Neuronal expression of an FMRFamide-gated Na+ channel and its modulation by acid pH.

The molluscan Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channels (FaNaCs) show both structural and functional similarities to the mammalian acid-sensing ion channels (ASICs). Both channel types are related to the epithelial sodium channels and, although the neuropeptide FMRFamide directly gates the FaNaCs, it also modulates the proton-gating properties of ASICs. It is not yet known whether protons can alter the gating properties of the FaNaCs. We chose to examine this possibility at a site of FaNaC expression in the nervous system of the mollusk Lymnaea stagnalis. We cloned a putative L. stagnalis FaNaC (LsFaNaC) that exhibited a high degree of sequence identity to the Helix aspersa FaNaC (HaFaNaC, 60%), and a weaker homology to the ASICs (ASIC3, 22%). In situ hybridization was used to map the LsFaNaC expression pattern in the brain and to identify the right pedal giant1 (RPeD1) neuron as a site where the properties of the endogenous channel could be studied. In RPeD1 neurons isolated in culture, we demonstrated the presence of an FMRFamide-gated sodium current with features expected for a FaNaC: amiloride sensitivity, sodium selectivity, specificity for FMRFamide and Phe-Leu-Arg-Phe-amide (FLRFamide), and no dependency on G-protein coupling. The sodium current also exhibited rapid desensitization in response to repeated FMRFamide applications. Lowering of the pH of the bathing solution reduced the amplitude of the FMRFamide-gated inward current, while also activating an additional sustained weak inward current that was apparently not mediated by the FaNaC. Acidification also prevented the desensitization of the FMRFamide-induced inward current. The acid sensitivity of LsFaNaC is consistent with the hypothesis that FaNaCs share a common ancestry with the ASICs.

Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.

BACKGROUND: Stimulation of beta(1)- and beta(2)-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the beta(3)AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. METHODS AND RESULTS: To better understand the role of beta(3)ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human beta(3)AR (TGbeta(3) mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous beta(1)- and beta(2)ARs, isoproterenol resulted in an increase in contractility in the TGbeta(3) mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human beta(3)AR agonist L-755,507 significantly increased contractility in the TGbeta(3) mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGbeta(3) mice was investigated in terms of beta(3)AR-G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGbeta(3) mice with L-755,507 resulted in a pertussis toxin-insensitive 1.33-fold increase in [(35)S]GTPgammaS loading and a 1.6-fold increase in adenylyl cyclase activity. CONCLUSIONS: Cardiac overexpression of human beta(3)ARs results in positive inotropy only on stimulation with a beta(3)AR agonist. Overexpressed beta(3)ARs couple to G(s) and activate adenylyl cyclase on agonist stimulation.

A human gene encoding morphine modulating peptides related to NPFF and FMRFamide.

FMRFamide-related peptides have been isolated from both invertebrates and vertebrates and exhibit a wide range of biological effects in rats. We show here that in humans 2 FMRFamide-related peptides are encoded by a single gene expressed as a spliced mRNA. The larger predicted peptide (AGEGLNSQFWSLAAPQRFamide) differs from the peptide isolated from bovines (AGEGLSSPFWSLAAPQRFamide) by the substitutions of 2 amino acids. The shorter predicted peptide (NPSF, SQAFLFQPQRFamide) is 3 amino acids longer than the bovine 8 amino-acid NPFF (FLFQPQRFamide) or the human NPFF peptide isolated from serum [5], suggesting that the encoded protein is subject to cleavage by a tripeptidyl peptidase or by a novel processing mechanism. On rat spinal cord, the larger peptide is indistinguishable in activity from the equivalent bovine peptide whereas the smaller extended peptide is inactive.

Teaching patient empathy: the ED visit program.

OBJECTIVE: Residency programs only are not challenged with developing competent emergency clinicians, but should strive to develop caring, empathetic, and community-minded physicians. An exercise was designed to help residents experience emergency department (ED) visits from the patient's perspective. METHODS: This study occurred in emergency medicine residency program at an urban teaching institution with an annual ED census of 94,000. On the first day of residency orientation, each resident was given a clinical scenario and registered through triage into the ED. Nurses were blinded to the study. The study concluded when the examining physician entered the exam room. Residents were then presented with a simulated bill based on their scenario. Residents completed a survey initially and at six months. Survey ratings were measured using a 100-mm visual analog scale (VAS) (0 = not at all; 100 = a great deal). RESULTS: Twenty-five residents participated over two years. Sixty-four percent had never been an ED patient before. Median length of stay was 139 minutes. This exercise was found to improve resident empathy for patients on initial survey, 66 mm (range 16-71), and at follow-up, 66 mm (range 23-91). Residents found the exercise useful both initially, 50 mm (range 4-86), and at follow-up, 49 mm (range 15-81). Ninety-two percent of the residents thought the goals of the exercise had been met. Residents also stated the study changed their approach to patient care (45 mm, range 4-76) and made them a better physician (49 mm, range 5-80). CONCLUSIONS: The ED visit study enhanced patient empathy within residents and was useful in improving patient care attitude.

