In vitro pharmacology of clinically used central nervous system-active drugs as inverse H(1) receptor agonists.

The human histamine H(1) receptor (H(1)R) is a prototypical G protein-coupled receptor and an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are also known to potently antagonize this receptor, underlying aspects of their side effect profiles. We have used the cell-based receptor selection and amplification technology assay to further define the clinical pharmacology of the human H(1)R by evaluating >130 therapeutic and reference drugs for functional receptor activity. Based on this screen, we have reported on the identification of 8R-lisuride as a potent stereospecific partial H(1)R agonist (Mol Pharmacol 65:538-549, 2004). In contrast, herein we report on a large number of varied clinical and chemical classes of drugs that are active in the central nervous system that display potent H(1)R inverse agonist activity. Absolute and rank order of functional potency of these clinically relevant brain-penetrating drugs may possibly be used to predict aspects of their clinical profiles, including propensity for sedation.

The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.

RATIONALE: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. OBJECTIVES AND METHODS: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. RESULTS: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. CONCLUSIONS: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

Developing best practice in arterial sheath removal for registered nurses.

The purpose of this study was to evaluate the consistency of nursing practice in the discontinuation of sheaths on designating nursing units. The sample population was randomly selected based on interventional cardiac procedures where sheaths remained in place after leaving the catheterization lab. The data collected demonstrated inconsistencies in current practice of sheath removal and specific device preference by the nursing staff. Changes to the policies and physician order set were revised to best practice standards. Annual competency training was developed for the staff as was written and visual education. The changes that were implemented throughout the project provided for a positive change in patient outcome, revenue savings, and patient satisfaction.

The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro.

The present study compares the development and quality of blastocysts derived from conventional oocyte insemination with those derived from intracytoplasmic sperm injection (ICSI). Oocytes were collected from patients undergoing ovarian stimulation with human menopausal gonadotrophins for IVF. Patients with normal semen were assigned to conventional oocyte insemination while those with progressive motility <20% and/or normal sperm morphology < or =4% were assigned to ICSI. Resulting embryos were cultured for up to 6 days. The mean number and percentage of embryos reaching the blastocyst stage and the mean number and percentage of blastocysts of high quality on days 5-6 were assessed for both treatment groups and compared. The influence of paternal factors (sperm concentration, motility, progressive motility, morphology) on blastocyst development and quality were assessed by regression analyses. Significantly more ICSI-derived embryos arrested at the 5- to 8-cell stage (P = 0.024) concomitant with the activation of the paternal genome than those derived from conventional oocyte insemination. Significantly fewer ICSI-derived embryos reached the blastocyst stage on days 5-6 (P<0.001) and significantly fewer ICSI-derived embryos were of high quality (P = 0.002) compared with conventional oocyte insemination. When treatment groups were combined and evaluated by regression analysis, progressive motility and sperm morphology were significantly correlated with diminished blastocyst development and quality (P < 0.05). From these data, we conclude that paternal factors and/or performing ICSI in cases of severe male factor infertility may have a detrimental effect on blastocyst development and their quality.

Cellular phone interference with external cardiopulmonary monitoring devices.

OBJECTIVES: To determine the potential effect (electromagnetic interference) of cellular telephones on external cardiopulmonary monitoring devices. METHODS: For this study, we tested 17 different medical devices with 5 portable telephones (4 digital, 1 analog) to assess the potential for electromagnetic interference. The telephones were tested in a normal operating mode to simulate a typical hospital environment with patients or their families using their cellular phones. The medical devices were connected to the appropriate simulators for proper operation while the tests were under way. The screens and alarms of the medical devices were monitored while the telephones were maneuvered in the y and z planes near the devices. Clinically important interference was defined as interference that may hinder interpretation of the data or cause the equipment to malfunction. RESULTS: Any type of interference occurred in 7 (41%) of the 17 devices tested during 54.7% of the 526 tests. The incidence of clinically important interference was 7.4%. CONCLUSIONS: Cellular telephones may interfere with the operation of external cardiopulmonary monitoring devices. However, most of the test results showed that the interference would rarely be clinically important.

Squamous cell carcinoma of the breast following silicone injection of the breasts.

