MicroRNA 29 modulates ß-cell mitochondrial metabolism and insulin secretion via underlying miR-29-OXPHOS complex pathways.

AIM: MicroRNAs (miRNAs) regulate ß-cell function, and ß-cell mitochondria and insulin secretion are perturbed in diabetes. We aimed to identify key miRNAs regulating ß-cell mitochondrial metabolism and novel ß-cell miRNA-mitochondrial pathways. METHODS: TargetScan (http://www.targetscan.org/) was used to predict if 16 miRNAs implicated in ß-cell function target 27 cis-eGenes implicated in mitochondrial activity. The expression of candidate miRNAs and insulin secretion after 24 and 1 h pre-incubation in 2.8, 11.1- and 16.7-mM glucose was measured in clonal INS-1 832/13 ß-cells. MiR-29 silenced INS-1 832/13 cells were assessed for insulin secretion (glucose, pyruvate, and K+), target cis-eGene expression (Ndufv3 and Ndufa10 components of mitochondrial complex I (CI)), OXPHOS (CI-V) protein expression, and mitochondrial OXPHOS respiration/activity. The expression of differentially expressed miR-29 miRNAs was evaluated in Goto-Kakizaki (GK) rat, db/db mouse and type 2 diabetic (T2D) human islets, as well as NMRI mouse islets cultured under glucolipotoxic conditions. RESULTS: MiR-29, miR-15 and miR-124 were predicted to regulate ~20 cis-eGenes, while miR-29 alone was predicted to regulate ≥12 of these in rat and human species. MiR-29 expression and insulin secretion were reduced in INS-1 832/13 cells after 24 h in elevated glucose. MiR-29 knockdown increased all tested insulin secretory responses, Nudfv3, Ndufa10, complex I and II expression, and cellular mitochondrial OXPHOS. MiR-29 expression was reduced in db/db islets but increased in GK rat and T2D human islets. CONCLUSION: We conclude miR-29 is a key miRNA in regulating ß-cell mitochondrial metabolism and insulin secretion via underlying miR-29-OXPHOS complex pathways. Furthermore, we infer reduced miR-29 expression compensatorily enhances insulin secretion under glucotoxicity.

Multivesicular exocytosis in rat pancreatic beta cells.

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 µmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 µm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.

SCRT1 is a novel beta cell transcription factor with insulin regulatory properties.

Here we show that scratch family transcriptional repressor 1 (SCRT1), a zinc finger transcriptional regulator, is a novel regulator of beta cell function. SCRT1 was found to be expressed in beta cells in rodent and human islets. In human islets, expression of SCRT1 correlated with insulin secretion capacity and the expression of the insulin (INS) gene. Furthermore, SCRT1 mRNA expression was lower in beta cells from T2D patients. siRNA-mediated Scrt1 silencing in INS-1832/13 cells, mouse- and human islets resulted in impaired glucose-stimulated insulin secretion and decreased expression of the insulin gene. This is most likely due to binding of SCRT1 to E-boxes of the Ins1 gene as shown with ChIP. Scrt1 silencing also reduced the expression of several key beta cell transcription factors. Moreover, Scrt1 mRNA expression was reduced by glucose and SCRT1 protein was found to translocate between the nucleus and the cytosol in a glucose-dependent fashion in INS-1832/13 cells as well as in a rodent model of T2D. SCRT1 was also regulated by a GSK3ß-dependent SCRT1-serine phosphorylation. Taken together, SCRT1 is a novel beta cell transcription factor that regulates insulin secretion and is affected in T2D.

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCalpha and PKCdelta.

AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. METHODS: Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.

A beta cell-specific knockout of hormone-sensitive lipase in mice results in hyperglycaemia and disruption of exocytosis.

