Chronic K-supplementation decreases myocardial [Na,K-ATPase] and net K-uptake capacity in rodents

The effects of high K intake on plasma K, myocardial K content and Na,K-ATPase concentration and on myocardial K uptake during KCl infusion were evaluated in rodents. Myocardial Na,K-ATPase was quantified in crude homogenates by K-dependent pNPPase activity in rats, and in intact samples by3H-ouabain binding in guinea pigs. Na, K-ATPase alpha isoform distribution was assessed by immunoblotting. Plasma K was monitored in anesthetized rats during intravenous infusion of 0.75 mmol KCl/100 g body weight/h. A significant increase in plasma K was observed after 2 days of K supplementation, 4.9+/-0.2 (mean+/-s.e.m.)v 3.0+/-0.2 mmol/l in weight matched controls ( P<0.01,n=5) and this difference remained stable. After 1 day, a significant myocardial K content increase was obtained, 86. 2+/-3.0v 76.7+/-1.9 micromol/g wet weight (P<0.05, n=5); after 4 days myocardial K stabilized 4.9+/-1.2 micromol/g wet weight above control level (P<0.05,n=5). From the 4th day, a significant decrease in myocardial K-dependent pNPPase activity was observed, 1.18+/-0.04v 1. 31+/-0.01 micromol/min/g wet weight in weight matched controls (P<0. 05,n=5); after 2 weeks the decrease was 29% (P<0.05,n=5), with a reduction in alpha1-isoform abundance by 24% (P<0.05,n=5), and a tendency to a decrease in alpha2 of 10% (n.s.,n=5). The measurements were validated by 3H-ouabain binding to myocardial samples from guinea pigs K-supplemented for 2 weeks, showing a decrease of 21% (P<0.05,n=5). During KCl infusion, the myocardial K content increase rate was reduced by 52% (P<0.05) in the K-supplemented rats. The observed effects of K-supplementation on plasma K, myocardial K content and myocardial K-dependent pNPPase activity were abolished within 2 days after reallocation to chow with normal K content. In conclusion, high K-intake is associated with significantly and reversible increased plasma and myocardial K content, and decreased myocardial Na,K-ATPase concentration and net myocardial K uptake capacity. Thus, the heart is protected from major increases in intracellular K concentrations during chronically-high K-intake.     

Prolongation of allograft survival by 1,25-dihydroxyvitamin D3

BACKGROUND: 1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, is now believed to play a significant role in the immune responses, both in vitro and in vivo, preventing the development of several autoimmune diseases. These studies suggest that 1,25-dihydroxyvitamin D3 may be effective in prolonging allograph survival. METHODS: To test the hypothesis that 1,25-dihydroxyvitamin D3 would prolong allograft survival, neonatal heart grafts were transplanted to allogeneic recipients receiving either 19-nor-1,25-dihydroxyvitamin D2 (200 ng/day) or 1,25-dihydroxyvitamin D3 (50 ng/mouse/day) orally through the diet. The efficacy of 1,25-dihydroxyvitamin D3 in prolonging graft survival in a vascularized model was determined by heterotopic ACI to Lewis heart transplants. RESULTS: The provision of exogenous 1,25-dihydroxyvitamin D3 or an analog, 19-nor-1,25-dihydroxyvitamin D2, to mice markedly prolonged the survival of neonatal mouse heart allografts. Similar results were obtained with a vascularized heterotopic heart transplant model in rats. Cyclosporine at a maximum 25 mg/kg dose for mice proved less effective than 1,25-dihydroxyvitamin D3. Graft survival in mice differing at class I and class II loci (B10.A(4R) --> C57BL/10) increased from 13.0+/-1.1 days to 51.0+/-5.6 days and was significantly better than cyclosporine monotherapy (33.2+/-3.6). Rat heart survival in a high responder strain combination (ACI --> Lewis) increased from 6.2+/-0.3 to 25.2+/-2.8 days. The increased survival of the transplants brought about with 1,25-dihydroxyvitamin D3 was not accompanied by hypercalcemia in rats. CONCLUSION: These results suggest that 1,25-dihydroxyvitamin D3 can be used as an effective agent in preventing graft rejection.     

