A large share of climate impacts of beef and dairy can be attributed to ecosystem services other than food production.

Domesticated ruminants supply nutrient-dense foods but at a large environmental cost. However, many ruminant production systems are multi-functional, providing ecosystem services (ES) other than direct provision of food. When quantifying the climate impact of ruminant products using life cycle assessment (LCA), provisioning ES (i.e. beef and milk) are generally considered the only valuable outputs and other ES provided are ignored, which risks overlooking positive contributions associated with ruminant production. Non-provisioning ES can be included in LCA by economic allocation, using compensatory payments (through agri-environmental schemes) as a proxy for the economic value of ES. For example, farmers can receive payments for maintenance of pastures, which supports e.g. pollination. However, the association between different payment schemes, the ES provided, and livestock production is not always straightforward and it can be difficult to determine which payment schemes to include in the allocation. This study examined how accounting for ES in quantification of climate impact for beef and milk production on Swedish farms was affected by different ways of coupling ES to livestock production through payment schemes. Quantification was done using LCA, attributing the climate impact to beef, milk, and other ES by economic allocation. This resulted in <1-48% and 11-31% of climate impacts being allocated to other ES, instead of beef and milk, respectively, affecting suckler farms most. The results were influenced by which payment schemes, representing different ES, that were included; when only payments directly related to livestock rearing were included, the difference in the climate impact was still large between farm types, while the difference decreased considerably when all environmental schemes were included. While emissions do not disappear, ES-corrected climate impact can potentially be useful as part of consumer communication or in decision-making, reducing the risk of overlooking ES provided by ruminant production in a simpler way than using separate indicators.

Effects of 17ß-estradiol on proliferation, cell viability and intracellular redox status in native human lens epithelial cells.

PURPOSE: The purpose of this study was to examine the effects of 17ß-estradiol on proliferation, cell death and redox status in cultured human lens epithelial cells (HLECs). METHODS: HLECs were exposed to 17ß-estradiol after which cell viability was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and the number of mitotic and apoptotic cell nuclei was determined after staining with Hoechst 33342. Apoptosis was also determined by measuring caspase-3 activity and propidium iodide was used to determine the proportion of non-viable cells. Pro- and antioxidative effects of 17ß-estradiol was investigated by measuring peroxides, superoxides and glutathione, using dichlorofluorescein diacetate (DCFH-DA), dihydroethidium (HET), and monochlorobimane (MCB), respectively. Effects on mitochondrial membrane potential were determined using 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The ability of 17ß-estradiol to prevent reactive oxygen species (ROS)-production in HLECs after exposure to 25 µM H2O2 for 24h was also measured. RESULTS: This study demonstrates increased mitotic activity in HLECs exposed to physiologic concentrations of 17ß-estradiol (1 nM). Pharmacological concentrations of 17ß-estradiol caused increased number of apoptotic cell nuclei and caspase-3 activation. Physiologic concentrations of 17ß-estradiol (0.1-10 nM) stabilized the mitochondrial membrane potential. Similar or slightly higher concentrations of 17ß-estradiol (0.01-1 µM) protected against H2O2-induced oxidative stress as evident by decreased levels of peroxides and superoxides. CONCLUSIONS: The present study demonstrates mitogenic and anti-oxidative effects of 17ß-estradiol at physiologic concentrations, whereas pharmacological levels induced oxidative stress and acted pro-apoptotic in cultured lens cells.

Acute effects of the sigma-2 receptor agonist siramesine on lysosomal and extra-lysosomal proteolytic systems in lens epithelial cells.