AIMS/HYPOTHESIS: The enzyme hormone-sensitive lipase (HSL) is produced and is active in pancreatic beta cells. Because lipids are known to play a crucial role in normal control of insulin release and in the deterioration of beta cell function, as observed in type 2 diabetes, actions of HSL in beta cells may be critical. This notion has been addressed in different lines of HSL knockout mice with contradictory results. METHODS: To resolve this, we created a transgenic mouse lacking HSL specifically in beta cells, and characterised this model with regard to glucose metabolism and insulin secretion, using both in vivo and in vitro methods. RESULTS: We found that fasting basal plasma glucose levels were significantly elevated in mice lacking HSL in beta cells. An IVGTT at 12 weeks revealed a blunting of the initial insulin response to glucose with delayed elimination of the sugar. Additionally, arginine-stimulated insulin secretion was markedly diminished in vivo. Investigation of the exocytotic response in single HSL-deficient beta cells showed an impaired response to depolarisation of the plasma membrane. Beta cell mass and islet insulin content were increased, suggesting a compensatory mechanism, by which beta cells lacking HSL strive to maintain normoglycaemia. CONCLUSIONS/INTERPRETATION: Based on these results, we suggest that HSL, which is located in close proximity of the secretory granules, may serve as provider of a lipid-derived signal essential for normal insulin secretion.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Endogenous beta-cell CART regulates insulin secretion and transcription of beta-cell genes.

Impaired beta-cell function is key to the development of type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide with insulinotropic and glucagonostatic properties. Here we studied the role of endogenous CART in beta-cell function. CART silencing in INS-1 (832/13) beta-cells reduced insulin secretion and production, ATP levels and beta-cell exocytosis. This was substantiated by reduced expression of several exocytosis genes, as well as reduced expression of genes important for insulin secretion and processing. In addition, CART silencing reduced the expression of a network of transcription factors essential for beta-cell function. Moreover, in RNAseq data from human islet donors, CARTPT expression levels correlated with insulin, exocytosis genes and key beta-cell transcription factors. Thus, endogenous beta-cell CART regulates insulin expression and secretion in INS-1 (832/13) cells, via actions on the exocytotic machinery and a network of beta-cell transcription factors. We conclude that CART is important for maintaining the beta-cell phenotype.

Sox5 regulates beta-cell phenotype and is reduced in type 2 diabetes.

Type 2 diabetes (T2D) is characterized by insulin resistance and impaired insulin secretion, but the mechanisms underlying insulin secretion failure are not completely understood. Here, we show that a set of co-expressed genes, which is enriched for genes with islet-selective open chromatin, is associated with T2D. These genes are perturbed in T2D and have a similar expression pattern to that of dedifferentiated islets. We identify Sox5 as a regulator of the module. Sox5 knockdown induces gene expression changes similar to those observed in T2D and diabetic animals and has profound effects on insulin secretion, including reduced depolarization-evoked Ca2+-influx and ß-cell exocytosis. SOX5 overexpression reverses the expression perturbations observed in a mouse model of T2D, increases the expression of key ß-cell genes and improves glucose-stimulated insulin secretion in human islets from donors with T2D. We suggest that human islets in T2D display changes reminiscent of dedifferentiation and highlight SOX5 as a regulator of ß-cell phenotype and function.

Dental plaque pH and micro-organisms during hyposalivation.

We have previously reported that minor gland and whole saliva flow rates and salivary proteins showed differences in individuals with primary Sjögren's syndrome or head and neck radiation therapy, compared with controls (Eliasson et al., 2005). We now hypothesize that pH and number of acidogenic micro-organisms in dental plaque as well as saliva buffering capacity also differ in these individuals. Plaque pH was measured by the microtouch method up to 60 min after a sucrose rinse. Plaque collected from the same sites was analyzed for counts of total and acidic micro-organisms. Compared with their controls, the irradiated group but not the Sjögren's syndrome group displayed significantly lower plaque pH, increased numbers of lactobacilli and Candida species, as well as reduced buffering capacity. Stepwise regression tests suggested that the buccal minor-salivary-gland secretion rate in the test groups and counts of mutans streptococci in the controls were of significant importance for dental plaque pH.

Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells.

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells.

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.

Minor gland saliva flow rate and proteins in subjects with hyposalivation due to Sjogren's syndrome and radiation therapy.

