Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024).

Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.

Replacement of the carboxylic acid group of prostaglandin f(2alpha) with a hydroxyl or methoxy substituent provides biologically unique compounds.

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Practice differences between male and female oral and maxillofacial surgeons: survey results and analysis.

PURPOSE: This study describes the personal and practice characteristics of oral and maxillofacial surgeons, with an emphasis on gender differences. Potential explanations for differences found are offered. MATERIALS AND METHODS: A 39-item questionnaire was designed to address areas of suspected differences between male and female oral and maxillofacial surgeons. It included items regarding training, certification, practice type and location, time spent working, practice composition, and personal characteristics. Identical questionnaires were sent to all of the 130 female oral and maxillofacial surgeons (OMSs) registered with AAOMS, as well as to 264 randomly sampled male OMSs. RESULTS: Of the 192 responses received, 109 were from male surgeons, and 83 were from females, 56.8% versus 43.2% of the total. For men, the response rate was 41.3%, whereas the rate was 68.3% for the women; overall, 48.7% of the 394 oral surgeons responded. Profiles of the "average" male and female OMS showed similarities with regard to race, training, and reasons for choosing an OMS career. Age, marital status, number of children, physical characteristics, and use of time outside of work indicated some significant personal differences, as did practice characteristics, including income, number of years in practice, hours spent working, and number of patients seen. Clear gender-based trends were found regarding opinions of physical and mechanical disadvantages, with women more strongly denying such problems. A variety of both positive and negative viewpoints were elicited by the free comment section. CONCLUSIONS: Although male and female OMSs are similar with regard to most variables investigated, some significant differences do exist.

Muscarinic receptor subtypes in human iris-ciliary body measured by immunoprecipitation.

PURPOSE: To determine the relative levels of the five muscarinic receptor subtypes in the anterior segment of the human eye. METHODS: Antisera selective for each of the five muscarinic receptor proteins were incubated with [3H]-QNB bound receptors solubilized from human iris sphincter, ciliary muscle, and ciliary processes. Precipitation of the radiolabeled receptor-antibody complexes and scintillation counting enabled quantitation of the subtypes in the various tissues. Reverse transcription-polymerase chain reaction was performed on the tissues and cultured smooth muscle cells derived from them. RESULTS: Approximately 60% to 75% of the muscarinic receptors in the human iris sphincter and ciliary body are the m3 subtype. Lower levels (5% to 10%) of the m2 and m4 receptors are present in these tissues. The m1 receptor (7%) was detected in the ciliary processes and iris sphincter and the m5 receptor (5%), which is usually found only in the central nervous system, was present in the iris sphincter. CONCLUSIONS: The m3 subtype is the predominant muscarinic receptor in the anterior segment of the human eye. The extensive heterogeneity of muscarinic receptors makes it difficult to predict whether subtype-selective drugs will have an improved efficacy and side-effect profile.

Molecular characterization and ocular hypotensive properties of the prostanoid EP2 receptor.

The cloning of the genes that encode for prostaglandin (PG) receptors has resolved much of the complexity and controversy in this area by confirming the classification proposed by Coleman, et al. Two issues that remained unresolved were (1) the inability of the EP2 agonist butaprost to interact with the cloned putative EP2 receptor and (2) molecular biological confirmation of a fourth PGE2-sensitive receptor, which was pharmacologically designated EP4. In order to provide clarification, we attempted to clone further PGE2-sensitive receptors. By using a cDNA probe that encodes for the human EP3A receptor, a cDNA clone that encoded for a novel PGE2-sensitive receptor was obtained by screening a human placenta library. This cDNA clone was transfected into COS-7 cells for pharmacological studies. The cDNA clone obtained from human placenta had only about 30% amino acid identity with cDNAs for other PG receptors, including those that encode for the previously proposed murine and human EP2 receptors. Radioligand binding studies on the novel EP receptor expressed in COS-7 cells revealed that selective EP2 agonists such as butaprost, AH 13205, AY 23626 and 19(R)-OH PGE2 all competed with 3H-PGE2 for its binding sites, whereas selective agonists for other PG receptor subtypes had minimal or no effect. This receptor was coupled to adenylate cyclase and EP2 agonists caused dose-related increases in cAMP. It appears that the cDNA described herein encodes for the pharmacologically defined EP2 receptor. Ocular studies revealed that AH 13205 decreased intraocular pressure in normal and ocular hypertensive monkeys by a mechanism that does not appear to involve inhibition of aqueous humor secretion.

Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype.

A cDNA that when expressed has the binding and functional characteristics of the pharmacologically defined EP2 prostaglandin (PG) receptor [Cardiovasc. Drug Rev. 11:165-179 (1993)] has been cloned from a human placenta library. This clone, known as Hup-4, encodes a protein of 358 amino acids that has only approximately 30% overall identity with other PG receptors, including mouse and human clones that have been designated as EP2 receptors [J. Biol. Chem. 268:7759-7762 (1993); Biochem. Biophys. Res. Commun. 197:263-270 (1993)]. In COS-7 cells transfected with Hup-4, PGE2 stimulated the formation of cAMP with an EC50 of approximately 50 nM. The EP2-selective agonists AH13205 and butaprost were also active, with EC50 values in the range of 2-6 microM. The order of potency of PGs for competition with binding of [3H]PGE2 to membranes prepared from COS-7 cells transfected with Hup-4 was PGE2 > or = PGE1 > 16,16-dimethyl-PGE2 > or = 11-deoxy-PGE1 > butaprost > AH13205 > 19(R)-OH-PGE2. Natural PGs and analogues that are selective for the FP (PGF2a), DP (PGD2), EP1 (sulprostone), EP3 (MB 28767), and EP4 (1-OH-PGE1) receptors were inactive or competed poorly with the binding of [3H]PGE2 (< 50% displacement of specific binding at 10 microM). Northern blot analysis showed the presence of a Hup-4 message of approximately 3.1 kilobases in mRNA from human lung and placenta. Reverse transcription-polymerase chain reaction studies also indicated that Hup-4 is probably expressed in human uterus and in HL-60 (human promyelocytic leukemia) cells. Our findings suggest that Hup-4 encodes the pharmacologically defined EP2 receptor, whereas the mouse and human cDNAs previously classified as EP2 may represent another EP receptor subtype or the recently defined EP4 subtype [Prostaglandins 47:151-168 (1994)].