PURPOSE: The aim of the present study was to examine the effects of the sigma-2 receptor agonist, siramesine, on morphology, growth, cell death, lysosomal function, and effects on extra-lysosomal proteolytic systems in human lens epithelial cells. METHODS: Human lens epithelial cells in culture were exposed to siramesine and examined for morphological changes using Nomarski optics or calcein. Lysosomes were evaluated using acridine orange and Magic Red (RR-cresyl violet). Nuclear morphology was studied using Hoechst 33342 and propidium iodide. Enzymatic activities in living cells or cell lysates were studied using fluorogenic substrates. RESULTS: Siramesine at low concentrations increased the cytoplasmic proteolytic activity of the proteasome and the calpain system. Effects were also observed with respect to lysosomal morphology, acidity and function. In addition, activation of caspase-3 and the appearance of nuclei with an apoptotic morphology was found. CONCLUSIONS: Siramesine at low concentrations affects lens epithelial cells with perturbations of the major proteolytic systems and lysosomal morphology, resulting in caspase activation and cell death. Siramesine may be a possible substance for the treatment or prevention of posterior capsular opacification (PCO).

Effects of dexamethasone on human lens epithelial cells in culture.

PURPOSE: Treatment with glucocorticoids is a well known risk factor for cataract development, although the pathogenic mechanism has not been elucidated. The aim of the study was to investigate the effects of glucocorticoids in cultured human lens epithelial cells. METHODS: Human lens epithelial cells (HLECs) were exposed to dexamethasone for 24 h. The number of viable cells was determined using the 3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide (MTT) assay, and proliferation was quantified using Ki-67. Apoptosis was investigated by measuring caspase-3 activity and by evaluating nuclear morphology of cells stained with Hoechst 33342. Mitochondria depolarization was measured using the potential-sensitive color, JC-1. Cells were assayed for changes in superoxide production using dihydroethidium (HET), for alterations in peroxide production using dichlorofluorescein diacetate (DCFH-DA), and for glutathione (GSH) variations using monochlorobimane (MCB). Caspase-3 activity was also measured in HLECs simultaneously exposed to dexamethasone and the glucocorticoid antagonist, RU486. RESULTS: Low doses of dexamethasone (0.1 microM) resulted in increased proliferation of HLECs. Apoptosis was increased in HLECs exposed to 1 microM, 10 microM, and 100 microM of dexamethasone as revealed by nuclear morphology studies. Apoptosis was also confirmed by measuring caspase-3 activation. No effect on superoxide production by dexamethasone was seen. There were no effects on GSH levels or mitochondrial depolarization either. Only the highest concentration of dexamethasone (100 microM) caused an increase in peroxide production. In HLECs incubated with the glucocorticoid antagonist, RU486, apoptosis was induced at a lower concentration of dexamethasone (0.1 microM) than with dexamethasone alone. CONCLUSIONS: Low doses of dexamethasone cause a moderate increase in proliferation of cultured HLECs. Slightly higher but still physiologically relevant concentrations of dexamethasone result in a dose-dependent increase in apoptosis. Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms. The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor. Effects on proliferation and/or dysregulation of apoptosis in lens epithelial cells may be an important factor in human steroid-induced posterior subcapsular cataract.

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume.

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle-van't Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15+/-0.01 using the new method, which was significantly smaller than the estimate of 0.37+/-0.02 obtained using the conventional method (p<0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.

Incidence of ventricular fibrillation during left coronary arteriography in pigs: comparison of a solution of the nonionic dimer iodixanol with solutions of five different nonionic monomers.

BACKGROUND: Solutions of iodine contrast media (CM) used for selective coronary arteriography (CA) should have minimal propensity to cause ventricular fibrillation (VF). Commonly used CM for CA are nonionic monomers or dimers. PURPOSE: To compare VF propensity of ready-to-use solutions of one nonionic dimer, iodixanol, and five nonionic monomers, iobitridol, iopamidol, iomeprol, iopromide, and ioversol. MATERIAL AND METHODS: Twenty milliliters of each CM was injected into the left coronary artery (LCA) through an inflated balloon catheter (0.5 ml/s) in 14 pigs; the longest period of injection was 40 s. If VF occurred before 40 s, the injection was stopped and the heart was defibrillated. After VF, there was a delay of 40 min before the next injection. Hemodynamic parameters and vector electrocardiography (VECG) were monitored. A CM with a lower frequency of VF and a longer period between start of injection and start of VF was considered to have a lower VF propensity. RESULTS: Following 14 injections, each of the five nonionic monomers caused 14 VF, whereas iodixanol caused three VF (P<0.01). When VF occurred after iodixanol, it occurred later than after the other CM (P<0.001). Iodixanol caused less prolongation in QRS time (P<0.01) and QTc time (P<0.05) than the other CM. Prolongations in QRS and QTc times caused by CM parallel the VF propensities of the CM. CONCLUSION: Ready-to-use solutions of the dimer iodixanol have lower VF propensity than solutions of the five monomeric CM. This is related to the fact that the solutions of the dimer iodixanol have lower osmolality, higher viscosity, and higher concentrations of NaCl and CaCl2 than solutions of the five monomers.