In this study, the secretion rate and IgA, albumin and lactoferrin concentrations in minor labial and buccal gland saliva were investigated in individuals with hyposalivation due to primary Sjogren's syndrome (pSS; 10 subjects) or head and neck radiation therapy (RT; 10 subjects) and in their matched controls. Whole saliva was similarly examined. The minor gland saliva flow was measured using the Periotron method. IgA, albumin and lactoferrin concentrations were analysed by ELISA techniques. A general finding was that the flow rate and protein concentrations were lower in labial than in buccal gland saliva. In both hyposalivation groups, the labial minor gland saliva secretion rate was lowered compared to their respective controls. The buccal gland saliva flow rate was significantly reduced in the RT group only. IgA and albumin concentrations were not different from the controls in the labial secretions. The concentration of lactoferrin was increased in the RT group. In buccal saliva, the concentrations of all proteins examined but pSS IgA, were increased compared to the controls. Reduced flow rate and increased protein concentrations were seen for whole saliva where the lactoferrin concentration was higher in RT than in pSS subjects. Thus, our findings suggested that minor gland saliva flow rate and protein concentrations are affected in RT and pSS subjects and to highest extent in the former.

Modulation of microRNA-375 expression alters voltage-gated Na(+) channel properties and exocytosis in insulin-secreting cells.

AIM: MiR-375 has been implicated in insulin secretion and exocytosis through incompletely understood mechanisms. Here we aimed to investigate the role of miR-375 in the regulation of voltage-gated Na(+) channel properties and glucose-stimulated insulin secretion in insulin-secreting cells. METHODS: MiR-375 was overexpressed using double-stranded mature miR-375 in INS-1 832/13 cells (OE375) or downregulated using locked nucleic acid (LNA)-based anti-miR against miR-375 (LNA375). Insulin secretion was determined using RIA. Exocytosis and ion channel properties were measured using the patch-clamp technique in INS-1 832/13 cells and beta-cells from miR-375KO mice. Gene expression was analysed by RT-qPCR, and protein levels were determined by Western blot. RESULTS: Voltage-gated Na(+) channels were found to be regulated by miR-375. In INS-1 832/13 cells, steady-state inactivation of the voltage-gated Na(+) channels was shifted by approx. 6 mV to a more negative membrane potential upon down-regulation of miR-375. In the miR-375 KO mouse, voltage-gated Na(+) channel inactivation was instead shifted by approx. 14 mV to a more positive membrane potential. Potential targets differed among species and expression of suggested targets Scn3a and Scn3b in INS-1 832/13 cells was only slightly moderated by miR-375. Modulation of miR-375 levels in INS-1-832/13 cells did not significantly affect insulin release. However, Ca(2+) dependent exocytosis was significantly reduced in OE375 cells. CONCLUSION: We conclude that voltage-gated Na(+) channels are regulated by miR-375 in insulin-secreting cells, and validate that the exocytotic machinery is controlled by miR-375 also in INS-1 832/13 cells. Altogether we suggest miR-375 to be involved in a complex multifaceted network controlling insulin secretion and its different components.

Quantitation of Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) by a two-site enzyme immunoassay, in parallel with EBV-DNA.

A two-site enzyme (TSE) immunoassay was developed for the quantitation of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) using a rabbit serum raised against a synthetic peptide derived from the BamHI K region of the viral genome. Comparison of 12 EBNA-positive and 3 negative cell lines proved that the test was EBV-specific. A dot-blot assay utilizing cloned and nick translated EBV-DNA BamHI M fragment confirmed the EBV-carrier status of the EBNA-positive lines. The results obtained with both the TSE immunoassay and dot-blot assay were in agreement with published values. In contrast to earlier reports, we could not demonstrate any correlation between the content of EBNA and the number of viral genome copies.

Factors Influencing the Efficacy of Platelet Transfusions in Acute Leukemia.

The efficacy of platelet transfusions was evaluated in a prospective study of 37 patients with acute leukemia. The patients received 495 platelet transfusions for chemotherapy-induced thrombocytopenia. Life table analysis was found to be useful for prediction of the platelet level after transfusion. Efficacy was positively correlated with platelet content in the concentrate, and inversely correlated with bleeding. Fever, contamination of the: concentrate by leukocytes, or previous platelet transfusions were not found to have any significant influence on the efficacy, as analyzed by multiple regression. Single-donor platelet preparations and multiple-donor preparations were equally efficient. It is nevertheless suggested that single-donor preparations should be preferred, due to reduced risk of blood-carried infections.