During the 1960s, injecting liquid silicone into the breasts for augmentation purposes was a common practice. Many women suffered complications, usually developing silicone mastopathy, but there have been reports of carcinoma as well. A case of squamous cell carcinoma of the breast is reported in a patient who had previously undergone injection of silicone into the breasts. Upon review of the literature, this is only the second reported case of squamous cell carcinoma of the breast following silicone injection. Squamous cell carcinoma of the breast is a very rare tumor comprising 0.04 to 0.075 percent of all breast malignancies. The tumor appears to develop from metaplasia of benign epithelial cells within the breast. Many theories are presented for the development of this metaplastic process. The clinical presentation, evaluation, and treatment of squamous cell carcinoma of the breast is quite similar to that of infiltrating ductal carcinoma of the breast of comparable stage and size.

Changes in lipid diffusibility in the hamster sperm head plasma membrane during capacitation in vivo and in vitro.

The technique of fluorescence recovery after photobleaching (FRAP) was employed on spermatozoa labeled with the fluorescent lipid analogue C14dil to provide two measures of lateral diffusion in the plane of the sperm plasma membrane during capacitation in vivo and in vitro: the diffusion coefficient (D) for C14dil and the fraction of C14dil that is free to diffuse (%R) within the domain. To evaluate changes in lipid diffusibility during capacitation in vivo, spermatozoa were recovered from the uterus within 30 min after ejaculation or from the oviduct at 2, 4, 6 and 8 hr after mating. To compare the changes which occur in vivo with those which occur during capacitation in vitro, caudal epididymal spermatozoa were incubated under capacitating or non-capacitating (control) conditions for 4 hr. Although transient changes in D occurred during the course of capacitation, there was no net change in D for either anterior (AH) or posterior head (PH) domains following capacitation in vitro or in vivo. Significant differences in the lipid diffusion coefficient between the two head domains were observed during the course of capacitation. A transient decrease in %R was observed for the AH domain during capacitation in vitro and incubation under control conditions, but no significant change in %R was observed in the AH domain during capacitation in vivo. A significant decline in %R of the PH domain was observed for spermatozoa during capacitation in vivo, in vitro and following incubation under non-capacitating conditions. These data indicate that the changes in the lipid diffusibility of the AH and PH domains which occur during capacitation in vivo exhibit both similarities and differences to those which occur during capacitation in vitro.

Sperm interaction with fallopian tube apical membrane enhances sperm motility and delays capacitation.

OBJECTIVE: To understand the effect of sperm contact with the apical plasma membrane of tubal epithelial cells on sperm motility, velocity, and capacitation. DESIGN: Prospective, controlled in vitro study. SETTING: University medical center. PATIENT(S): Women of reproductive age undergoing hysterectomy for benign gynecologic indications and normozoospermic donors of proved fertility. MAIN OUTCOME MEASURE(S): Sperm motility as measured manually, velocity as measured by computer-assisted sperm motility analysis, and capacitation status as measured by the chlortetracycline fluorescence assay. RESULT(S): Sperm incubated with apical membrane vesicles had a significantly higher motility at 12 (87.4% +/- 3.4% versus 69.2% +/- 4.8% [mean +/- SEM]), 24 (85.2% +/- 3.1% versus 60.5% +/- 7.2%) and 48 hours (78.9% +/- 5.3% versus 42.4% +/- 11.3%) compared with control (sperm incubated with human tubal fluid media in the absence of apical membrane vesicles) (n = 4). Progressive velocity was significantly higher at 12 (78.2 +/- 1.4 versus 61.7 +/- 16.1 microns/s) and 24 (66.2 +/- 3.9 versus 34.4 +/- 9.8 microns/s) hours (n = 4). Incubation with apical membrane vesicles significantly slowed the transition from uncapacitated to capacitated chlortetracycline fluorescence pattern (n = 5). CONCLUSION(S): Contact with the apical plasma membrane of tubal epithelial cells enhances sperm motility and delays sperm capacitation in vitro.

The use of iodixanol as a density gradient material for separating human sperm from semen.

