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A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M(-1)) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.

We used eight adjustable preparations in which the canine atrial tricuspid rings were cut and reconnected electronically by sensing activation on one side of the cut and pacing the other after an adjustable delay. A long delay resulted in a long cycle length (CL) and excitable gap (EG) during reentry. Decreasing delay decreased CL and EG. d-Sotalol (4 mg/l) significantly increased effective refractory period (ERP) and action potential duration with no effects on conduction time during constant 400-msec pacing. During reentry, d-sotalol increased action potential durations more than CLs, so it decreased diastolic intervals. It decreased EG by increasing ERP more than CL. Although d-sotalol increased action potential duration more at longer delays with longer CLs, showing reverse use-dependence, it terminated sustained tachycardias by increasing ERP only for the short delays when the initial EG was short. In 5 of 8 experiments, longer equilibration with d-sotalol produced fixed block at a vulnerable site, so reentry could not be induced at any delays. Fixed block could be transiently reversed by ACh and resolved after washout of d-sotalol. We conclude that d-sotalol terminated reentry by two mechanisms: 1) It terminated sustained reentry by increasing ERP when the initial EG was sufficiently short. 2) In some preparations, it caused fixed block at a vulnerable site, which prevented reentry regardless of the initial EG.

beta 1- and beta 2-adrenergic receptors coexist in atria of humans and a variety of other species. The significance of these receptors and the quantitative extent to which beta 2-adrenergic receptors contribute to the electrophysiologic effects of catecholamines acting at beta-adrenergic receptors have not been determined. In the present study, the beta 1-selective antagonist ICI 89,406 and the beta 2-selective antagonist ICI 118,551 were used to determine the relative densities of beta 1- and beta 2-adrenergic receptors on membranes prepared from dog atria and the contribution that each subtype makes to isoproterenol-induced shortening of action potential duration measured at 75% repolarization (APD75). Computer-aided nonlinear regression analysis of the inhibition of the specific binding of [125I]iodopindolol and the inhibition of isoproterenol-stimulated APD75 shortening by these antagonists showed that a two-site model fits the data better than a one-site model (P less than 0.0001). The affinity constants for each selective antagonist determined by inhibition of APD75 shortening were similar to those determined in studies of the inhibition of the specific binding of [125I]iodopindolol to beta 1- and beta 2-adrenergic receptors. beta 2-Adrenergic receptors made up approximately equal to 25% of the total number of beta-adrenergic receptors but mediated approximately equal to 50% of the electrophysiologic effect of isoproterenol. The inhibition of the binding of [125I]iodopindolol and of the APD75 shortening by propranolol, a nonselective antagonist, was best fit by a one-site model. In other experiments, dogs were treated with reserpine and atropine to eliminate complications caused by the presence of endogenous norepinephrine and acetylcholine. Results obtained with atria from these animals were similar to those obtained in studies with control atria. These data suggest that beta 2-adrenergic receptors as well as beta 1-adrenergic receptors are involved in mediating the electrophysiologic effects of catecholamines in dog atrial muscle and that both beta 1- and beta 2-adrenergic receptors are present on atrial muscle cells.

The major enzymes involved in the degradation of heme were identified in human platelets. It was determined that heme oxygenase activity levels in umbilical cord blood platelets were higher, whereas biliverdin reductase activity levels were comparable with that found in platelets from adults. In membranes prepared from adenosine diphosphate-activated platelets, UDP-glucuronic acid-dependent bilirubin conjugation was detected, whereas activity was negligible in unactivated platelets and undetected in serum and heat-inactivated platelets, and in platelets prepared from umbilical cord blood. Platelet fractions were analyzed by Western blot and shown to express heme oxygenase, biliverdin reductase, and UDP-glucuronosyltransferases, and there was concordance with known developmental profiles found in other tissues. Heme oxygenase expression was higher, whereas UGT expression was lower, in neonatal compared with adult platelets. These data suggest that platelets are involved in multiple steps of heme and bilirubin metabolism and that developmental regulation of these enzymes may be similar to that in other human tissues.