Minor gland and whole saliva in postmenopausal women using a low potency oestrogen (oestriol).

Many women undergo hormone replacement therapy in order to relieve menopausal and postmenopausal symptoms. Oral discomfort is common among these symptoms and studies have shown that the stimulated whole saliva flow rate is increased after combined oestradiol and progesterone replacement therapy. There is, however, no data regarding the effect of other oestrogens or of oestrogen alone on whole and minor gland saliva. In the present study, the flow rate from minor salivary glands (buccal, labial and palatal) and the secretion rate and buffer capacity of whole saliva was examined in 18 postmenopausal women (61-76 years) prior to, and during 1 year of a low potency oestrogen (oestriol) use. The ability of whole saliva to aggregate and mediate bacterial adherence as well as subjective feelings of dry mouth was also examined. For comparison, the same variables were examined in nine peri- and postmenopausal, non-medicated women (reference group, 53-61 years). During hormone treatment, the labial saliva flow was significantly increased and the complaints of dry mouth reduced. Increased stimulated whole saliva flow was seen in both the hormone and reference groups. This was also true for the stimulated whole saliva buffer capacity, which was increased parallel to the flow rate. The secretion rates were generally lower in the hormone group compared to the reference group throughout the study period. Except for stimulated whole saliva, statistical analysis at baseline revealed no age-related reduction of the saliva flow rates. The ability of whole saliva to mediate aggregation of Actinomyces naeslundii was significantly decreased after hormone treatment. Thus, the present findings indicate that a low dose oestrogen (oestriol) may affect the flow rate of labial salivary glands and the bacterial aggregation activity of whole saliva.

Studies on human minor salivary gland secretions using the Periotron method.

Secretions from minor salivary glands were estimated in 127 individuals by the Periotron method of measuring fluid output from different mucosal sites, and outputs were related to different variables. Large intra- and interindividual variations in secretions (expressed as microliter/cm2 per min) were observed, with means of 0.9 for the palatal, 4.8 for the labial and 16.0 for the buccal mucosal sites. Age had no influence on the secretion rate, but women had 10-20% lower values from all three sites than men (p < 0.05). Individuals wearing upper dentures or using tobacco had 300 and 27% increased palatal secretion rates, respectively (p < 0.001, p < 0.05). In addition, those being treated with diuretics had 15% lower rates of secretion from buccal mucosal glands (p < 0.05), and those complaining of oral dryness had 21% lower fluid output from the labial mucosa (p < 0.05). These results support the use of minor salivary glands in combination with the Periotron method to study mucosal secretions and functions.

Human minor and major gland saliva proteins and ability to mediate Actinomyces naeslundii adherence.

Bacteria-binding components and the ability to mediate bacterial adhesion to the tooth surface have been thoroughly studied in major salivary gland secretions. Our knowledge on the bacteria binding activity in minor gland saliva is, however, limited. In this study, proteins were examined in parallel in minor (palatal, buccal and labial) and major (parotid and submandibular/sublingual) salivary gland secretions in one subject using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The adherence of early colonizing Actinomyces naeslundii to pellicles formed from the secretions on hydroxyapatite beads was also examined. Amylase, IgA, proline-rich proteins and the high-molecular-weight glycoproteins, agglutinins, were detected in all saliva tested. Carbohydrate-reactive antibodies recognized the low-molecular weight mucin, MUC 7 in submandibular/sublingual saliva only. A. naeslundii strain 12104 adhered to all pellicles and especially to the buccal gland saliva pellicles. Strain LY7 adhered in highest numbers to the submandibular/sublingual saliva pellicles. It also bound in considerable numbers to parotid and palatal saliva pellicles but not to the ones formed from buccal and labial gland saliva. Our findings indicate that several bacteria-binding components are secreted in both minor and major gland saliva. The adherence-promoting ability of the various gland secretions differs, however.

Candesartan, an Insurmountable Antagonist of Angiotensin II-mediated Contractile Effects in Isolated Vascular Preparations: Comparison with Irbesartan, Losartan and its Active Metabolite (EXP-3174).

