Determination of low plasma timolol concentrations following topical application of timolol eye drops in humans by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for determination of timolol in human plasma following administration of two drops of a 5% timolol ophthalmic solution. A 4% butyl alcohol—hexane extract of an alkalized sample of plasma was chromatographed on a reversed-phase column and the components in the column effluent were monitored by coulometric detection. The extraction efficiency of timolol was 69.02 ± 4.16% (mean ± S.D.) and its detection limit was 107.2 pg/ml. The effect of mobile phase pH, buffer concentration and the working potential of the detector on column performance and the electrochemical response are described.     

Peripheral blood steroid concentrations in the preovulatory rabbit.

Levels of 4 steroids in peripheral serum samples obtained from groups of mature, female New Zealand rabbits were determined and these steroids concentrations were related to the time after coital stimulation. Only females showing vaginal spermatozoa were included. Blood samples were collected directly from the heart from 13 rabbits sac rificed immediately after mating (Oh) and from 8 at 2 hours' postcoitus, 10 at 6 hours' postcoitus, and 8 at 12 hours' postcoitus. Serum concentrations of progesterone, 17alpha-hydroxyprogesterone, testosterone, and 17beta-estradiol were determined by radioimmunoassay. Blood levels of all these steroids except progesterone rose to approxima tely twice Oh concentrations by 2 hours' postcoitus, then declined to about Oh levels by 6 and 12 hours after mating. Blood progesterone levels showed a 20-fold rise over Oh concentrations 2 hours' postcoitus and remained elevated at 6 hours and then declined to Oh levels by 12 hours. Previously published patterns of postcoital determinations are similar. Postcoital levels of 17alpha-hydroxyprogesterone in the rabbit had not been published previously.     

Symplasma formation and decidualization in the pseudopregnant rabbit after intraluminal instillation of tricaprylin.

A pure neutral triglyceride, tricaprylin, was injected (Day 0) into isolated segments of the uterus of rabbits in which pseudopregnancy had been induced by i.v. injection of hCG 6 days before. The animals were killed in groups of 3 on Days 1, 2, 3, 4 and 6. Saline, simple ligation of the uterus or uterine trauma were used as control treatments in other uterine segments in the same animals. Extensive symplasma formation in the uterine epithelium of the tricaprylin-treated segments was observed on Days 2 and 3; this had degenerated by Day 4 and was absent by Day 6. Decidualization of the stroma began on Days 3 and 4 and was absent by Day 6. The symplasma formed earlier on the antimesometrial side than it did laterally or mesometrially. Decidualization was maximal mesometrially, intermediate laterally, and absent antimesometrially. Ligation with saline and trauma, but not ligation alone, produced symplasma formation. Decidualization occurred after trauma but not after saline injection or ligation alone.     

Elevated cyclic GMP concentrations in rabbit bone marrow culture and mouse spleen following erythropoietic stimulation.

The effects of erythropoietin and hypoxia on cyclic nucleotide concentrations in erythroid tissue were evaluated. A rabbit bone marrow culture system and a mouse spleen model provided evidence that erythropoietin and an hypoxic stimulus which increases erythropoietin production may enhance erythropoiesis by initiating reciprocal changes in erythroid cell cyclic nucleotide levels. Cyclic GMP appears to be the active signal in mediating the response to erythropoietin, whereas cyclic AMP may be a passive signal allowing full expression of the cyclic GMP response. Whether the type of response mediated by cyclic nucleotides is proliferative, differentiative or both is not clear, but our data and that of other investigators suggest that cyclic GMP mediates the proliferative actions of erythropoietin.     

Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva

We determined net fluid secretion rate across the pigmented rabbit conjunctiva in the presence and absence of pharmacological agents known to affect active Cl- secretion and Na+ absorption. Fluid flow across a freshly excised pigmented rabbit conjunctiva mounted between two Lucite half chambers was measured by a pair of capacitance probes in an enclosed cabinet maintained at 37 degrees C and a relative humidity of 70%. Fluid transport was also measured in the presence of compounds known to affect active Cl- secretion (cAMP, UTP, and ouabain), Na+ absorption (D-glucose), or under the Cl--free condition on both sides of the tissue. Net fluid secretion rate across the pigmented rabbit conjunctiva in the serosal-to-mucosal direction at baseline was 4.3+/-0.2 microl/hr/cm2 (mean +/- s.e.m.). Net fluid secretion rate was increased approximately two-fold by mucosally applied 1 mM 8-Br cAMP (8.4+/-0.4 microl/hr/cm2) and 10 microM UTP (9.8+/-0.6 microl/hr/cm2), but was abolished by either serosally applied 0.5 mM ouabain (0.3+/-0.1 microl/hr/cm2) or under the Cl--free conditions (0.06+/-0.04 microl/hr/cm2). Mucosal addition of 20 mM D-glucose decreased net fluid secretion rate to 1.0+/-0.5 microl/hr/cm2. In conclusion, the pigmented rabbit conjunctiva appears to secrete fluid secondary to active Cl- secretion. This net fluid secretion is subject to modulation by changes in active Cl- secretion rate and in mucosal fluid composition such as glucose concentration.     

Role of tachykinins in the bronchoconstriction induced by HCl intraesophageal instillation in the rabbit

Gastroesophageal acid reflux (GER) is a common disorder associated with the exacerbation of asthma. In this study we investigated the effects on the airways of intraoesophageal HCl instillation in the rabbit and the role of tachykinins in these effects. In anaesthetized New Zealand rabbits bronchopulmonary functions [total lung resistance (R(L)) and dynamic compliance (C(dyn))] were calculated before and after HCl intraoesophageal instillation. Infusion of HCl induced a significant bronchoconstriction (P < 0.05) in the terms of R(L) and C(dyn) changes, that were increased by phosphoramidon pre-treatment and reduced by capsaicin pre-treatment. Moreover, a pre-treatment with SR 48968, a tachykinin NK2 receptor antagonist, or SR 140333, a NK1 receptor antagonist, significantly inhibited the bronchoconstriction induced by intraoesophageal HCl infusion in terms of R(L) and C(dyn)changes. Finally, the HCl induced bronchoconstriction was unaffected by SR 142801, a tachykinin NK3 receptor antagonist.In conclusion these results suggest that bronchoconstriction induced by intraoesophageal HCl infusion is mainly dependent on the release of tachykinins and that both NK1 and NK2 tachykinin receptors are involved.     

Structure of cross-linked rabbit muscle phosphofructokinase in solution.

Cross-linked rabbit muscle phosphofructokinase in the active tetrameric and octameric state was studied in solution by hydrodynamic methods and small angle x-ray scattering techniques. The translational diffusion coefficients were determined by means of inelastic light scattering and were found to be 3.60 (+/- 0.02) x 10(-7) cm2 . s-1 for the tetramer and 2.54 (+/- 0.15) x 10(-7) cm2 . s-1 for the octamer. From small angle x-ray scattering measurements the radius of gyration, the specific inner surface area, and the volume were determined for both enzyme forms, revealing that the octameric cross-linked form is approximately spherical, with a diameter of 120.0 A, whereas the tetrameric form is asymmetric having an axial ratio of 2. By comparison of the scattering curves with triaxial geometric bodies which are equivalent in scattering, the tetrameric enzyme is described as a rectangular prism, with overall dimensions of A = 131.0 A, B = 131.0 A, and C = 65.0 A, and the octameric form as that of a cube with A = B = C = 120.0 A. The shape of the protomer, having a radius of gyration of 24.8 A, in the tetramer and octamer is similar to that for the native tetramer at pH 10 in the presence of 5 mM fructose 6-phosphate or 15 mM fructose 1,6-bis-phosphate. From the different shapes of the scattering curves of the native phosphofructokinase at pH 7.5 in the presence of 15 mM ATP and of the cross-linked tetramer or octamer, it can be inferred that the shapes of the protomers are different: in the presence of ATP the protomers are elongated, having an axial ratio of 1.8 to 2.0; the cross-linked state reveals a spherical protomer of radius 33.0 A, similar to that of the native enzyme at pH 7.5 in the presence of fructose 6-phosphate or fructose 1,6-bisphosphate.     