Intracellular effects of NSAIDs/ASA in oxidatively stressed human lens epithelial cells in culture.

The aim of the study was to examine the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/acetylsalicylic acid (ASA) on human lens epithelial cells (HLECs) during oxidative stress. HLECs were exposed to H2O2 in the absence or presence of indomethacin, diclofenac, celecoxib (NSAIDs) or ASA for 24 h. HLECs were assayed for changes in superoxide and peroxide production and for variations in glutathione. Mitochondrial depolarization was measured using the membrane potential-sensitive dye JC-1. The results of the study include reduction in superoxide and peroxide production as well as reduction in glutathione depletion in oxidatively stressed HLECs incubated with low concentrations of NSAIDs/ASA. However, no protection against H2O2-induced mitochondrial depolarization by NSAIDs/ASA could be seen. In conclusion, NSAIDs/ASA display reactive oxygen species-scavenging properties in H2O2-exposed HLECs in culture.

The proteasome and intracellular redox status: implications for apoptotic regulation in lens epithelial cells.

PURPOSE: This study aimed to investigate redox regulation of the proteasome as well as the effect of proteasome inhibition on intracellular oxidative status and apoptosis. METHODS: Oxidative stress was induced in cultured human lens epithelial cells (HLECs) and intact mouse lenses by 100 microM H2O2. HLECs were also exposed to the reduced and the oxidized forms of glutathione (GSH/GSSG) and the reducing agent dithiotreitol (DTT). The chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured using synthetic fluorogenic substrates. Superoxide as well as peroxide production, mitochondrial membrane potential, and the level of GSH was measured in HLECs after proteasome inhibition by MG-132 or lactacystin. Apoptosis was determined by measuring caspase-3 activation and by studying apoptotic nuclei after staining with Hoechst 33342. RESULTS: All three peptidase activities of the proteasome were inhibited by 100 microM H2O2 and by the oxidized form of glutathione (GSSG), whereas the reduced form (GSH) stimulated chymotrypsin-like and peptidylglutamyl peptidase activities in HLECs lysates. Intact mouse lenses exposed to 100 microM H2O2 exhibited loss of transparency and trends of decreased chymotrypsin-like proteasome activity as well as decreased GSH levels. Inhibition of the proteasome in cultured HLECs caused significant increase in apoptosis and disturbed intracellular redox balance. Simultaneous addition of exogenous GSH completely abolished the increased apoptosis seen after MG-132 treatment. CONCLUSIONS: This study supports the hypothesis that intracellular proteolytic and oxidative regulatory systems are tightly coupled. The current data also indicate that apoptosis by proteasome inhibition is mediated through oxidative mechanisms.

Proteolysis of tubulin and microtubule-associated proteins 1 and 2 by calpain I and II. Difference in sensitivity of assembled and disassembled microtubules.

Calpain I and II (EC 3.4.22.17) are Ca2+-activated neutral thiol-proteases. Isolated brain tubulin and microtubule-associated proteins were found to be good substrates for proteolytic degradation by brain calpain I and II. The assembly of microtubules was totally inhibited when the calpains were allowed to act on microtubule proteins initially, and a complete disassembly was found after addition of calpain I to assembled microtubules. The high-molecular weight microtubule-associated proteins were degraded within a few minutes following incubation with calpain as shown by SDS-polyacrylamide gel electrophoresis and electron microscopy. When calpain was added to pre-formed microtubules, either in the presence or in the absence of microtubule-associated proteins, the proteolysis was significantly reduced. When tubulin was pre-assembled by taxol, the formation of proteolytic fragments was decreased indicating that assembly alters the availability of tubulin sites for proteolytic cleavage by calpain. Digested tubulin spontaneously formed aberrant polymers. No considerable change of apparent net charge was seen, thus indicating that calpain cleaves off fragments containing neutral amino acid residues and/or that the fragments of tubulin remain associated as an entity with the same charge as native tubulin. The results suggest that the calpains act as irreversible microtubule regulators.

