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Objective To improve hand hygiene in two outpatient health care clinics through the introduction of a gel sanitizer and an informational poster. Methods In this interventional study, health care workers at two outpatient clinics were observed for frequency of hand hygiene (attempts versus opportunities). Gel sanitizer and informational posters were introduced together as an intervention. Direct observation of the frequency of hand hygiene was performed during baseline, intervention, and follow-up. A poststudy survey of health care workers was also distributed and collected. Results In both clinics, the frequency of hand hygiene was poor at baseline (11% and 21%) but improved significantly after intervention (36% and 54%) and was maintained through the follow-up period (32% and 51%). Throughout the study, postcontact hand hygiene was observed significantly more often than precontact hand hygiene. In both clinics, health care workers reported a preference for soap and water; yet observations showed that when the intervention made gel sanitizer available, sanitizer use predominated. Fifty percent of the surveyed health care workers considered the introduction of gel sanitizer to be an effective motivating tool for improving hand hygiene. Conclusions Hand hygiene performance by health care workers in outpatient clinics may be improved through promoting the use of gel sanitizer and using informational posters. Compared with surveys, direct observation by trained observers may provide more accurate information about worker preferences for hand hygiene tools.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs for the suppression of inflammation and pain. However, the analgesic properties of NSAIDs are also associated with significant negative side effects, most notably in the gastrointestinal (GI) tract. Increasingly, evidence indicates that the ulcerogenic properties of some NSAIDs are not exclusively the result of inhibition of cyclooxygenase isoforms in the GI tract, and other mechanisms, including inhibition of cell migration and epithelial restitution, are being explored. Recently, microarray analysis was used to identify potential novel targets of NSAID activity in intestinal epithelial cells. Treated cells exhibited significant reductions in the gene expression of pleiotrophin (PTN), a cytokine and growth factor known to participate in angiogenesis and bone growth. This report aimed to confirm the microarray results reported previously, and to measure protein expression of PTN in intestinal epithelial cells. Furthermore, we also examined the effects of exogenous PTN on cell migration in the presence and absence of either NSAIDs with variable ulcerogenic potential or PTN-specific siRNA. Our results demonstrated that indomethacin and NS-398, two NSAIDs with ulcerogenic potential significantly decrease both gene and protein expressions of PTN in IEC-6 cells and protein expression in IEC-6-Cdx2 cells. Additionally, cell migration experiments with PTN siRNA showed that PTN is an important mediator of IEC-6 cell migration, and addition of exogenous PTN partially restores the deficits in cell migration caused by treatment with indomethacin and NS-398. Finally, measurement of PTN protein expression in the GI tract of horses treated with phenylbutazone showed that PTN expression is reduced by NSAIDs in vivo. Our results show that PTN is an important mediator of cell migration in IEC-6 cells, and PTN is a potential target through which NSAIDs may inhibit cell migration, epithelial restitution, and wound healing in the GI tract.

What are veterinary medical and public-health professionals doing to remedy the immediate and impending shortages of veterinarians in population health and public practice? This question was addressed at the joint symposium of the Association of American Veterinary Medical Colleges and the Association of Schools of Public Health, held in April 2007. Thinking locally, faculty and students at Kansas State University (KSU) asked similar questions after attending the symposium: What are we doing within the College of Veterinary Medicine to tackle this problem? What can we do better with new collaborators? Both the professional veterinary curriculum and the Master of Public Health (MPH) at KSU provide exceptional opportunities to address these questions. Students are exposed to public health as a possible career choice early in veterinary school, and this exposure is repeated several times in different venues throughout their professional education. Students also have opportunities to pursue interests in population medicine and public health through certificate programs, summer research programs, study abroad, and collaborations with contributing organizations unique to KSU, such as its Food Science Institute, National Agricultural Biosecurity Center, and Biosecurity Research Institute. Moreover, students may take advantage of the interdisciplinary nature of public-health education at KSU, where collaborations with several different colleges and departments within the university have been established. We are pleased to be able to offer these opportunities to our students and hope that our experience may be instructive for the development of similar programs at other institutions, to the eventual benefit of the profession at large.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well documented gastrointestinal (GI) toxicity. At this time, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of NSAIDs. Monolayers of intestinal epithelial cells (IEC-6) were treated with NSAIDs that either exhibit (indomethacin, NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]) or lack (SC-560 [5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]) inhibitory effects on IEC-6 migration. Bioinformatic analysis of array data identified the calpain cysteine proteases and their endogenous inhibitor calpastatin as potential targets of NSAIDs shown previously to retard IEC-6 migration. Accordingly, quantitative real-time reverse transcription polymerase chain reaction and immunoblotting were performed to assess the effects of NSAIDs on the expression of mRNA and protein for calpain 8, calpain 2, calpain 1, and calpastatin. In treated IEC-6 monolayers, NS-398 decreased the expression of mRNA for calpain 2 and calpain 8. Both NS-398 and indomethacin decreased the protein expression of calpains 8, 2, and 1. None of the NSAIDs affected expression of calpastatin mRNA or protein. The calpain inhibitors, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-Nle-CHO, retarded IEC-6 cell migration in a concentration-dependant fashion, and these inhibitory effects were additive with those of indomethacin and NS-398. Our experimental results suggest that the altered expression of calpain proteins may contribute to the adverse effects of NSAIDs on intestinal epithelial restitution.