Determination of aspirin and salicylic acid in transdermal perfusates

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of aspirin and salicylic acid in transdermal perfusates. The compounds were separated on a C8 Nucleosil column (5 microm, 250x4.6 mm) using a mobile phase containing a mixture of water-acetonitrile-orthophosphoric acid (650:350:2, v/v/v) and a flow-rate of 1 ml/min. The transdermal samples were in phosphate-buffered saline (PBS) and could be injected directly onto the HPLC system. The method was reproducible with inter-day R.S.D. values of no greater than 3.46 and 2.60% for aspirin and salicylic acid, respectively. The method was linear over the concentration range 0.2-5.0 microg/ml and had a limit of detection of 0.05 microg/ml for both compounds. For certain samples, it was necessary to ensure that no transmembrane leakage of the aspirin prodrugs had occurred. In these cases, a gradient was introduced by increasing the acetonitrile content of the mobile phase after the salicylic acid had eluted. The method has been applied to the determination of aspirin and salicylic acid in PBS following in vitro application of the compounds to mouse skin samples.     

Development of orally active oxytocin antagonists: studies on 1-(1-[4-[1-(2-methyl-1-oxidopyridin-3-ylmethyl)piperidin-4-yloxy]-2- methoxybenzoyl]piperidin-4-yl)-1,4-dihydrobenz[d][1,3]oxazin-2-one (L-372,662) and related pyridines

The previously reported oxytocin antagonist L-371,257 (2) has been modified at its acetylpiperidine terminus to incorporate various pyridine N-oxide groups. This modification has led to the identification of compounds with improved pharmacokinetics and excellent oral bioavailability. The pyridine N-oxide series is exemplified by L-372,662 (30), which possessed good potency in vitro (Ki = 4.1 nM, cloned human oxytocin receptor) and in vivo (intravenous AD50 = 0.71 mg/kg in the rat), excellent oral bioavailability (90% in the rat, 96% in the dog), good aqueous solubility (>8.5 mg/mL at pH 5.2) which should facilitate formulation for iv administration, and excellent selectivity against the human arginine vasopressin receptors. Incorporation of a 5-fluoro substituent on the central benzoyl ring of this class of oxytocin antagonists enhanced in vitro and in vivo potency but was detrimental to the pharmacokinetic profiles of these compounds. Although lipophilic substitution around the pyridine ring of compound 30 gave higher affinity in vitro, such substituents were a metabolic liability and caused shortfalls in vivo. Two approaches to prevent this metabolism, addition of a cyclic constraint and incorporation of trifluoromethyl groups, were examined. The former approach was ineffective because of metabolic hydroxylation on the constrained ring system, whereas the latter showed improvement in plasma pharmacokinetics in some cases.     

Reduced beta-adrenergic sensitivity in patients with type 1 diabetes and hypoglycemia unawareness

OBJECTIVE: We tested the hypothesis that impaired tissue sensitivity to catecholamines contributes to hypoglycemia unawareness in subjects with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 21 subjects with type 1 diabetes underwent a standardized insulin infusion protocol to produce a stepwise decrease in plasma glucose to 45-min plateaus of 4.3, 3.6, 3.0, and 2.3 mmol/l. Glycemic thresholds, maximum responses for adrenergic and neuroglycopenic symptoms, and counterregulatory hormones were determined. Patients were classified as hypoglycemia unaware if the initiation of adrenergic symptoms occurred at a plasma glucose level 2 SD below that of nondiabetic volunteers. beta-Adrenergic sensitivity was measured as the dose of isoproterenol required to produce an increment in heart rate of 25 beats per minute above baseline (I25) in resting subjects. RESULTS: Subjects with type 1 diabetes and hypoglycemia unawareness experienced the onset of adrenergic symptoms at a lower plasma glucose level than did those with awareness (2.5+/-0.1 vs. 3.7+/-0.1 mmol/l, P < 0.001), whereas neuroglycopenic symptoms occurred at similar glucose levels (2.7+/-0.2 vs. 2.8+/- 0.1 mmol/l). The plasma glucose levels for counterregulatory hormone secretion (epinephrine 2.9+/-0.2 vs. 4.1+/-0.2 mmol/l; norepinephrine 2.7+/-0.1 vs. 3.2+/-0.2 mmol/l; cortisol 2.5+/-0.2 vs. 3.3+/-0.2 mmol/l, P < 0.01) were also lower in subjects with unawareness. The maximal epinephrine (1,954+/-486 vs. 5,332+/- 1,059 pmol/l, P < 0.01), norepinephrine (0.73 +/- 0.14 vs. 1.47+/-0.21 nmol/l, P = 0.04), and cortisol (276+/-110 vs. 579+/-83 nmol/l, P < 0.01) responses were reduced in the unaware group. I25 was greater in unaware subjects than in subjects without unawareness (1.5+/-0.3 vs. 0.8+/-0.2 microg), where I25 was not different from that of controls (0.8 +/-0.2 microg). CONCLUSIONS: We conclude that subjects with type 1 diabetes and hypoglycemia unawareness have reduced beta-adrenergic sensitivity, which may contribute to their impaired adrenergic warning symptoms during hypoglycemia.     