Ibuprofen overdose: the first two years of over-the-counter sales.

Experience during 14 years of prescription only use indicates that the non-steroidal anti-inflammatory drug (NSAID) ibuprofen is of low toxicity in acute overdose. In August 1983 ibuprofen was licensed for over-the-counter (OTC) use in the UK and it was recognised that this change could have an impact upon the epidemiology of analgesic overdose in this country. The London centre of the National Poisons Information Service (NPIS) began a new prospective survey of ibuprofen overdose at the time of OTC release. The first 2 years of this survey detected a marked increase in enquiries concerning ibuprofen overdose but there was no evidence to contradict the former claims of low toxicity. The importance of continued monitoring is stressed.

Renal handling of silicon in normals and patients with renal insufficiency.

We have compared the renal handling of silicon in 16 patients with renal insufficiency to 14 normal individuals. Silicon, phosphate and creatinine were measured in plasma and urine samples. The renal insufficiency group showed significant increases in plasma silicon (1.28 +/- 0.19 vs. 0.17 +/- 0.03 mg/liter), creatinine (5.19 +/- 0.85 vs. 0.89 +/- 0.03 mg/dl) and phosphate (1.33 +2- 0.11 vs. 1.07 +/- 0.4 mmol/liter). Fractional phosphate excretion was increased in the renal insufficiency group (0.55 +/- 0.07 vs. 0.14 +/- 0.01). In contrast, the fractional excretion of ultrafiltrable silicon was not significantly different between groups (0.78 +/- 0.07 vs. 0.87 +/- 0.06). It is concluded that renal insufficiency does not alter the tubular handling of silicon and that regulatory control of silicon excretion is unlikely.

Silicon accumulation in dialysis patients.

Plasma silicon measurements have been obtained in patients with end-stage renal disease on chronic dialysis therapy. The mean +/- SE values for normal plasma silicon concentrations are .15 +/- .02 mg/L. All dialysis groups showed marked elevations in their plasma silicon that correlated with the silicon content of their respective dialysis fluids. The values of two different hemodialysis groups and a peritoneal dialysis group were 4.6 +/- .4, 2.5 +/- .2, and 1.9 +/- 1.2 mg/L, respectively. The silicon content of their respective dialysis fluids were 4.0 +/- .7, 0.5 +/- .4, and 0 +/- .1 mg/L. The ultrafiltrability of plasma silicon through Cuprophane membranes was 79 +/- 2%. Hemodialysis patients drinking high-silicon well water showed significantly higher plasma silicon levels than patients drinking lower silicon municipal water. It is concluded that use of dialysis fluid with elevated silicon levels and the consumption of water containing high concentrations of silicon are two important determinants of silicon levels in a dialysis population. We observed no overt effects of silicon accumulation on the health status of our dialysis patients.

The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing.

We recently characterized a novel protein, GIT1, that interacts with G protein-coupled receptor kinases and possesses ADP-ribosylation factor (ARF) GTPase-activating protein activity. A second ubiquitously expressed member of the GIT protein family, GIT2, has been identified in data base searches. GIT2 undergoes extensive alternative splicing and exists in at least 10 and potentially as many as 33 distinct forms. The longest form of GIT2 is colinear with GIT1 and shares the same domain structure, whereas one major splice variant prominent in immune tissues completely lacks the carboxyl-terminal domain. The other 32 potential variants arise from the independent alternative splicing of five internal regions in the center of the molecule but share both the amino-terminal ARF GTPase-activating protein domain and carboxyl-terminal domain. Both the long and short carboxyl-terminal variants of GIT2 are active as GTPase-activating proteins for ARF1, and both also interact with G protein-coupled receptor kinase 2 and with p21-activated kinase-interacting exchange factors complexed with p21-activated kinase but not with paxillin. Cellular overexpression of the longest variant of GIT2 leads to inhibition of beta(2)-adrenergic receptor sequestration, whereas the shortest splice variant appears inactive. Although GIT2 shares many properties with GIT1, it also exhibits both structural and functional diversity due to tissue-specific alternative splicing.

beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking.

The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling.

Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.

Myomodulin gene of Lymnaea: structure, expression, and analysis of neuropeptides.

The myomodulin family of neuropeptides is an important group of neural cotransmitters in molluscs and is known to be present in the neural network that controls feeding behavior in the snail Lymnaea. Here we show that a single gene encodes five structurally similar forms of myomodulin: GLQMLRLamide, QIPMLRLamide, SMSMLRLamide, SLSMLRLamide, and PMSMLRLamide, the latter being present in nine copies. Analysis of the organization of the gene indicates that it is transcribed as a single spliced transcript from an upstream promoter region that contains multiple cAMP-responsive elements, as well as putative elements with homology to tissue-specific promoter-binding sites. The presence in nervous tissue of two of the peptides, GLQMLRLamide and PMSMLRLamide, is confirmed by mass spectrometry. In situ hybridization analysis indicates that the gene is expressed in specific cells in all ganglia of the CNS of Lymnaea, which will allow physiological analysis of the function of myomodulins at the level of single identified neurons.