Calpain and calpastatin in normal and Alzheimer-degenerated human brain tissue.

The Ca2(+)-dependent neutral proteases calpain I and II as well as their specific inhibitor, calpastatin, were isolated from normal and Alzheimer-degenerated frozen human brain tissue. In the Alzheimer group calpain I activity was higher in cortex than in mesencephalon. The calpastatin activity was lower in cortex in both groups. This may implicate a higher Ca2(+)-dependent proteolysis in cortex compared to mesencephalon. In the Alzheimer group the cortical calpain II level decreased with an increasing degree of neuropathological changes. In the control group, the level of calpastatin decreased as the number of plaques and tangles increased. Evidence was obtained for a correlation of net calpain activity and the extent of neuropathological changes in cortex.

Increased proteolytic activity in lymphocytes from patients with early onset Alzheimer's disease.

The levels of calpains (m-calpain and mu-calpain) in peripheral blood lymphocytes from patients with Alzheimer's disease were determined via Western blotting. The Ca-dependent proteolytic activity and the calpastatin activity were estimated using incubation with exogenous substrate. Evidence was obtained for an increased Ca-dependent proteolytic activity in lymphocytes from patients with early onset Alzheimer's disease. There was also an increased level of membrane-bound mu-calpain in this group of patients. The observed changes may be caused by a general dysregulation of Ca homeostasis in peripheral cells of early onset Alzheimer victims.

Calpastatin immunoreactivity in the monkey and human brain of control subjects and patients with Parkinson's disease.

Parkinson's disease is characterized by a selective loss of dopaminergic neurons in the nigrostriatal pathway. However, not all dopaminergic neurons degenerate in this disease, and calcium has been suspected of playing a role in this differential vulnerability. An overexpression of the calcium-dependent protease calpain II has recently been reported in the parkinsonian substantia nigra, suggesting that a rise in intracellular calcium concentrations may be involved in the mechanism leading to cell death. The proteasic activity of calpain is regulated by an endogenous inhibitory protein called calpastatin. Because little is known about the distribution of calpastatin in the primate brain, we first analyzed immunohistochemically the calpastatin expression in normal human and monkey brain. A ubiquitous distribution of calpastatin immunostaining was observed in both cases, but its expression was variable from one region to another. In the basal ganglia, staining was intense in the striatum, in the pallidal complex, and in some nuclei of the thalamus. The cerebellum was stained intensely, particularly in the granular and Purkinje cell layers. A dense, heterogeneous staining was observed in the hippocampal formation, mostly in the pyramidal and granular layers. The distribution of staining was similar in the different cerebral cortices studied, and it was most intense in layer V. In the brainstem, staining was particularly prominent in the substantia nigra pars reticulata and compacta, the central gray substance, the superior colliculus, and the cuneiform nucleus, and staining was moderate in the tegmenti pedonculopontinus nucleus and the griseum pontis. In the second part of the study, the authors compared calpastatin expression in the mesencephalon between patients with Parkinson's disease and control subjects. Sequential double staining revealed that some dopaminergic neurons coexpress calpastatin, the proportion of double-stained neurons ranging between 52% and 76% among the different dopaminergic cell groups. Quantitative analysis of the number of calpastatin-stained neurons evidenced a loss of both calpastatin-positive and calpastatin-negative neurons in the substantia nigra of patients with Parkinson's disease. These data suggest that calpain II overexpression in Parkinson's disease is not compensated for by a concomitant increase in calpastatin expression.

Increased M-calpain expression in the mesencephalon of patients with Parkinson's disease but not in other neurodegenerative disorders involving the mesencephalon: a role in nerve cell death?