Candesartan is a new angiotensin II type 1 (AT 1 ) receptor blocker that produces more effective 24-h blood pressure lowering than losartan in hypertensive patients. In in vitro tissue preparations, candesartan displays insurmountable antagonism of the responses to angiotensin II. Both irbesartan and EXP-3174, the active metabolite of losartan, have also previously been described in some studies as insurmountable AT 1 -receptor blockers, whereas losartan exhibits surmountable blockade of the AT 1 -receptor. We compared the properties of candesartan, irbesartan, losartan and EXP-3174 in isolated vascular preparations of rat portal vein and rabbit aortic strips. The concentrations of the different AT 1 -receptor antagonists that were effective in these in vitro preparations were also correlated to the non-protein bound plasma concentrations obtained in clinical use. Preparations of the rabbit aorta and the rat portal vein were dissected, mounted on a force-displacement transducer and submerged in oxygenated Krebs' buffer at 37°C. The vessel strips were pre-stretched to a passive force of 5 and 20 mN for portal vein and aorta, respectively. The response to angiotensin II, measured as the mean force development in response to increasing concentrations of angiotensin II, was recorded in the absence and presence of candesartan, 0.003-10 nmol/l, irbesartan, 1-100 nmol/l, losartan, 1-100 nmol/l, and EXP-3174, 0.01-10 nmol/l, for a period of 90 min. In rabbit aortic strips, candesartan caused a non-parallel shift and suppression of the angiotensin II concentration-response curve, with complete suppression of the response to angiotensin II at a dose of candesartan of 1 nmol/l. In contrast, irbesartan, losartan and EXP-3174 all caused a parallel shift of the concentration-response curve. No suppression of the angiotensin II response was seen with losartan, while its active metabolite caused saturable suppression of the maximal response at higher concentrations. For irbesartan, some degree of suppression of the maximal response could not be excluded at the highest concentration studied. Similar concentration-response curves were obtained in rat portal vein. Data on protein binding for the different AT 1 -receptor blockers are variable in the literature. Plasma protein binding for the different AT 1 -receptor blockers was determined (in triplicate) by liquid chromatography with fluorescence detection after equilibrium dialysis (6 h) of cold drug at a concentration of 1500 nmol/l. Protein binding was high (see Table) and, for candesartan, losartan and its active metabolite EXP-3174, in accordance with previously reported levels. For irbesartan, a large discrepancy in protein binding between previously reported and the present experimental data, obtained from two different non-associated laboratories, was found. The higher plasma protein binding for irbesartan found in the present study may explain why high doses of irbesartan seem to be needed for clinical efficacy. It appears unlikely that losartan exerts any significant inhibitory effect at therapeutic plasma levels, and the main AT 1 -blocking effect observed after oral losartan is probably exerted by EXP-3174. It is concluded that AT 1 -receptor blockers differ in their ability to inhibit angiotensin II-mediated vascular contraction, and that the antagonistic characteristics are similar in vessel preparations of different origins and with different degrees of AT 1 -receptor reserve.

A study on primiparous sows of the ability to show standing oestrus and to ovulate after weaning. Influences of loss of body weight and backfat during lactation and of litter size, litter weight gain and season.

The ability to show standing oestrus and to ovulate within 10 days of weaning was studied in 240 purebred Swedish Yorkshire primiparous sows, fed according to a conventional feeding regime during lactation. The sows were weighted and backfat depth was recorded at farrowing and at weaning. Oestrus control was performed daily and blood samples for determination of plasma progesterone were drawn regularly in 205 sows. The distribution among the sows of the first standing oestrus after weaning had 2 peaks. The first peak occurred within 10 days of weaning and the second 24-30 days after weaning. Twelve per cent of the sows ovulated without showing standing oestrus within 10 days of weaning and 4% had an anovulatory first oestrus within the same time. Significant differences in age at farrowing and in loss of weight and backfat during lactation were found between sows which both showed standing oestrus and ovulated within 10 days of weaning and sows which neither showed standing oestrus, nor ovulated within the same time. The season during which weaning occurred significantly influenced the ability to show standing oestrus and ovulate within 10 days of weaning. Among the sows which both showed standing oestrus and ovulated within 10 days of weaning, significant positive correlations were found between weight loss, litter size, litter weight gain and the interval from weaning to first standing oestrus.