Non-steroidal anti-inflammatory drugs (NSAIDs) contribute to gastrointestinal ulcer formation by inhibiting epithelial cell migration and mucosal restitution; however, the drug-affected signaling pathways are poorly defined. We investigated whether NSAID inhibition of intestinal epithelial migration is associated with depletion of intracellular polyamines, depolarization of membrane potential (E(m)) and altered surface expression of K(+) channels. Epithelial cell migration in response to the wounding of confluent IEC-6 and IEC-Cdx2 monolayers was reduced by indomethacin (100 microM), phenylbutazone (100 microM) and NS-398 (100 microM) but not by SC-560 (1 microM). NSAID-inhibition of intestinal cell migration was not associated with depletion of intracellular polyamines. Treatment of IEC-6 and IEC-Cdx2 cells with indomethacin, phenylbutazone and NS-398 induced significant depolarization of E(m), whereas treatment with SC-560 had no effect on E(m). The E(m) of IEC-Cdx2 cells was: -38.5+/-1.8 mV under control conditions; -35.9+/-1.6 mV after treatment with SC-560; -18.8+/-1.2 mV after treatment with indomethacin; and -23.7+/-1.4 mV after treatment with NS-398. Whereas SC-560 had no significant effects on the total cellular expression of K(v)1.4 channel protein, indomethacin and NS-398 decreased not only the total cellular expression of K(v)1.4, but also the cell surface expression of both K(v)1.4 and K(v)1.6 channel subunits in IEC-Cdx2. Both K(v)1.4 and K(v)1.6 channel proteins were immunoprecipitated by K(v)1.4 antibody from IEC-Cdx2 lysates, indicating that these subunits co-assemble to form heteromeric K(v) channels. These results suggest that NSAID inhibition of epithelial cell migration is independent of polyamine-depletion, and is associated with depolarization of E(m) and decreased surface expression of heteromeric K(v)1 channels.

Long QT syndrome (LQTS) is a condition characterized by prolongation of ventricular repolarization and is manifested clinically by lengthening of the QT interval on the surface ECG. Whereas inherited forms of LQTS associated with mutations in the genes that encode ion channel proteins are identified only in humans, the acquired form of LQTS occurs in humans and companion animal species. Often, acquired LQTS is associated with drug-induced block of the cardiac K + current designated I K r . However, not all drugs that induce potentially fatal ventricular arrhythmias antagonize I K r , and not all drugs that block I K r are associated with ventricular arrhythmias. In clinical practice, the extent of QT interval prolongation and risk of ventricular arrhythmia associated with antagonism of I K r are modulated by pharmacokinetic and pharmacodynamic variables. Veterinarians can influence some of the potential risk factors (eg, drug dosage, route of drug administration, presence or absence of concurrent drug therapy, and patient electrolyte status) but not all (eg, patient gender/genetic background). Veterinarians need to be aware of the potential for acquired LQTS during therapy with drugs identified as blockers of HERG channels and I K r .

This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

The major objective of this study was to elucidate the molecular bases for K(+) current diversity in porcine granulosa cells (GC). Two delayed rectifier K(+) currents with distinct electrophysiological and pharmacological properties were recorded from porcine GC by using whole-cell patch clamp: 1) a slowly activating, noninactivating current (I(Ks)) antagonized by clofilium, 293B, L-735,821, and L-768,673; and 2) an ultrarapidly activating, slowly inactivating current (I(Kur)) antagonized completely by clofilium and 4-aminopyridine and partially by tetraethylammonium, charybdotoxin, dendrotoxin, and kaliotoxin. The molecular identity of the K(+) channel genes underlying I(Ks) and I(Kur) was examined using reverse transcription-polymerase chain reaction and immunoblotting to detect K(+) channel transcripts and proteins. We found that GC could express multiple voltage-dependent K(+) (Kv) channel subunits, including KCNQ1, KCNE1, Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kvbeta1.3, and Kvbeta2. Coimmunoprecipitation was used to establish the hetero-oligomeric nature of granulosa cell Kv channels. KCNE1 and KCNQ1 were coassociated in GC, and their expression coincided with the expression of I(Ks). Extensive coassociation of the various Kv alpha- and beta-subunits was also documented, suggesting that the diverse electrophysiological and pharmacological properties of I(Kur) currents may reflect variation in the composition and stoichiometry of the channel assemblies, as well as differences in post-translational modification of contributing Kv channel subunits. Our findings provide an essential background for experimental definition of granulosa K(+) channel function(s). It will be critical to define the functional roles of specific GC K(+) channels, because these proteins may represent either novel targets for assisted reproduction or potential sites of drug toxicity.

A 9-year-old, spayed female domestic shorthair cat presented for polyphagia, polydipsia, and polyuria following chronic methylprednisolone acetate therapy for pruritus. Initial diagnostics were consistent with uncomplicated diabetes mellitus. Serum calcium was within reference range. Within 12 hours the cat developed depression, anorexia, vomiting, and severe dehydration. Laboratory analysis indicated marked hypercalcemia as measured by both ionized and total calcium concentration. No underlying neoplastic or inflammatory process was identified. An adrenocorti-cotropic hormone stimulation test was indicative of adrenocortical insufficiency. The hypercalcemia resolved with glucocorticoid supplementation and correction of the dehydration. The diabetes mellitus and adrenal insufficiency both resolved within 9 weeks.