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra and, to a lesser extent, the ventral tegmental area and catecholaminergic cell group A8. However, among these dopaminergic neurons, those expressing the calcium buffering protein calbindin are selectively preserved, suggesting that a rise in intracellular calcium concentrations may be involved in the cascade of events leading to nerve cell death in Parkinson's disease. We therefore analysed immunohistochemically the expression of the calcium-dependent protease calpain II (m-calpain) in the mesencephalon of patients with Parkinson's disease, progressive supranuclear palsy or striatonigral degeneration, where nigral dopaminergic neurons degenerate, and matched controls without nigral involvement. Calpain immunoreactivity was found in fibers and neuronal perikarya in the substantia nigra, the ventral tegmental area, catecholaminergic cell group A8 and the locus coeruleus. In patients with Parkinson's disease but not with the other neurodegenerative disorders, m-calpain immunoreactivity was detected in fibers with an abnormal morphology and in Lewy bodies. Sequential double staining revealed that most of these m-calpain-positive fibers and neuronal perikarya co-expressed tyrosine hydroxylase, indicating that most m-calpain neurons are catecholaminergic. Quantitative analysis of m-calpain staining in the substantia nigra and locus coeruleus revealed an increased density of fibers and neuronal perikarya in parkinsonian patients in both structures. These data suggest that increased calcium concentrations may be associated with nerve cell death in Parkinson's disease.

Proteolysis in human lens epithelium determined by a cell-permeable substrate.

PURPOSE: To develop a system for continuous evaluation of proteolytic activity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells. METHODS: Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface. RESULTS: After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin. CONCLUSIONS: Human lens epithelium responds to increased Ca levels from external or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.

Caspase and proteasome activity during staurosporin-induced apoptosis in lens epithelial cells.

PURPOSE: To determine what caspases are activated during staurosporin-induced apoptosis in cultured bovine lens epithelial cells (BLECs), to study the time course of caspase activation in relation to morphologic changes, and to investigate the effect of caspase and/or proteasome inhibition on apoptosis. METHODS: BLECs were incubated with staurosporin at different concentrations or for different times. Phosphatidylserine (PS) externalization was detected by annexin-V labeling, nuclear morphology was studied by staining with Hoechst 33342 stain (Hoechst, Frankfurt, Germany), and the percentage of apoptotic cells was determined by the TdT-dUTP terminal nick-end labeling (TUNEL) assay. The activity of caspase-1, -2, -3, -4, -8, and -9 as well as the chymotrypsin-like activity of the proteasome was measured by the use of fluorogenic peptide substrates. Inhibition of the proteasome was performed by incubation with 10 microM lactacystin, and caspases were inhibited by 1 microM Z-DEVD-FMK or 20 microM Z-VAD-FMK. RESULTS: Staurosporin treatment caused a dose- and time-dependent increase in the number of apoptotic cells and in caspase-3 activity. Activation of caspase-2, -4, -8, and -9 was also seen. Caspase activity was increased after 3 hours' incubation with 1 microM staurosporin, which is also the time when most cells became annexin-V-positive. Nuclear changes indicative of apoptosis, viewed with both Hoechst and TUNEL staining, appeared after 4 to 6 hours of staurosporin incubation. Incubation of BLECs with lactacystin caused reduction of proteasome activity and increased apoptosis, evidenced in both the TUNEL assay and caspase-3 activation. Preincubation of lens epithelial cells with caspase inhibitors caused complete inhibition of lactacystin- or staurosporin-induced caspase-3 activation (Z-DEVD-FMK/Z-VAD-FMK) and also of caspase-2, -4, -8, and -9 (Z-VAD-FMK), but the reduction in TUNEL-positive cells was only partial. PS translocation and DNA fragmentation after staurosporin treatment occurred despite complete caspase blockade. CONCLUSIONS: Staurosporin-induced apoptosis in BLECs involves activation of several caspases. Inhibition of the proteasome causes caspase-3 activation and apoptosis. Both staurosporin- and lactacystin-induced apoptosis can be executed in a caspase-independent manner. The present data are useful for understanding of proteolytic mechanisms during apoptosis in lens epithelial cells, which may be an important event in normal lens development as well as in some types of cataract.