Circadian variations in concentrations of plasma thyroxine and triiodothyronine in man.

The plasma concentrations of thyroxine (T4), triiodothyronine (T3), and total protein (Pr) were measured at 2-h intervals in 8 male subjects during two 24-h periods. Plasma T4 and T3 levels varied significantly during the day. T4 values were highest at 0900 hours and thereafter declined rapidly reaching lowest levels at 1500-1700 hours (mean decrement, 13.2% of 0.00-hour value). Plasma T3 was highest at 0900 hours and lowest at 1700-1900 hours (mean decrement, 16.7% of 0900-hour value). Fluctuations observed in Pr were not significant. Variations in plasma T4 and T3 appeared concordant with respect to time, since no significant variation was detected in T3:T4 plasma concentration ratios. In view of previous studies that have demonstrated circadian variations in the binding of thyroid hormones by plasma proteins, it is suggested that the observed temporal variations in plasma concentrations of T3 and T4 reflect parallel changes in the capacity or affinity of specific plasma binding proteins of these iodothyronines.     

Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells

We have previously reported that the pigmented rabbit conjunctiva is a Cl- secreting tissue, subject to cAMP, Ca2+, and PKC modulation. The present study was conducted to characterize, at the cellular and molecular levels, cAMP-regulated Cl- channels in rabbit conjunctival epithelial cells. cAMP-inducible Cl- channel properties were evaluated by monitoring the whole-cell currents using patch clamp techniques. Results showed that 10 microM forskolin significantly stimulated a glibenclamide-inhibitable whole-cell conductance by approximately five-fold. Furthermore, reduction of the Cl- concentration in the bathing solution through partial substitution of NaCl with Na-isethionate resulted in a rightward shift of the reversal potential for both baseline and forskolin-stimulated whole-cell currents from 0 to values close to the theoretical Cl- reversal potential predicted by the Nernst equation. Western blot analysis with a monoclonal antibody recognizing the epitope in the C-terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) showed a positive band at its molecular weight, approximately 170 kD. Immunostaining under confocal microscopy revealed a CFTR specific signal in the apical sections of primary conjunctival epithelial cells. In addition, RT-PCR detection amplified a cDNA fragment 100% identical to the predicted portion of the cloned rabbit CFTR message. The stage is thus set for determining the extent of CFTR contribution to cAMP-regulated Cl- conductance in pigmented rabbit conjunctival epithelial cells.     

Photoperiodic variations in plasma concentration of testosterone in the rabbit

The photoperiodic variations of testosterone plasmatic levels in male rabbits have been studied. The animals were subjected to the influence of three circadian rhythms: 12/12, 14/10 and 10/14 h of light/darkness. The hormone assay was carried out using an enzymeimmunoassay method (Competitive ELISA). Blood samples were of two different types: seriated and non-seriated, to find if these might be any differences in the results. It is concluded that testosterone plasmatic levels are within physiologic values (0.3-10.0 ng/ml), although differences depending on the light/darkness cycle and on the type of blood extraction are remarkable; for this reason the selection of the circadian rhythm will depend on the experimentation that is to be carried out.     

Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.     

Sex, seasonal, and stress-related variations in elasmobranch corticosterone concentrations.