Molecular cloning and expression of human EP3 receptors: evidence of three variants with differing carboxyl termini.

1. The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library. 2. Three of these variants, i.e. the EP3A, EP3E and EP3D, share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP3A1) has a nucleotide coding sequence identical to EP3A but has a completely different 3' untranslated sequence. 3. The carboxyl termini of the three isoforms differ most obviously in length with the EP3A being the longest (41 amino acids) and the EP3E being the shortest (16 amino acids). They also differ in content with the EP3A containing 9 serine and threonines in its carboxyl terminus and the EP3E none. 4. Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg-1 protein of [3H]-prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP3A coupled more efficiently to the inhibition of adenylyl cyclase than did the EP3E. 5. PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3' untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.

Stability of reconstituted methohexital sodium.

The purpose of this study was to determine the stability of reconstituted solutions of methohexital sodium over a 6-week period. Stability of methohexital was examined using reversed-phase high-performance liquid chromatography. The results indicate that reconstituted methohexital is extremely stable for up to 6 weeks when stored at 4 degrees C. When stored at room temperature, reconstituted solutions of methohexital contained increasing levels of degradation products and showed a corresponding decrease in methohexital over a 6-week period. However, the rate of degradation of the drug was slow, with less than 10% of the methohexital undergoing breakdown. In addition, tests for microbial contamination of the solutions stored at room temperature and under refrigeration were negative for up to 6 weeks. This study demonstrates that methohexital, when stored under refrigeration for up to 6 weeks, is virtually chemically identical to a freshly reconstituted solution of the drug. When stored at room temperature, there is some degradation of the drug, but it is not known whether the small amount of degradation is clinically significant. This study emphasizes the importance of obtaining scientific data to support changes in guidelines related to handling and storage of drugs.

Prediction of lactate threshold (LT) and fixed blood lactate concentrations (FBLC) from 3200-m running performance in women.

The present study evaluated the accuracy of predicting velocity and oxygen consumption (VO2) at the LT lactate threshold and FBLC fixed blood lactate concentrations from a 3200-m time trial in women. Forty-four women (mean age = 31.1 yrs, mean ht = 164.9 cm, mean wt = 65.0 kg) completed a treadmill protocol for the determination of LT and FBLC and a 3200-m time trial. Velocity and VO2 values at LT, FBLC of 2.0 2.5, and 4.0 mM, and peak were determined. Mean VO2 and velocity ranged from 27.8 +/- 10.8 ml/kg.min-1 at LT to 42.5 ml/kg.min-1 at peak and from 129.8 +/- 44.0 m.min-1 at LT to 187.0 +/- 52.4 m.min-1 at peak, respectively. Results indicated that a 3200-m time trial (mean time = 20.6 +/- 6.6 min) was a good predictor of VO2 and velocity at LT, FBLC, and peak. Correlation coefficients (using a quadratic model) for velocity ranged from R = 0.96 to R = 0.98 with SEE ranging from 9.0 to 13.1 m.min-1. Correlation coefficients for VO2 ranged from R = 0.94 to R = 0.96 with SEE ranging from 2.8 to 3.6 ml/kg.min. The validity of these regression equations was examined in 13 women who completed a 12-month running program (VO2 LT, VO2 at FBLC of 2.0, 2.5 and 4.0 mM, and VO2 peak increased by 34.7, 19.9, 16.9, 11.9, and 5.4%, respectively, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A new copia-like transposable element found in a Drosophila rDNA gene unit.

We have discovered a member of a new family of copia-like transposable elements inserted into the non-transcribed spacer between two ribosomal genes (rDNA). This family, which we call 3S18, consists of at least 15 elements which are scattered throughout the Drosophila melanogaster genome. The elements of this family are approximately 6.5 kb long and have 0.5 kb terminal direct repeats. All of the elements appear to have the same restriction sites. The element is mobile as the size pattern of homologous fragments varies among different strains. In situ hybridization results confirm the scattered location and transposable qualities of 3S18. The element is not transcribed into abundant RNA.

Mapping of chromosomal IS5 elements that mediate type II F-prime plasmid excision in Escherichia coli K-12.

Three IS5 elements were mapped in overlapping chromosomal segments on a series of F-prime plasmids by restriction analysis and hybridization. IS5A was located clockwise of proA near 6 min, IS5B was located clockwise of purE near 12 min, and IS5C was tentatively located near 14 min on the Escherichia coli K-12 map. The physical structures of nine type II F-prime plasmids that contain chromosomal DNA from this region indicated that these plasmids were excised from the chromosome by recombination between pairs of IS5 elements.