Iodixanol, a new nonionic density gradient material with relatively low osmolality and high density, was evaluated to determine its suitability for the separation of human sperm from semen for their subsequent therapeutic use. Using a three-layer iodixanol gradient (approximately 1.17/1.15/1.05 g/mL), sperm were centrifuged at 1000g for 30 min and collected from the 1.05/1.15 interface. Using this method, a mean of 78% of the motile and 99% of the morphologically normal sperm originally present in the semen were recovered at the interface. There was no significant increase in the percentage of motile or morphologically normal sperm in the final preparation compared to the original semen. Sperm survived iodixanol density gradient centrifugation well, showing only modest declines in motility (18%) and velocity (35%) during a subsequent 24-h incubation period. When iodixanol was compared to Percoll density gradient centrifugation with semen from the same ejaculate, there was no significant difference between methods with regard to sperm yield, enrichment of motile or morphologically normal sperm in the preparation or sperm survival following separation. Iodixanol provides a suitable, nontoxic alternative to Percoll for the preparation of human sperm for therapeutic use.

Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro.

Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca2+]i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca2+]i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca2+]i. In the first experiment, changes in [Ca2+]i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC monolayers after 4 h of culture compared to [Ca2+]i in spermatozoa still attached to OEC. In addition, [Ca2+]i was determined in spermatozoa incubated with OEC-conditioned medium for 6 h compared to that in spermatozoa incubated in control medium. [Ca2+]i was higher in motile spermatozoa released from OEC than in spermatozoa still attached to OEC after 4 h of incubation. Incubation in OEC-conditioned medium resulted in lower sperm [Ca2+]i only at 4 h of incubation, but not at 0.5, 2, or 6 h of incubation. In the second experiment, a suspension of apical plasma membrane vesicles (AMV) isolated from isthmic oviductal epithelium was used to study the specific effect of sperm contact with OEC membranes on sperm viability, capacitation, and [Ca2+]i. Direct membrane contact between spermatozoa and AMV prolonged sperm viability, delayed capacitation, and maintained low [Ca2+]i in spermatozoa. These results indicated that membrane contact between equine spermatozoa and OEC is required to maintain low [Ca2+]i, delay capacitation, and prolong viability of spermatozoa in vitro. Modulation of capacitation rate for spermatozoa stored in the isthmic sperm reservoir might ensure the availability of a competent sperm population at the time of fertilization.

Role of direct contact between spermatozoa and oviductal epithelial cells in maintaining rabbit sperm viability.

In many species, including the rabbit, spermatozoa attach to the apical plasma membrane of oviductal epithelial cells. Attachment to oviductal epithelial cells has a beneficial effect on the maintenance of sperm viability during storage in the oviduct, but the mechanism by which this occurs is unknown. The present study was conducted to determine the role of direct contact between spermatozoa and the apical plasma membrane of oviductal cells in maintaining sperm viability. To accomplish this, an apical plasma membrane fraction was isolated from homogenized rabbit oviducts and kidney (control) by differential precipitation. When viewed by electron microscopy, this fraction was composed of closed, roughly spherical apical membrane vesicles (AMV) 50-300 nm in diameter. Analysis of anovulatory, preovulatory, and periovulatory oviductal AMV by one-dimensional PAGE revealed 19 major bands. Densitometry revealed quantitative changes in these bands in relation to reproductive stage. Rabbit spermatozoa were incubated for 48 h in the presence of anovulatory, preovulatory, and periovulatory oviductal AMV. As controls, spermatozoa were incubated with rabbit kidney AMV or in defined medium (DM) alone. There was no significant (p < 0.05) decline in viability for spermatozoa incubated with periovulatory oviductal AMV during the 48-h incubation period. In contrast, viability dropped significantly by 12 h for spermatozoa incubated with preovulatory and anovulatory oviductal AMV, kidney AMV, or DM alone. Periovulatory oviductal AMV were significantly more effective at maintaining sperm viability than preovulatory or anovulatory oviductal AMV. Sperm viability in the presence of kidney AMV was not significantly different from that in DM alone. From these data it can be concluded that direct membrane contact between spermatozoa and oviductal epithelial cells plays a role in maintaining sperm viability. Furthermore, this effect appears to be tissue specific and related to reproductive stage.

A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction.