Serum corticosterone was previously studied in numerous elasmobranch fishes (sharks, skates and rays), but the role of this steroid, widespread throughout many taxa, has yet to be defined. The goal of this study was to test whether corticosterone varied in response to acute and chronic capture stress, and across the reproductive cycle in the bonnethead shark, Sphyrna tiburo, and Atlantic stingray, Dasyatis sabina. Serum corticosterone in S. tiburo increased following capture and again 24 h post-capture, possibly caused by interference with 1alpha-hydroxycorticosterone, the primary stress hormone in elasmobranchs. Higher serum concentrations in males compared to females were observed in both species. Variations in corticosterone also occurred during the reproductive cycle in both species. Consistent with other taxa, elevations in male bonnethead sharks and stingrays coincided with peak testicular development and mating. Elevations in female bonnethead sharks occurred from the time of mating through sperm storage into early gestation. In contrast, corticosterone levels in female stingrays were low during their protracted mating season, but elevated through late gestation and parturition. These results indicate that corticosterone has a limited role, if any, in acute and chronic stress associated with capture in S. tiburo, but likely has physiological functions associated with its glucocorticoid properties across the reproductive cycle of both species.     

Circadian variations and extraadrenal effect of ACTH on insulinemia in rabbit.

The present experiment was designed to study the action of ACTH1-24 on insulin secretion during the circadian cycle in normal rabbits and to provide evidence that ACTH1-24 has an extra-adrenal effect on this secretion. In normal rabbits intravenous administration of three doses of ACTH1-24 (1, 10, 100 micrograms/kg) at 10 a. m. increased plasma insulin levels. Hyperglycemia only occurred with doses of 10 and 100 micrograms/kg. A maximum insulin response was already obtained at 1 micrograms/kg. The same experiment performed at 12 p. m. also induced hyperinsulinemia which was only noted at 10 and 100 micrograms/kg; hyperglycemia was only observed after stimulation by the highest dose (100 micrograms/kg). ACTH was therefore more effective during the day; however, at 12 p. m. plasma insulin levels were the highest, but only with the maximum dose of ACTH (100 micrograms/kg). The effect of ACTH1-24 was evaluated throughout the day on normal and adrenalectomized rabbits. In normal animals injection of ACTH1-24 increased plasma glucose and insulin levels both together. In the contrary, in rabbits deprived of adrenal glands, ACTH1-24 induced high insulinemia along with hypoglycemia. We could, therefore, reasonably conclude that ACTH stimulates directly the pancreatic secretion of insulin.     

d-Ibuprofen in ocular inflammation induced by paracentesis of the rabbit eye

The anti-inflammatory compound d-ibuprofen has been investigated for anti-inflammatory and cyclooxygenase inhibitory activity in ocular inflammation induced by paracentesis of eyes of living rabbits. d-Ibuprofen is the dextro-rotary isomer of ibuprofen, a potent non-steroidal anti-inflammatory agent. Eyes pretreated topically with d-ibuprofen 0.8% showed a significant inhibition of aqueous protein (73.0%) and prostaglandin E2 (96.4%) increase after paracentesis as compared to paracentesized untreated fellow eyes and control eyes. In aqueous humor no significant correlation between the increase in prostaglandin E2 and protein could be established after paracentesis. These results indicate that d-ibuprofen could be a useful ocular anti-inflammatory agent as cyclooxygenase inhibitor.     

Properties of Go cells: variations in the proliferative response following isoprenaline.

The proliferative behaviour induced in the acinar cells of the rat submaxillary gland in response to isoprenaline has been used to examine the transit time of cells from a quiescent (Go) state into the S phase. Cumulative 3H-TdR labelling index curves were constructed to determine the mean time interval (Gis time) between stimulation with isoprenaline and entry into the S phase. Data were collected for the proliferative wave induced by three sequential injections of isoprenaline, and the effects of varying the interval between the second and third injections of isoprenaline, and of changing the dose of the drug, were examined. Intervals of 28, 52 and 76 hr between isoprenaline injections resulted in mean Gis times of 16-2, 20-9 and 25-6 hr respectively. It was concluded that the Gis time depended on the recent history of cells with respect to stimulation, but not division. The results are considered in terms of two models, in one of which the time to leave Go is variable, whilst in the other the cells leave Go immediately the stimulus is applied.