Retinoic acid produces rod photoreceptor selective apoptosis in developing mammalian retina.

PURPOSE: All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neural retinal cells, induces progenitor cells to adopt the rod cell's fate. Retinoic acid (RA) also produces apoptotic cell death in developing tissues. The effects of retinoids on mouse retinal development were examined. METHODS: Retinas were explanted on postnatal day (PN)1 and cultured with or without the retinal pigment epithelium (RPE) attached. Retinas were cultured for 3 weeks in the absence or presence of 100 or 500 nM ATRA or 9CRA. Morphologic development and apoptotic cell death were examined using cell-specific immunocytochemical markers, the TdT-dUTP terminal nick-end labeling (TUNEL) method, and a caspase assay. RESULTS: Retinal explants, with and without RPE, had similar age-dependent increases in opsin expression. In contrast, explants with RPE had less apoptosis during the first week than retinas without RPE. In explants with RPE, ATRA or 9CRA produced rod-selective apoptotic cell death in which 20% to 25% were lost by PN7 with no further loss by PN21. 9CRA-treated explants without RPE had a decreased number of apoptotic cells and a higher number of (rhod)opsin-positive cells at PN3. CONCLUSIONS: Factors in RPE appear to regulate rod apoptosis in developing retina. Retinoids produce rod-selective apoptotic cell death during normal rod differentiation. In contrast, retinoids accelerate the expression of opsin in retinas without RPE. These differential effects of RA on rod photoreceptors-apoptosis and differentiation-are similar to those observed in other developing tissues and play an important role in both normal and pathologic development.

Characterization of pigment aggregating alpha 2-adrenoceptors of fish melanophores by use of different agonists after partial irreversible receptor inactivation.

1. The affinity for, and the intrinsic efficacy on, postsynaptic melanosome aggregating alpha 2-adrenoceptors of fish melanophores was studied for B-HT 920, clonidine, medetomidine, noradrenaline, phenylephrine and UK-14,304. Investigations were carried out by evaluating the effects of progressive, irreversible inactivation of the alpha 2-adrenoceptors by benextramine. 2. The double reciprocal plots of equieffective concentrations of B-HT 920, clonidine, noradrenaline and phenylephrine were linear, which indicated that these compounds exerted their effects, mainly, through interaction with one receptor site. 3. The affinity for the alpha 2-adrenoceptor selective agonist B-HT 920, was found to be about 1000 times higher than the affinity for the alpha 1-adrenoceptor selective agonist phenylephrine. 4. The corresponding plot of equieffective concentrations of medetomidine was not linear, which may indicate that this imidazole compound exerted its effect through more than one receptor site. However, when phenoxybenzamine was used in place of the more selective benextramine, a linear relationship was obtained.

Ionic mechanisms contributing to the vasorelaxant properties of iodinated contrast media: a comparison of iohexol and iodixanol in the rabbit isolated aorta.

1. We have used rings of rabbit thoracic aorta to investigate the vasorelaxant properties of two different classes of non-ionic iodinated radiographic contrast media (IRCM) and the mechanisms, underlying their mode of action. Iohexol (a triiodinated monomer) was compared with iodixanol (a hexaiodinated dimer). 2. Iohexol and iodixanol both relaxed phenylephrine (0.3 microM) constricted rabbit aorta in a concentration-dependent manner that did not depend on the presence of an intact endothelium. When expressed as a function of iodine concentration, iodixanol caused significantly less relaxation than iohexol. However, the extent of relaxation was similar for both IRCM when expressed on a molar basis. Furthermore, increasing the molarity of the buffer to comparable levels with mannitol evoked only a small (approximately 15%) relaxation of phenylephrine-induced tone. 3. Ouabain (10 microM) significantly inhibited both iohexol- and iodixanol-induced relaxations by approximately 30%. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA, 100 nM) significantly inhibited iohexol-induced relaxation to the same extent as ouabain, but did not alter the vasorelaxant effect of iodixanol. Co-incubation with ouabain and EIPA had an additive effect in the case of iohexol, increasing inhibition of relaxation to approximately 60%, whereas inhibition of iodixanol-induced relaxation by the combination of ouabain plus EIPA did not differ from that of ouabain alone. 4. Replacing NaCl with N-methyl-D-glucamine (NMDG) to lower extracellular [Na+] and thereby inhibit Na(+)-Ca2+ exchange, attenuated the relaxation evoked by iohexol or by iodixanol (by approximately 25%) in each case. 5. We conclude that iohexol- and iodixanol-induced vasorelaxation in rabbit aorta is mediated through a direct action on vascular smooth muscle that is not simply a consequence of altered osmolality. It involves modulation of the Na(+)-K+ ATPase and, in the case of iohexol, Na(+)-H+ exchange. Both agents also appear to modulate Na(+)-Ca2+ exchange, through direct and/or indirect mechanisms. This is the first study to show specific pharmacological differences between monomeric and dimeric contrast media in vascular smooth muscle.