The overall purpose of this study was to evaluate the pharmacodynamic response to warfarin in cats. The specific aim was to determine if a log-linear indirect response model (Nagashima et al., 1969) used to describe the in vivo effect of warfarin in humans could be applied to cats. The pharmacokinetics of racemic warfarin were described using a non-compartmental approach. The relationship between prothrombin complex activity (PCA) and normalized prothrombin time (PTR) was defined for feline plasma under our experimental conditions, and determined to be: %PCA=12.38+648 e-PTR/0.492. These data were then integrated and used to predict the warfarin dose associated with therapeutic anti-coagulation defined as an International Normalized Ratio (INR) of 2.0-3.0. The maximum prothrombinopenic response to warfarin in cats after a single intravenous dose of 0.5 mg/kg occurred at 24-48 h. Pharmacodynamic modeling suggested that each cat had a narrow therapeutic range of the steady-state concentration of total warfarin required to appropriately block prothrombin complex synthesis (median: 265.2-358.7 ng/mL). The median daily dose range predicted to yield therapeutic concentrations of warfarin was 0.061-0.088 mg/kg per day. Wide inter-individual variations in both pharmacokinetics and pharmacodynamic response suggest that a more optimal dosing of warfarin may be possible with the development of individual pharmacokinetic/pharmacodynamic algorithms, analogous to those currently employed in human patients.

The purpose of this study was to determine the dispositions of S-warfarin and R-warfarin in normal cats following intravenous and oral administrations of racemic warfarin. Citrated blood samples were collected from 10 cats prior to and at times 5, 15, and 30 min, 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, 72, 96, and 120 h following a single intravenous bolus of 0.5 mg/kg of racemic warfarin. After a 21-day washout period, samples were then similarly collected in three groups of four cats for 120 h following oral administration of 0.1, 0.25, and 0.5 mg/kg racemic warfarin. S-warfarin and R-warfarin were detected using a high-performance liquid chromatography assay validated for cat plasma. Drug concentration-time curves were subjected to non-compartmental analysis. Median pharmacokinetic parameters associated with the intravenous administration of 0.5 mg/kg racemic warfarin were as follows: t1/2 (S:28.2, R:18.3 h), area under the plasma concentration-time curve (AUC; S:33.0, R:24.6 h*microg/mL), area under the moment curve (AUMC; S:1889, R:527.8 h*h*microg/mL), and mean residence time (MRT; S:38.7, R:20.9 h). For each parameter, S-warfarin was significantly different from R-warfarin (P0.05). Warfarin was absorbed rapidly after oral administration, and the dosage did not affect the time to maximum concentration (S:0.87, R:0.75 h). Oral dosage significantly influenced maximum plasma concentration (ng/mL, S:1267, R:1355 at 0.5 mg/kg; S:614.9, R:679.4 at 0.25 mg/kg; S:250.5, R:367.6 at 0.1 mg/kg), AUC (h*microg/mL, S:45.12, R:30.91 at 0.5 mg/kg; S:22.98:, R:18.99 at 0.25 mg/kg; S:3.922, R:3.570 at 0.1 mg/kg) and AUMC (h*h*microg/mL, S:2135, R:1062 at 0.5 mg/kg; S:943.1, R:599.9 at 0.25 mg/kg; S:132.2, R:59.03 at 0.1 mg/kg), but not t1/2 (S:23.5, R:11.6 h) nor MRT (S:26.3, R:13.5 h). Both warfarin enantiomers were highly (96.5%) protein-bound. Quantitation of the warfarin content in commercially available tablets indicated an unequal distribution of the drug throughout the tablet.

CK(4)-M2GlyR, an aqueous soluble peptide derived from the transmembrane M2 segment of the glycine-gated Cl(-) channel found in postsynaptic membranes of the central nervous system, has previously been shown to increase transepithelial Cl(-) and fluid secretion of epithelial monolayers. The goal of this study was to determine whether CK(4)-M2GlyR exerts these effects via formation of a novel chloride conductance pathway, modulation of endogenous chloride channel activity, or a combination of these effects. Ionic currents were recorded from isolated epithelial cells before and after treatment with the peptide using the whole-cell configuration of the patch-clamp technique. CK(4)-M2GlyR increased whole-cell Cl(-) currents in all epithelial cell lines that were studied, including: Madin-Darby canine kidney cells, a human colonic epithelial cell line (T84), and airway epithelial cells derived from a human cystic fibrosis patient (IB3-1). No evidence was found for modulation of endogenous Cl(-) channels by CK(4)-M2GlyR based on both the electrophysiological properties of the observed currents and the pharmacological profile of the CK(4)-M2GlyR-induced current. These results suggest that CK(4)-M2GlyR increases Cl(-) permeability in epithelial cells directly, by forming a distinct conduction pathway in cell membranes.

To improve hand hygiene in two outpatient health care clinics through the introduction of a gel sanitizer and an informational poster.In this interventional study, health care workers at two outpatient clinics were observed for frequency of hand hygiene (attempts versus opportunities). Gel sanitizer and informational posters were introduced together as an intervention. Direct observation of the frequency of hand hygiene was performed during baseline, intervention, and follow-up. A poststudy survey of health care workers was also distributed and collected.In both clinics, the frequency of hand hygiene was poor at baseline (11% and 21%) but improved significantly after intervention (36% and 54%) and was maintained through the follow-up period (32% and 51%). Throughout the study, postcontact hand hygiene was observed significantly more often than precontact hand hygiene. In both clinics, health care workers reported a preference for soap and water; yet observations showed that when the intervention made gel sanitizer available, sanitizer use predominated. Fifty percent of the surveyed health care workers considered the introduction of gel sanitizer to be an effective motivating tool for improving hand hygiene.Hand hygiene performance by health care workers in outpatient clinics may be improved through promoting the use of gel sanitizer and using informational posters. Compared with surveys, direct observation by trained observers may provide more accurate information about worker preferences for hand hygiene tools.