Differential item functioning in the Danish translation of the SF-36

Statistical analyses of Differential Item Functioning (DIF) can be used for rigorous translation evaluations. DIF techniques test whether each item functions in the same way, irrespective of the country, language, or culture of the respondents. For a given level of health, the score on any item should be independent of nationality. This requirement can be tested through contingency-table methods, which are efficient for analyzing all types of items. We investigated DIF in the Danish translation of the SF-36 Health Survey, using two general population samples (USA, n = 1,506; Denmark, n = 3,950). DIF was identified for 12 out of 35 items. These results agreed with independent ratings of translation quality, but the statistical techniques were more sensitive. When included in scales, the items exhibiting DIF had only a little impact on conclusions about cross-national differences in health in the general population. However, if used as single items, the DIF items could seriously bias results from cross-national comparisons. Also, the DIF items might have larger impact on cross-national comparison of groups with poorer health status. We conclude that analysis of DIF is useful for evaluating questionnaire translations.     

Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage

The relative contributions of protein tyrosine kinases (PTKs) and protein kinase C isoenzymes (PKCs), a family of serine/threonine kinases, in integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa) exposure are the subject of much controversy. In the present study we measured the effect of the PTK inhibitor herbimycin A and the PKC inhibitor bisindolylmaleimide I on 125I-fibrinogen binding to alpha(IIb)beta3 and on aggregation/secretion induced by different agonists. Dose-response studies showed complete inhibition of alpha(IIb)beta3 exposure by 30 micromol/L (ADP stimulation) and 35 to 40 micromol/L (alpha-thrombin stimulation) herbimycin A. In contrast, inhibition of exposure by bisindolylmaleimide I varied from none (for ADP and epinephrine), to 30% (for platelet-activating factor), and to approximately 80% (for alpha-thrombin). Studies with a submaximal dose of herbimycin A (approximately 50% inhibition of the ADP-response) and a maximal dose of bisindolylmaleimide I showed that optical aggregation had a similar sensitivity to the inhibitors as alpha(IIb)beta3 exposure with minimal interference by secreted ADP. Thus, the relative contributions of tyrosine and serine/threonine kinases in alpha(IIb)beta3 exposure and aggregation differ among the different agonists, with an exclusive role for PTKs in ADP- and epinephrine-induced responses and a role for both PTKs and PKCs in responses induced by platelet-activating factor and alpha-thrombin.     

Membrane-permeant esters of phosphatidylinositol 3,4,5-trisphosphate

Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A reliable means to introduce these lipids into intact cells would be of great value for showing the physiological roles of this pathway and for testing the specificity of pharmacological inhibitors of the kinases. We have stereospecifically synthesized di-C8-PIP3/AM and di-C12-PIP3/AM, the heptakis(acetoxymethyl) esters of dioctanoyl- and dilauroylphosphatidylinositol 3,4,5-trisphosphate, in 14 steps from myo-inositol. The ability of these uncharged lipophilic derivatives to deliver phosphatidylinositol 3,4,5-trisphosphate across cell membranes was demonstrated on 3T3-L1 adipocytes and T84 colon carcinoma monolayers. Insulin stimulation of hexose uptake into adipocytes was inhibited by the kinase inhibitor wortmannin and was largely restored by di-C8-PIP3/AM, which had no effect in the absence of insulin. Thus phosphatidylinositol 3,4,5-trisphosphate or a metabolite was necessary but not sufficient for stimulation of hexose transport. In T84 epithelial monolayers, di-C12-PIP3/AM mimicked epidermal growth factor in inhibiting chloride secretion and potassium efflux, suggesting that phosphatidylinositol 3,4, 5-trisphosphate was sufficient to modulate these fluxes and mediate epidermal growth factor's action.