Female hamsters were artificially inseminated at the time of ovulation with an equal concentration and volume of capacitated sperm suspension in one uterus and uncapacitated sperm suspension in the contralateral uterus. When oviducts were examined 3.5-4.0 h after insemination, a significantly (paired t-test, p less than 0.05) lower number of spermatozoa were found in the oviduct from the side inseminated with capacitated sperm suspension compared to the side inseminated with uncapacitated sperm suspension. The reduction in the number of spermatozoa entering the oviduct on the side inseminated with capacitated sperm suspension was particularly evident when nearly all the spermatozoa in the suspension were hyperactivated. These results suggest that hamster spermatozoa require a progressive linear type of motility pattern to pass efficiently through the uterotubal junction and that under normal conditions in vivo, fertilizing spermatozoa initiate hyperactivated motility after entering the oviduct.

Effect of ovulation on sperm transport in the hamster oviduct.

When hamsters mate shortly after the onset of oestrus (4.5-6 h before the onset of ovulation), spermatozoa are stored in the caudal isthmus of the oviduct until near the time of ovulation. At this time, a few spermatozoa ascend to the ampulla to fertilize the eggs. Superovulation resulted in a significant increase in the number of spermatozoa in the caudal isthmus at 6 h post coitus (p.c.) and in the ampulla and bursal cavity at 12 h p.c. Precocious ovulation resulted in a highly significant reduction in the total number of spermatozoa in the oviduct at 3 and 6 h p.c. This effect was completely overcome by intrauterine artificial insemination, suggesting lack of cervical patency as the block to sperm transport in precociously ovulated animals. Ligation of the ampulla-infundibulum junction in naturally ovulating hamsters resulted in significantly fewer spermatozoa in the caudal isthmus and ampulla at 12 h p.c. Preclusion of ovulation also resulted in fewer spermatozoa in the caudal isthmus and ampulla at 12 h p.c., suggesting that the products of ovulation stimulate sperm transport in the oviduct.

Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct.

Female hamsters were mated shortly after the onset of oestrus. At 3 or 6 h after mating, the right oviduct was flushed in situ with 30, 90 or 180 microliters medium to remove spermatozoa from the lumen, leaving only those firmly attached to the isthmic mucosa of the oviduct. When eggs were recovered from oviducts at 20 h after flushing the majority were fertilized, indicating that the spermatozoa that were firmly attached to the mucosa were capable of detaching and ascending to the ampulla to fertilize eggs. Neither the time of flushing nor the volume of flushing medium had a significant effect on the percentage of spermatozoa that remained in the isthmus after flushing. These results suggest that there is no change in the surface of the oviduct mucosa that causes the release of spermatozoa from the caudal isthmus near the time of ovulation. When incapacitated spermatozoa were introduced into the oviduct, many of them attached to oviductal mucosa, while capacitated spermatozoa did not. This indicates that it is a change in the sperm surface, rather than the mucosal surface, that causes the release of spermatozoa, i.e. spermatozoa remain attached to the isthmic mucosa until they become capacitated and then detach and migrate to the ampulla to fertilize the eggs.

The viability of hamster spermatozoa stored in the isthmus of the oviduct: the importance of sperm-epithelium contact for sperm survival.

When hamsters mate shortly after the onset of estrus, spermatozoa are stored in the lower oviduct (isthmus) during the preovulatory period. The present study was performed to determine what proportion of the spermatozoa in the isthmus survive until fertilization. Females were mated 5 to 6.5 h before ovulation. When spermatozoa in the isthmus were observed through the wall of oviducts excised 2 h after the onset of mating, spermatozoa were seen free in the lumen, attached to the mucosal surface of the wall, and in crypts. The vast majority of spermatozoa in the lumen were immotile, whereas most of those attached to the mucosal surface of the wall and almost all of the those in the crypts exhibited flagellar movement. This suggested that attachment to the mucosa and/or storage in the crypts is beneficial to the survival of spermatozoa. Sequential flushing of an oviduct at various times (2-8 h) after mating was used to remove spermatozoa from the lumen (first flush), from the mucosal surface (second flush), and from the crypts (third flush). The highest number of spermatozoa was always contained in the first flush, the next highest in the second flush, and the smallest in the third flush. When Trypan blue was included in the flushing medium to differentiate live and dead spermatozoa, the first flush recovered the smallest percentage of liver spermatozoa (2-22%), the second flush slightly more (16-37%), and the third flush the highest (51-69%), regardless of the time after mating. These data indicate that the majority of spermatozoa stored in the hamster isthmus die before ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Capacitation status of hamster spermatozoa in the oviduct at various times after mating.