Cloning and expression of a fish alpha 2-adrenoceptor.

1. Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an alpha 2-adrenoceptor pharmacology. We now report the cloning of the alpha 2-F, a fish skin alpha 2-receptor from the cuckoo wrasse (Labrus ossifagus). 2. Degenerate oligonucleotides corresponding to conserved regions of the human alpha 2-adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human alpha 2-adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3. A clone (pTB17BS) consisting of approximately 5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and approximately 2 kb of 5'untranslated sequence. The deduced amino acid sequence of this protein showed 47-57% identity with the human alpha 2-adrenoceptors and thus appeared to encode a fish alpha 2-adrenoceptor. 4. In the 5'-untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the alpha 2-F might be regulated by cyclic AMP, calcium and/or steroids. 5. The alpha 2-F was expressed in COS-7 cells and radioligand binding studies were performed with [3H]-rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 +/- 0.1 nM and a Bmax of 5.7 +/- 1.0 pmol mg-1 of protein.6. Competition curves for the displacement of specific [3H]-rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine >p-aminoclonidine> B-HT 920> (- )-noradrenaline;for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin.7. These results show that alpha2-F has characteristics of both the human alpha2-CIO and alpha2-C4 and that it might represent an ancestral alpha2-adrenoceptor subtype.

Iodinated radiographic contrast media inhibit shear stress- and agonist-evoked release of NO by the endothelium.

1 We have used isolated arterial preparations from the rabbit and dog to investigate whether non-ionic iodinated radiographic contrast media (IRCM) modulate nitric oxide (NO) release. The tri-iodinated monomers iopromide and iohexol were compared with the hexa-iodinated dimer iodixanol. 2 The vasodilator effects of iohexol (300 mg ml-1) and iodixanol (320 mg ml-1) were assessed in cascade bioassay. Increasing concentrations of iohexol or iodixanol caused concentration-dependent relaxations of the detector tissue which were insensitive to 100 microM NG-nitro L-arginine methyl ester (L-NAME) and 10 microM indomethacin, whereas viscosity-associated relaxations induced by the 'inert' agent dextran (MW 80,000; 1-4%) were attenuated by inhibition of NO synthesis. 3 Relaxations of endothelium-intact rings to acetylcholine (ACh) were attenuated by preincubation with iohexol or iodixanol, whereas relaxations to sodium nitroprusside (SNP) in endothelium-denuded rings were unaffected. Inhibitory activity did not correlate with either molarity or iodine concentration. Mannitol caused inhibition of both ACh- and SNP-induced responses. 4 In isolated perfused arteries the depressor responses to iodixanol (320 mg ml-1) and iopromide (300 mg ml-1) administered as close arterial bolus attained a plateau with maximal dilatations of approximately 25% and approximately 60%, respectively. Addition of 100 microM NG-nitro L-arginine (L-NOARG) and/or 10 microM indomethacin to the perfusate had no effect on the responses to either agent. 5 We conclude that IRCM exert direct effects on the endothelium that inhibit NO production rather than its action on vascular smooth muscle. Shear stress-induced stimulation of NO production by IRCM is unlikely to contribute to their vasodilator activity in vivo when administered during angiography despite high intrinsic viscosity.