Public health is a demanding field that draws from a number of disciplines: medicine, veterinary medicine, nutrition, nursing, law enforcement, research, policy-making, engineering, and public outreach and education. The nature of this field requires multi-media educational resources that can introduce diverse learners to this curricular and real-world complexity. This chapter describes how a virtual team of faculty members from four Kansas institutions of higher learning collaborated to create an online course titled “Introduction to Public Health”. Drawing from the larger environment, the online course uses global and culturally sensitive techniques to reach a wide learner base and to make use of local resources for its geographically diverse learners. The online learning environment constructed is tied in a deliberate fashion to the values and practices of public health. The online course is built to be more widely adoptable by a range of faculty members from different related fields. As a result, students from rural, urban, and suburban backgrounds will have an opportunity to learn public health and related careers early in their undergraduate education. Public universities and community colleges will have access to a new collaborative model for course development and delivery. This course build involved a consistent tactic of building learning adaptable to local and culturally variant conditions for global transferability, with some of these practices transferable to other online course builds. This chapter explores the uses of research and design in a specific case to enhance the global inheritability of the shared course.

Potassium channels are membrane-spanning proteins that regulate the flow of potassium ions across the cell membrane. Because of the electrochemical gradient for potassium ions in almost all cell types, opening of potassium channels causes an efflux, of potassium ions that in turn tends to make the interior of the cell more negative than its extracellular environment. During the last 5 years, there has been a rapidly expanding base of information about the structure, function, and pharmacologic regulation of this type of ion channel, and the integration of this information is particularly important to cardiac electrophysiology, where control of potassium efflux is relevant to the design of class-III antiarrhythmic agents and to the understanding of genetic basis of certain disorders. This brief review highlights the functional importance of various potassium channels to the electrophysiology of the heart and presents relevant molecular information about the structures that constitute this important family of integral membrane proteins.

International audience; Non-steroidal anti-inflammatory drugs (NSAIDs) are used frequently worldwide for the alleviation of pain despite their capacity to cause adverse gastrointestinal (GI) side effects. GI toxicity, once thought to be the result of non-specific inhibition of cyclooxegenase (COX) enzymes, is now hypothesized to have multiple other causes that are COX independent. In particular, NSAIDs inhibit intestinal epithelial restitution, the process by which barrier function in intestinal mucosa is restored at sites of epithelial wounds within hours through cell spreading and migration. Accordingly, recent evidence indicates that the expression of calpain proteases, which play a key role in cell migration, is decreased by NSAIDs that inhibit cell migration in intestinal epithelial cells (IEC). Here, we examine the effect of NSAIDs on calpain activity and membrane expression in IEC-6 cells. Indomethacin, NS-398, and SC-560 inhibited calpain activity and decreased expression of calpain 2 in total membrane fractions and in plasma membranes involved in cell attachment to the substrate. Additionally, we demonstrated that inhibition of calpain activity by NSAIDs or ALLM, a calpain inhibitor, limits cell migration and in vitro wound healing of IEC-6 cells. Our results indicate that NSAIDs may inhibit cell migration by decreasing calpain activity and membrane-associated expression of calpain 2. Our results provide valuable insight into the mechanisms behind NSAID-induced GI toxicity and provide a potential pathway through which these negative side effects can be avoided in future members of the NSAID class.

Objective: Intracellular application of proteolytic agents modifies the function of many voltage gated ion channels. The presence of a trypsin sensitive inhibitory domain in a channel protein may be important for G protein dependent activation. Guinea pig ventricular IK is modulated by a direct G protein pathway. The aim was to determine if guinea pig ventricular IK is also modified by intracellularly applied trypsin. Methods: Whole cell and excised inside out configurations of patch clamp were used to record IK from guinea pig ventricular myocytes before and after cytosolic application of trypsin (1 mg·ml−1). We used previously reported effects of trypsin on the L type calcium current (ICa) to monitor dialysis time and enzyme activity in whole cell experiments where IK and ICa were measured concomitantly. Results: Addition of trypsin to the solution bathing the cytosolic face of excised membrane patches had no effect on the amplitude or kinetics of Ik. When added to the pipette solution and introduced by cell dialysis, trypsin had no effect on whole cell Ik, even when significant effects on the amplitude and kinetics of ICa were evident. Conclusions: Guinea pig ventricular IK is not enhanced or otherwise altered by intracellularly applied trypsin. Therefore direct phosphorylation independent enhancement of IK by guanine nucleotides cannot depend on interactions between G protein subunits and trypsin sensitive inhibitory channel domains. The lack of trypsin modification of cardiac ventricular IK suggests that the structure of the endogenous delayed rectifier K+ channel may be different than that of other voltage gated channels. Cardiovascular Research 1992; 26 :1131-1136