Female hamsters were mated shortly after the onset of oestrus or immediately after ovulation. At various times after mating, spermatozoa were flushed from the isthmus of the oviduct using a modified Tyrode's medium supplemented with 20% hamster serum. Cumulus oophorus-free eggs were introduced into the suspensions of isthmic spermatozoa. Some eggs were removed every 30 min and examined for evidence of fertilization. For females mated shortly after the onset of oestrus, spermatozoa recovered from the oviducts 8 h after mating (about 1.5 h after ovulation) could penetrate eggs within 30 min and were considered fully capacitated. When spermatozoa were recovered at earlier times (1, 2, 4 and 6 h after mating) they required additional time (2, 1.5, 1 and 1 h respectively) in vitro before penetrating eggs. Therefore, when mating occurs shortly after the onset of oestrus, spermatozoa in the oviduct do not appear to become fully capacitated until about the time of ovulation. For females mated immediately after ovulation, spermatozoa recovered from the oviducts at 4 h after mating could penetrate eggs within 30 min. Spermatozoa recovered at 1 and 3 h after mating required 2 and 1 h respectively in vitro before penetrating eggs. These results suggest that sperm capacitation proceeds at a faster rate when mating occurs after ovulation.

Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster.

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.

Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination.

A group of female hamsters was mated with males of proven fertility either several hours before or during ovulation. Another group of females was artificially inseminated several hours before ovulation. Females were killed at various times after the onset of mating or artificial insemination, oviducts were fixed and sectioned serially, and spermatozoa were counted individually as to their location in the oviduct. Regardless of the type or time of insemination, the vast majority of spermatozoa that entered the oviduct remained in the lower segments of the isthmus (the intramural and caudal isthmus) without ascending to the ampulla. The lower segments of the oviduct, particularly the caudal isthmus, appeared to be acting as a "sieve" and/or "sperm reservoir." In females mated or artificially inseminated prior to ovulation, virtually no spermatozoa reached the cephalic isthmus or ampulla until the commencement of ovulation. Although a few spermatozoa reached the ampulla by 1 h after the onset of mating, they were the exception rather than the rule. When females were mated during ovulation, spermatozoa spent a minimum of about 3 h in the caudal isthmus before ascending to the ampulla. The number of spermatozoa that entered the oviduct after artificial insemination was considerably lower than in naturally mated animals, but this low number was apparently large enough to ensure complete fertilization.

Distribution of fluid in bronchovascular bundles with permeability lung edema induced by alpha-naphthylthiourea in dogs. A morphometric study.

We utilized light microscopic morphometry to examine the distribution of fluid in bronchovascular bundles of different sizes. Permeability edema was induced in 10 anesthetized dogs with 27 mg/kg of alpha-naphthylthiourea. Eight dogs served as controls. After moderately severe edema, diagnosed on chest radiographs and with decreasing arterial pO2, lobes were fixed with glutaraldehyde and formaldehyde or by freeze substitution. Postmortem wet weight to dry weight ratios were 7.82 +/- 0.62 (mean +/- SE) in the edematous lungs and 4.38 +/- 0.25 in the controls. Bronchovascular bundles were photographed and grouped as follows: bundles composed of separated arteries and bronchioles, bundles with connected arteries and bronchioles, and bundles with connected arteries and bronchi. The transparencies were projected on a tablet interfaced to a computer and the following areas were determined: T, the total bundle area; V, the vessel (artery) area; B, the airway (bronchiole or bronchus) area; A1, the tight periarterial adventitial sheath area; A2, the loose periarterial interstitial area; and A3, the bronchiolar/bronchial interstitial area. In addition, edema ratios for arteries (A2/V) and airways (A3/B) were calculated. We found that (a) A1 was very small and did not change with edema; (b) A2 in all bundles increased 10-fold with edema (p less than 0.01), whereas A3 increased 2- to 3-fold; (c) A2/V increased 9- to 15-fold in the edematous bundles (p less than 0.01) and (d) A3/B did not change in separated bundles (p greater than 0.05) but was approximately double after edema in the connected bundles with bronchioles and bronchi (p less than 0.01). We conclude that edema in bronchovascular bundles accumulates preferentially in the loose periarterial interstitium and does not appear to accumulate around smaller bronchioles. These data may be explained by anatomical factors and by gradients of interstitial pressure.