Four injections (intraperitoneal) of 3 mg/kg amphetamine (2 hr apart) produced pronounced hyperthermia and sustained decreases in dopamine levels and tyrosine hydroxylase (TH) protein levels in the striatum of 15-month-old male rats. A partial recovery of striatal dopamine levels was observed at 4 months after amphetamine. In contrast, TH mRNA and TH protein levels in the midbrain were unaffected at all time points tested up to 4 months after amphetamine treatment. The number of TH-immunopositive cells in the midbrain was also unchanged at 4 months after amphetamine, even though the number of TH-positive axons in the striatum remained dramatically decreased at this time point. Interestingly, TH-immunopositive cell bodies were observed 4 months after amphetamine in the lateral caudate/putamen, defined anteriorly by the genu of the corpus collosum and posteriorly by the junction of the anterior commissures; these striatal TH-positive cells were not observed in saline- or amphetamine-treated rats that did not become hyperthermic. In addition, low levels (orders of magnitude lower than that present in the midbrain) of TH mRNA were detected using reverse transcription-polymerase chain reaction in the striatum of these amphetamine-treated rats. Our results suggest that even though there is a partial recovery of striatal dopamine levels, which occurs within 4 months after amphetamine treatment, this recovery is not associated with increased TH gene expression in the midbrain. Furthermore, new TH-positive cells are generated in the striatum at this 4-month time point.

In adipocyte membranes, cholera toxin may ADP-ribosylate the islet-activating protein (IAP) substrate, under certain conditions. Covalent modification is maximal in the absence of a guanosine triphosphate; in the presence of 5'-guanylylimidodiphosphate, incorporation of [32P]ADP-ribose is markedly reduced. ADP-ribosylation by cholera toxin has similar functional consequences as does IAP-mediated modification, i.e. the biphasic response of isoproterenol-stimulated adenylate cyclase to GTP and the inhibition by N6-phenylisopropyladenosine is abolished, and only the stimulatory phase remains. In contrast, membranes treated with cholera toxin in the presence of GTP display both the stimulatory and inhibitory responses to GTP. The binding of the adenosine analog [3H]N6-phenylisopropyladenosine is increased in the presence of GTP. Treatment of the membranes with IAP, but not with cholera toxin in the absence of GTP, reverses this GTP effect on [3H]N6-phenylisopropyladenosine binding. However, [3H]N6-phenylisopropyladenosine binding is still sensitive to GTP in membranes treated with cholera toxin in the presence of GTP. In adipocyte and cerebral cortical membranes, the IAP substrate appears as a 39,000/41,000-Da doublet which does not appear to reflect protease activity. On two-dimensional polyacrylamide gels, these two proteins migrate with approximate pI values 6.0 and 5.6, respectively. Although both behave similarly under all conditions explored in this study, it is unknown whether both, or only one, are involved in inhibition of adenylate cyclase activity. These results extend the already striking homology between the adenylate cyclase complex and the visual system. Ni, as well as transducin, may be ADP-ribosylated by cholera toxin and by IAP, and, in all cases, there are functional consequences.

Exogenous glutamate will evoke dopamine (DA) release from striatal slices in vitro. To further characterize glutamate-evoked DA release from striatal slices, experiments were designed to: 1) determine if sufficient endogenous glutamate can be released in vitro to presynaptically mediate [3H]DA release in the absence of Mg++ and 2) reevaluate how K+ depolarization affects glutamate-evoked [3H]DA release. Removal of Mg++ to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated DA release increased 15 mM K(+)-evoked [3H]DA release to about 200% of control. The potentiation of this release was probably not mediated by NMDA receptors because it was not blocked by the glutamate receptor antagonists MK-801, 6,7-dinitroquinoxalinedione (DNQX) or kynurenate. Furthermore, the removal of Mg++ increased DA release substantially (200%) in the presence of 5 microM sulpiride and 10 microM nomifensine, indicating that DA reuptake and DA D2 autoreceptors are not primarily responsible for increased DA release. In the absence of Mg++, depolarization produced by 20 mM or greater [K+] inhibited DA released by exogenous glutamate, whereas a much higher [K+] was necessary to evoke endogenous glutamate release. In the presence of 1.5 mM Mg++, a reduction of the "Mg++ blockade" of NMDA receptors by 15 mM K+ depolarization during glutamate-evoked DA release was evaluated with and without the DA reuptake inhibitor nomifensine and the DA D2 antagonist sulpiride. DA released by K+ depolarization (Mg++ present) was markedly increased by 1 mM glutamate, but this effect was only partially reversed by kynurenate or high concentrations of either MK-801 (25 microM) or DNQX (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