Drug delivery of ocular therapeutics presents several challenges. There are many difficulties in attaining and sustaining adequate therapeutic levels in the eye while avoiding systemic toxicity. Although there are several routes of administration available for the eye (e.g., topical, subconjunctival, retrobulbar, intracameral, and systemic), topical application of therapeutic agents to the eye offers several advantages including localized drug effects with limited entry into systemic circulation, better accessibility than can be achieved by systemic delivery, avoidance of first-pass hepatic metabolism, convenience, and simplicity. Despite the benefits of topical application, many factors limit the bioavailability of therapeutic agents. For instance, the maximum volume that can be contained within the conjunctival cul-de-sac and precorneal tear film is approximately 30 µL, and the solutes are eliminated rapidly from the precorneal area by lacrimal secretions. The cornea is the outermost transparent portion of the eye and provides the primary barrier through which ocular absorption must occur.1 The anatomic structure of the cornea results in inefficient delivery of drugs to the deeper structures of the eye due to the relatively small surface area and its intrinsic nature as a barrier. Three main strategies are used to increase the bioavailability of ophthalmic drugs including increasing the contact time of the drug with the cornea, improving drug penetration characteristics, and enhancement of the corneal permeability (reviewed in Ref. 2). Corneal epithelial cells constitute the major rate-limiting barrier to drug absorption after topical application. There are two major routes for the movement of compounds through the corneal epithelium: transcellular and paracellular. Since epithelial cell membranes are barriers rich in lipid, lipophilic agents show the best intrinsic penetration through the transcellular pathway. In the absence of an active transport process, however, hydrophilic compounds are limited substantially to permeation through the paracellular pathway where movement is blocked by tight junctions that join adjacent cells near their mucosal surface. The tight junctions define the apical and basolateral membrane components and they serve as a semi-permeable barrier to the flow of solutes through the paracellular pathway. In some tissues, the permeation rates across the tight junctions change in conjunction with various events such as leukocyte transmigration and nutrient uptake in the intestine. 3,4 Tight junctions are also regulated by hormones in some tissues, such as transforming growth factor, glucocorticoids, and progesterone in mammary epithelium.5,6 Thus, therapeutic modulation of corneal tight junctions may allow alteration of the paracellular pathway that would enhance ophthalmic drug concentrations. A synthetic channel-forming peptide, designated NC-1059, has been shown to transiently modulate the barrier function of tight junctions in Madin-Darby canine kidney (MDCK) epithelial cell monolayers.7 This functionality was also observed with epithelial cells derived from the urogenital duct, gut, and air-way of other species.8 In initial studies that targeted the development of a synthetic ion channel, the peptide used was based on the second transmembrane segment of the glycine receptor α-subunit (M2GlyR). This peptide forms a selective pathway in epithelial cells that allows for anion secretion,9 but exhibits relatively low bioavailability due to solution aggregation. Modification of the M2GlyR peptide increased aqueous solubility while maintaining the channel-forming properties as well as anion selectivity.10,11 A peptide was then designed in which the transmembrane segment had an N-terminal tetra-lysine segment and a central leucine that was flanked by a palindrome that is based on the N-terminal portion and was designated NC-1059 (KKKK-AARVGLGITTVLVTTIGLGVRAA). NC-1059 elicited an increase in short circuit current (Isc, a sensitive indicator of net ion transport), remains monomeric in aqueous solution,10 and exhibits no obvious indication of cytotoxicity.7 Thus, NC-1059 represents a possible means of manipulating the epithelial barrier of the cornea at the tight junctions. Studies that assess ophthalmic bioavailability can be conducted in a variety of assay systems. Historically, drug permeation studies have been conducted in vivo with the use of rabbits. However, an in vitro system has many advantages over conventional techniques including the opportunity to use tissues derived from humans and the opportunity to minimize time-consuming, expensive, and controversial animal studies. 12 THCE cells13 provide a model system to asses drug permeation, as has been reported recently.12 These cells have been used to assess tight junctions,14,15 to characterize corneal wound healing,16–18 and to test for cytotoxicity.19 Additional electrophysiological studies verified that THCE cells provided a viable option for the assessment of drug permeation across a confluent epithelial layer. The goal of this study was to determine whether a synthetic channel-forming peptide, NC-1059, can modulate the barrier function of corneal epithelium in vitro using THCE cells. After establishing critical basal parameters for the cultured corneal epithelial cells, experiments were conducted to test for an NC-1059-associated enhancement in gradient driven drug permeation across confluent monolayers. The outcomes show that methotrexate and surrogate drugs of various sizes and chemistries more readily gain access across the corneal epithelial barrier in the presence of or after brief exposure to NC-1059.

A number of FSH Receptor (FSH-R) isoforms with distinct structural motifs and signaling paradigms have been described, including a single transmembrane domain variant that functions as a growth factor type receptor (FSH-R3). This study tested the hypothesis that FSH can stimulate ovarian cancer cell proliferation by acting on FSH-R3, using the tumorigenic mouse ovarian surface epithelial cell (MOSEC) line ID8. FSH enhanced ID8 proliferation in a concentration-dependent fashion. Moreover, FSH-treatment of ID8 elicited intracellular events consistent with activation of FSH-R3 and distinct from those associated with activation of the canonical G-protein coupled FSH-R isoform (FSH-R1). Specifically, the FSH-R3 signaling pathway included cAMP-independent activation of ERK downstream of an SNX-482 sensitive component likely to be the Cav2.3 calcium channel. Northern analysis using probes specific for exons 7 and 11 of FSH-R identified consistently only one 1.9 kb transcript. Immunoblot analysis confirmed expression of FSH-R3 but not FSHR-1 in ID8. Together, these data suggest that FSH-R3 signaling promotes proliferation of ovarian cancer cells.

