'-nucleotidase by ADP, adenosine was produced very rapidly by smooth muscle cells. Comparisons of the apparent Km values of ADPase and 5'-nucleotidase (determined from experiments in which each substrate was used as the initial substrate with Km values observed when each substrate was supplied from the upstream reaction) suggest that the local concentrations of substrate supplied from the preceding reactions are very much higher than those in the bulk phase. This enhancement of efficiency overcomes the effect of the feed-forward inhibition to give rise to very rapid adenosine production from ADP or ATP. These observations are in marked contrast to our previous findings with endothelial cells (Gordon, E. L., Pearson, J. D., and Slakey, L. L. (1986) J. Biol. Chem. 261, 15496-15504), on which feed-forward inhibition causes a profound lag in adenosine production from adenine nucleotides and on which there are no apparent surface effects on substrate delivery.

Mechanisms underlying retarded emergence of conditioned responding following inhibitory training: evidence for the comparator hypothesis.

The comparator hypothesis posits that conditioned responding is determined by a comparison at the time of testing between the associative strengths of the conditioned stimulus (CS) and stimuli proximal to the CS at the time of conditioning. The hypothesis treats all associations as being excitatory and treats conditioned inhibition as the behavioral consequence of a CS that is less excitatory than its comparator stimuli. Conditioned lick suppression by rats was used to differentiate four possible sources of retarded responding to an inhibitory CS. These include habituation to the unconditioned stimulus (US), latent inhibition to the CS, blocking of the CS-US association by the conditioning context, and enhanced excitatory associations to the comparator stimuli. Prior research has demonstrated the first three phenomena. Therefore, we employed parameters expected to highlight the fourth one--the comparator process. In Experiment 1, our negative contingency training was shown to produce a conditioned inhibitor that passed inhibitory summation and retardation tests. In Experiment 2 we found transfer of retardation from an inhibitory CS to a novel stimulus when the location where retardation-test training occurred was excitatory, which is indicative of contextual blocking and/or comparator effects. In Experiment 3, extinction of the conditioning context was found to attenuate retardation regardless of whether extinction occurred before or after the CS-US pairings of the retardation test. This indicates that much of the present retardation was due to the comparator process rather than to contextual blocking. Experiment 4 demonstrated that habituation to the US did not contribute to retardation in the present case. Collectively, these studies suggest that retardation following inhibitory training can be explained without recourse to any of the traditional mechanisms of conditioned inhibition.

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface.

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).

Malleability of conditioned associations: path dependence.

Using conditioned suppression of barpressing to investigate the stability of a conditioned stimulus-unconditioned stimulus (CS-US) association, we gave water-deprived rats either a few pairings of the CS with a strong footshock US or many pairings with a weak footshock US so that barpress suppression in response to the CS was equated. Experiment 1 established training parameters that yielded this equivalence. Specifically, rapid acquisition to a preasymptotic level of responding with strong shock produced suppression comparable to the asymptotic level reached more slowly with weak shock. Experiment 2 showed that although equivalent performance was obtained from extensive conditioning with a weak shock or limited conditioning with strong shock, only extensive conditioning with weak shock resulted in retarded acquisition of an association between that same CS and a footshock level perceived as midway between the two initial training shock intensities as implied by asymptotic performance in Experiment 1. Experiment 3 demonstrated that the observed retardation in animals given many conditioning trials with weak shock was CS specific. Collectively, these findings indicate that the malleability of learned behavior is not simply a function of initial associative strength but is dependent on path during initial acquisition.

Hydrolysis of diadenosine 5',5''-P',P''-triphosphate (Ap3A) by porcine aortic endothelial cells.

Diadenosine triphosphate is present in platelet-dense granules and released quantitatively on platelet aggregation. We have found that intact porcine aortic endothelial cells can efficiently hydrolyze extracellular diadenosine triphosphate. The products of diadenosine triphosphate hydrolysis are adenosine monophosphate and adenosine diphosphate. Adenosine diphosphate is a potent stimulus of platelet aggregation. Since platelet-dense granules contain high concentrations of adenosine triphosphate and adenosine diphosphate, we examined endothelial cell hydrolysis of a mixture of diadenosine triphosphate and adenosine triphosphate. We find that the presence of adenosine triphosphate severely inhibits the hydrolysis of diadenosine triphosphate. Thus, although endothelial cells can rapidly clear extracellular diadenosine triphosphate, during platelet aggregation the hydrolysis of diadenosine triphosphate may be slow due to the presence of high concentrations of other adenine nucleotides. This phenomenon may be important physiologically if, as current evidence implies, diadenosine triphosphate is involved in the maintenance of hemostasis.

A micromethod for the quantitation by radioimmunoassay of cyclic AMP in samples containing immuno-cross reactive compounds and other interfering substances.

A procedure for measuring cyclic AMP in samples containing interfering substances, especially high concentrations of other adenine nucleotides, is described. Samples are purified by sequential chromatography on phenyl boronate followed by Dowex - 1. The final elution is with 0.1 normal hydrochloric acid; the samples are immediately ready for acetylation and radioimmunoassay. Cyclic AMP can be detected at 0.5 nanomolar without need to concentrate the sample at any step. The method has been successfully applied to samples containing 100 micromolar ATP, ADP, or AMP in tissue culture medium.

Heat stroke and fluphenazine therapy: report of a case.

A fatal case of heat stroke in a 21-year-old man with chronic schizophrenia is reported. Phenothiazine therapy is thought to have been a factor of major importance in the induction of this syndrome. These cases may become the responsibility of the medical examiner, and the importance of obtaining a complete drug history is stressed.