The adenosine receptor of rat cerebral-cortical membranes was examined by radiation inactivation. In control membranes the receptor is distributed between high- and low-affinity states, that can be preferentially expressed by Mg2+ ions and guanine nucleotides respectively. Upon exposure to increasing doses of radiation, the high-affinity receptor decayed linearly as a function of radiation dose. This decay rate corresponded to a target size of 63,000 Da, when compared with the decay of the muscarinic cholinergic receptor that was also measured in these membranes.

Differentiating rat neurons express high levels of the protooncogene product pp60c-src, a 60-kDa tyrosine kinase of unknown function encoded by c-src. pp60c-src was found to be concentrated at least 9-fold in membranes from a subcellular fraction of nerve growth cones, the motile tips of outgrowing neuronal processes. Indirect immunofluorescence staining of cultured chick retinal explants showed pp60c-src in neuronal growth cones and processes, with the antigen particularly concentrated in growth cones of long neurites. pp60c-src in growth cone membranes was an active tyrosine-specific protein kinase with elevated tyrosine-specific protein kinase activity and reduced electrophoretic mobility characteristic of the form of pp60c-src in central nervous system neurons. pp60c-src was present at lower levels in subcellular fractions from mature rat brain but synaptosomal membranes were not enriched. Preferential localization of an active form of pp60c-src in nerve growth cone membranes and persistence of pp60c-src in mature neurons suggest that this tyrosine kinase is important in growth cone-mediated neurite extension and synaptic plasticity.

BackgroundWe redesigned a Renal Supportive Care (RSC) service in 2012 for patients with chronic kidney disease (CKD) managed without dialysis. We audited the service to evaluate impact on symptoms and care pathways.MethodsThe RSC service allows consultations over a wide geographical area and includes a RSC nurse, a Renal consultant and a Palliative Medicine consultant. The main focus is continuing care of CKD, symptom management and Advanced Care Planning.A retrospective case-note audit was performed from April 2012 until October 2014 on all known patients who had chosen non-dialytic management. Data collected included demographics, RSC input, Palliative Care Outcome Score (POS), anticipatory care plan (ACP) information and mortality data.Results98 patients were managed with non-dialytic care during the audit period. 72% (71/98) received RSC consultations. 72% (57/79) of patients who received a RSC consultation had a full assessment of symptoms using the POS with an improvement in total symptom burden from a median score of 11 to 8, between the first and last consultation (p=0.03).For patients with RSC input, 79% (56/71) had an ACP compared to 19% (5/27) without (p<0.001). Preferred Place of Care (PPC) was documented in 68% (48/71) and 26% (7/27) in each respective group (p<0.001). PPC was community in 100%. DNA CPR was documented in 84% (57/68) of patients with RSC input and 47% (8/17) without (p=0.001).During the audit, 62% (61/98) of patients died. 29% (11/38) of patients with an ACP died in an acute hospital versus 43% (10/23) of those without (p=0.25). 24% (8/34) of patients with documented PPC died in acute hospitals versus 48% (13/27) without documentation (p=0.04).ConclusionsThe Renal Supportive Care team achieved symptom management and Advanced Care Planning for many patients with CKD managed without dialysis and may help achieve preferred place of care.