Background Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels. Methods Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size. Results 4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production. Conclusion 4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Objective—To determine whether ether-a-go-go (ERG) potassium channels are expressed in equine gastrointestinal smooth muscle, whether ERG channel antagonists affect jejunal muscle contraction in vitro, and whether plasma cisapride concentrations in horses administered treatment for postoperative ileus (POI) are consistent with ERG channels as drug targets. Sample Population—Samples of intestinal smooth muscle obtained from 8 horses free of gastrointestinal tract disease and plasma samples obtained from 3 horses administered cisapride for treatment of POI. Procedure—Membranes were prepared from the seromuscular layer of the duodenum, jejunum, ileum, cecum, large colon, and small colon. Immunoblotting was used to identify the ERG channel protein. Isolated jejunal muscle strips were used for isometric stress response to ERG channel blockers that included E-4031, MK-499, clofilium, and cisapride. Plasma concentrations of cisapride were determined in 3 horses administered cisapride for treatment of POI after small intestinal surgery. Results—Immunoblotting identified ERG protein in all analyzed segments of the intestinal tract in all horses. The selective ERG antagonist E-4031 caused a concentration- dependent increase in jejunal contraction. Clofilium, MK-499, and cisapride also increased jejunal contraction at concentrations consistent with ERG channel block; effects of E-4031 and cisapride were not additive. Peak plasma cisapride concentrations in treated horses were consistent with ERG block as a mechanism of drug action. Conclusions and Clinical Relevance—The ERG potassium channels modulate motility of intestinal muscles in horses and may be a target for drugs. This finding may influence development of new prokinetic agents and impact treatment of horses with POI. (Am J Vet Res 2003;64:267–272)

Randomized crossover studies were performed to determine the effect of coadministration of d-alpha-tocopheryl polyethylene glycol 1,000 succinate (vitamin E TPGS) on the oral bioavailability of cyclosporine A (CsA) formulated as either Sandimmune (Novartis Pharmaceuticals) or Neoral (Novartis Pharmaceuticals). Healthy dogs were given a single oral dose of each cyclosporine formulation with and without vitamin E TPGS. Blood samples were collected from each dog before and at various intervals for 24 hours after drug administration. Whole-blood CsA concentrations were determined by high-pressure liquid chromatography. Noncompartmental pharmacokinetic analysis showed that coadministration of vitamin E TPGS increased the oral bioavailability of Sandimmune. The bioavailability of Neoral was greater than that of cyclosporine. Concurrent administration of vitamin E TPGS had no consistent effect on the bioavailability of Neoral.

This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

In dogs and in humans, potassium channels formed by ether-a-go-go-related gene 1 protein ERG1 (KCNH2) and KCNQ1 α-subunits, in association with KCNE β-subunits, play a role in normal repolarization and may contribute to abnormal repolarization associated with long QT syndrome (LQTS). The molecular basis of repolarization in horse heart is unknown, although horses exhibit common cardiac arrhythmias and may receive drugs that induce LQTS. In horse heart, we have used immunoblotting and immunostaining to demonstrate the expression of ERG1, KCNQ1, KCNE1, and KCNE3 proteins and RT-PCR to detect KCNE2 message. Peptide N-glycosidase F-sensitive forms of horse ERG1 (145 kDa) and KCNQ1 (75 kDa) were detected. Both ERG1 and KCNQ1 coimmunoprecipitated with KCNE1. Cardiac action potential duration was prolonged by antagonists of either ERG1 (MK-499, cisapride) or KCNQ1/KCNE1 (chromanol 293B). Patch-clamp analysis confirmed the presence of a slow delayed rectifier current. These data suggest that repolarizing currents in horses are similar to those of other species, and that horses are therefore at risk for acquired LQTS. The data also provide unique evidence for coassociation between ERG1 and KCNE1 in cardiac tissue.

Objectives— To determine the serum and synovial fluid concentrations of ceftiofur sodium after intraarticular (IA) and intravenous (IV) administration and to evaluate the morphologic changes after intraarticular ceftiofur sodium administration. Study Design— Strip plot design for the ceftiofur sodium serum and synovial fluid concentrations and a split plot design for the cytologic and histopathologic evaluation. Animals— Six healthy adult horses without lameness. Methods— Stage 1: Ceftiofur sodium (2.2 mg/kg) was administered IV. Stage 2: 150 mg (3 mL) of ceftiofur sodium (pHavg 6.57) was administered IA into 1 antebrachiocarpal joint. The ceftiofur sodium was reconstituted with sterile sodium chloride solution (pH 6.35). The contralateral joint was injected with 3 mL of 0.9% sterile sodium chloride solution (pH 6.35). Serum and synovial fluid samples were obtained from each horse during each stage. For a given stage, each type of sample (serum or synovial fluid) was collected once before injection and 12 times after injection over a 24-hour period. All horses were killed at 24 hours, and microscopic evaluation of the cartilage and synovium was performed. Serum and synovial fluid concentrations of ceftiofur sodium were measured by using a microbiologic assay, and pharmacokinetic variables were calculated. Synovial fluid was collected from the active joints treated during stage 2 at preinjection and postinjection hours (PIH) 0 (taken immediately after injection of either the ceftiofur sodium or sodium chloride), 12, and 24, and evaluated for differential cellular counts, pH, total protein concentration, and mucin precipitate quality. Results— Concentrations of ceftiofur in synovial fluid after IA administration were significantly higher (P = .0001) than synovial fluid concentrations obtained after IV administration. Mean peak synovial fluid concentrations of ceftiofur after IA and IV administration were 5825.08 μg/mL at PIH .25 and 7.31 μg/mL at PIH 4, respectively. Mean synovial fluid ceftiofur concentrations at PIH 24 after IA and IV administration were 4.94 μg/mL and .12 μg/mL, respectively. Cytologic characteristics of synovial fluid after IA administration did not differ from cytologic characteristics after IA saline solution administration. White blood cell counts after IA ceftiofur administration were ≤3,400 cells/μL. The mean synovial pH of ceftiofur treated and control joints was 7.32 (range, 7.08–7.5) and 7.37 (range, 7.31–7.42), respectively. Grossly, there were minimal changes in synovium or cartilage, and no microscopic differences were detected (P = .5147) between ceftiofur-treated joints and saline-treated joints. The synovial half-life of ceftiofur sodium after IA administration joint was 5.1 hours. Conclusions— Synovial concentrations after intraarticular administration of 150 mg of ceftiofur sodium remained elevated above minimal inhibitory concentration (MIC90) over 24 hours. After 2.2 mg/kg IV, the synovial fluid ceftiofur concentration remained above MIC no longer than 8 hours. Clinical Relevance— Ceftiofur sodium may be an acceptable broad spectrum antimicrobial to administer IA in septic arthritic equine joints.

Decreased Ito in Dogs with Inherited Arrhythmias.Introduction: A colony of inbred German shepherd dogs with inherited ventricular arrhythmias has been established. Methods and Results: The inward rectifier (IKI), the slow delayed rectifier (IKs), and the transient outward current (IKI) were recorded from epicardial myocytes, and IKI, was recorded from Purkinje myocytes isolated from the left ventricles of dogs mildly or severely affected with arrhythmias, and unaffected relatives. There were no differences between unaffected and severely affected dogs in the densities of either IKI or IKI. Peak Ito density at +1–40 mV was reduced by 49% in epicardial myocytes from severely affected dogs. Ito density was also reduced in a subset of Purkinje myocytes. Boltzmann analysis of steady-state inactivation showed no differences between groups in slope factor. V1/2 the half-inactivation voltage, was shifted by +6.2 mV in epicardial cells from severely affected versus unaffected dogs. In addition, the time constant for Ito decay was reduced in mildly and severely affected dogs compared to unaffected dogs. Conclusion: Altered density and inactivation of Ito are associated with the presence of severe ventricular arrhythmias in inbred dogs at risk for sudden death.

Two components of delayed rectifier potassium channels are known to contribute repolarizing currents in cardiac tissue. One component is characterized by very slow activation kinetics and insensitivity to class III antiarrhythmic agents such as E4031. This component, referred to as IKs, has been linked to a gene that encodes a unique and small (130 amino acids) protein that has been shown to induce channel activity in heterologous expression systems remarkably similar to native IKs. In this chapter, several lines of experimental evidence are reviewed that strongly suggest that, despite the similarity between native and recombinant forms of minK or IKs channel activity, a sufficient number of differences have emerged that raise the possibility that the minK gene product, while contributing to the functioning IKs channel, is not sufficient to account for important functional channel properties. Thus these data suggest that the channel underlying IKs is likely to be heteromultimeric with subunit components that are yet to be identified.

We tested for direct G protein regulation of delayed rectifier K+ (IK) channels, by measuring IK currents in guinea pig ventricular cells using patch-clamp procedures. In excised inside-out patches, IK was enhanced by adding guanosine triphosphate or guanosine 5'-O-(3-thiotriphosphate) to the cytoplasmic side, even in the presence of phosphorylation inhibitors. Enhancement of patch IK did not require extracellular agonist; however, enhancement of IK was also seen when isoproterenol was included in the pipette solution. Whole cell IK currents were increased by isoproterenol when phosphorylation pathways were blocked. These data demonstrate that guanine nucleotides and beta-adrenergic agonists can enhance IK by a phosphorylation-independent pathway. Our findings are consistent with a direct coupling of the beta-adrenergic receptor to the cardiac IK channel via a membrane-delimited G protein pathway, in addition to the well-established indirect pathway.

In heart cells, as in other excitable tissues, potassium channel currents are important in maintaining the normal cell resting potential and controlling the duration of the action potential. These channels have particularly important roles in heart because of the complex pattern of electrical activity that exists in different cell types ranging from the sino-atrial (SA) node in the right atrium to the Purkinje fiber in the ventricle. The regular pattern of impulse conduction and muscle activation must be maintained over a wide range of stimulation frequencies because heart rate is under direct sympathetic and parasympathetic control, and under these conditions proper relationships must be maintained between diastolic filling times and systolic ejection.

When the patch-clamp technique was used, a slowly activating, time-dependent outward current was identified in both cell-attached and excised membrane patches obtained from guinea pig ventricular myocytes. This macroscopic patch current was present in approximately 50% of patches studied and could be observed both in the presence and absence of unitary single channel activity (i.e., ATP-sensitive K+ channels). The time course of activation of the patch current resembled that of the whole cell delayed-rectifier K+ current (IK) recorded under similar ionic conditions, and the patch current and IK were activated over a similar membrane potential range. The time-dependent patch current could be eliminated when the Nernst potential for K+ equaled that of the pulse voltage. The patch current was inhibited by external addition of the tertiary ammonium compound LY 97241 (50 microM) and was augmented after internal application of the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (500 nM). Deactivating tail currents with kinetics similar to those of IK could be recorded to cell-attached and excised patches. Unitary single channel events underlying the time-dependent patch current could not be resolved despite various attempts to increase single channel conductance. Thus our results suggest that a major component of delayed rectification in guinea pig ventricular cells is due to the activity of a high-density, extremely low conductance K+ channel.

Anesthesia was induced in 14 greyhounds with a mixture of diazepam or midazolam (0.28 mg/kg) and ketamine (5.5 mg/kg), and maintained with halothane. There were no significant differences in weight, age, or duration of anesthesia between the treatment groups. Time to intubation with diazepam-ketamine (4.07 +/- 1.43 min) was significantly longer than with midazolam-ketamine (2.73 +/- 0.84 min). Heart rate, respiratory rate, PaCO2, and arterial pH did not vary significantly during anesthesia in either treatment group. Arterial blood pressures, PaO2, halothane vaporizer setting, and body temperature changed significantly from baseline values in both treatment groups during anesthesia. Times to sternal recumbency and times to standing were not significantly different. These data suggest that both diazepam-ketamine and midazolam-ketamine are useful anesthetic combinations in greyhounds. In combination with ketamine, midazolam offers little advantage over diazepam.

The effects of halothane on isoproterenol-induced delayed after-depolarizations (DADs) were investigated in canine ventricular myocytes. In addition, the effects of halothane on the intracellular free calcium transient were determined in fura-2-loaded myocytes exposed to isoproterenol to explore the mechanisms underlying halothane effects on DADs. Isoproterenol (100 nM) induced DADs and/or undriven action potentials in myocytes stimulated electrically with the use of trains of 10 stimuli delivered at basic drive cycle lengths of 200-1,000 ms. Isoproterenol (100 nM) increased the peak ratio (350/380 nm excitation) of stimulated myocyte calcium transients; furthermore, isoproterenol induced a second spontaneous component in the calcium transients of 62% of treated myocytes (n = 72). Halothane (1.5%, 0.53 mM) significantly decreased the amplitude of isoproterenol-induced DADs (P less than 0.01). Halothane not only reduced the peak ratio of the stimulated calcium transient, but also eliminated the second spontaneous component in myocytes previously exposed to isoproterenol (n = 14). Elevated extracellular calcium concentrations (5 mM) restored the amplitudes of DADs and the second components of the calcium transients in myocytes exposed to isoproterenol and halothane. These data suggest that halothane opposes isoproterenol-induced DADs by altering intracellular calcium stores. The authors' findings do not support a role for DAD-induced triggered activity in the genesis of anesthetic-catecholamine dysrhythmias.

Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels. Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size. 4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production. 4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Alterations in Purkinje-to-muscle conduction may play a role in the development of cardiac arrhythmias. We compared the effects of halothane on impulse propagation in Purkinje fibers with its effects on impulse propagation across the Purkinje-muscle (P-M) junction. In canine Purkinje fibers, halothane (3%) significantly depressed conduction (P less than 0.05). Exposure to halothane altered conduction velocity (theta) in a manner predicted by cable theory; a significant correlation was noted between depression of Vmax and depression of the square of conduction velocity (theta 2) in Purkinje fibers exposed to 3% halothane (n = 11, r = 0.78, P less than 0.01). Halothane (3%) significantly slowed impulse propagation across the P-M junction (P less than 0.05). Vmax and the square of apparent P-M conduction velocity were not significantly correlated (n = 7, r = 0.34, P = 0.45). The data demonstrate that alteration of active membrane properties can account for halothane's slowing of conduction in Purkinje fibers but not for its slowing of conduction across the P-M junction. The data also suggest that a reduction in cell-to-cell coupling may contribute to depression of Purkinje-to-muscle conduction by halothane.

Halothane effects on action potential characteristics and conduction were determined in canine Purkinje fibers, before and during alpha-adrenergic stimulation. Halothane significantly decreased effective refractory period and action potential duration in Purkinje fibers. alpha-Adrenergic stimulation restored effective refractory period and action potential duration in Purkinje fibers exposed to halothane via an alpha 1-adrenoceptor mediated effect antagonized by prazosin. Halothane significantly slowed propagation of impulses initiated at a basic cycle length of 500 ms, and conduction of premature impulses, in Purkinje fibers. In the presence of halothane, alpha-adrenergic stimulation had no additional effects on normal impulse propagation; however, alpha-adrenergic stimulation significantly slowed the conduction of premature impulses in Purkinje fibers exposed to halothane. These data refute a previous report that alpha-adrenergic stimulation enhances halothane's negative dromotropic effect in Purkinje fibers paced at a basic drive cycle length. alpha-Adrenergic prolongation of the conduction times of premature stimuli in Purkinje fibers exposed to halothane is a new finding.

Ovarian tissues were obtained from cyclic goats during the early, mid and late breeding season. Immunoreactive oxytocin was measured by RIA in tissue extracts after chromatography on octadecylsilica cartridges. Luteal oxytocin concentrations were significantly greater during the early breeding season than during the mid or late breeding season. Oxytocin is luteolytic in goats. High concentrations of luteal oxytocin may be related to the high incidence of short estrus at the onset of the breeding season.