Small cardioactive peptide gene: structure, expression and mass spectrometric analysis reveals a complex pattern of co-transmitters in a snail feeding neuron.

The small cardioactive peptides (SCPs) are an important group of neural cotransmitters in molluscs where they are known to play both central and peripheral modulatory roles in the control of feeding behaviour. Here we show that in the snail Lymnaea the SCP gene exists in one interrupted copy that produces a single species of transcript which encodes a prepropeptide containing two structurally related SCPs SGYLAFPRMamide (SCP(A)) and pQNYLAFPRMamide (SCP(B)). In situ hybridization was used to localize expression specifically to the soma of several types of motoneurons in the feeding system of Lymnaea, including the giant B2 foregut motoneurons. The peptide content of individual B2 cell bodies was analysed by matrix-assisted laser desorption/ionization mass spectrometry and the structures of the SCPs predicted from the cloned gene were confirmed in these cells by post-source decay fragmentation analysis. Identical stimulatory activity for the two SCP peptides was demonstrated by their application to the isolated foregut, suggesting that their co-release from the B2 cells may play an important part in the co-modulation of gut motility, together with acetylcholine and the myomodulin family of peptides.

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79.

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.

Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity.

Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.

Isolation and expression of a cDNA coding for rat kidney cytosolic cysteine conjugate beta-lyase.

The role of rat kidney cysteine conjugate beta-lyase in the production of nephrotoxic thiols from S-cysteine conjugates of xenobiotics has been well established. However, the factors controlling the cellular distribution and substrate specificity of the enzyme have yet to be elucidated. As an approach to this we have isolated a cDNA for cysteine conjugate beta-lyase from a rat kidney cDNA library, using a combination of immunological and hybridization screening. A full length cDNA was sequenced and its identity was confirmed by deduced molecular weight, deduced amino acid composition, the presence of a consensus pyridoxal phosphate (PLP) binding site in the deduced amino acid sequence, kidney-specific expression of the corresponding mRNA, and the expression of beta-lyase and glutamine transaminase K activities in tissue culture cells transfected with the cDNA. The cDNA coded for a protein of 48 kDa containing the sequence Ser-Ala-Gly-Lys-Ser-Phe, which corresponds closely to the PLP binding site in other PLP-containing enzymes. Use of the cDNA to detect beta-lyase mRNA sequences in rat liver and kidney RNA demonstrated that expression was kidney specific and that the mRNA size (2.1 kilobases) was in good agreement with the size of the cDNA. When the cDNA was inserted into the expression vector pUS1000 and transfected into COS-1 tissue culture cells, a 7-10-fold increase in cytosolic beta-lyase and glutamine transaminase K activities could be detected. The use of beta-lyase cDNA for the elucidation of the mechanism of action of this enzyme and for the development of in vitro systems to examine xenobiotic cysteine conjugate toxicity is discussed.

Neural modulation of gut motility by myomodulin peptides and acetylcholine in the snail Lymnaea.

Families of peptide neuromodulators are believed to play important roles in neural networks that control behaviors. Here, we investigate the expression and role of one such group of modulators, the myomodulins, in the feeding system of Lymnaea stagnalis. Using a combination of in situ hybridization and antibody staining, expression of the myomodulin gene was confirmed in a number of identified behaviorally significant neuronal types, including the paired B2 motor neurons. The B2 cells were shown to project axons to the proesophagus, where they modulate foregut contractile activity. The presence of the five myomodulin peptide structures was confirmed in the B2 cells, the proesophagus, and the intervening nerve by mass spectrometry. Using a sensitive cell culture assay, evidence that the B2 cells are cholinergic also is presented. Application of four of the five myomodulin peptides to the isolated foregut increased both contraction frequency and tonus, whereas the main effect of acetylcholine (ACh) application was a large tonal contraction. The fifth myomodulin peptide (pQIPMLRLamide) appeared to have little or no effect on gut motility. Coapplication of all five myomodulin peptides gave a greater increase in tonus than that produced by the peptides applied individually, suggesting that corelease of the peptides onto the gut would produce an enhanced response. The combined effects that the myomodulin peptides and ACh have on foregut motility can mimic the main actions of B2 cell stimulation.

Reducing reactions to blood donation with applied muscle tension: a randomized controlled trial.

BACKGROUND: Unpleasant blood donation-related symptoms may discourage otherwise healthy, altruistic individuals from becoming repeat donors. This study examined a behavioral technique called applied muscle tension (AMT) that might reduce reactions. STUDY DESIGN AND METHODS: A total of 605 donors at mobile clinics were assigned to either an AMT treatment condition, a no-treatment control condition, or a placebo control condition. AMT involves repeated tensing of major muscles and was taught using an instructional video. Participants in the placebo control group watched the same video but were told to practice the technique only from the time they got on the donation chair until insertion of the needle, without being told that reactions are unlikely during this period. RESULTS: There were no differences between men assigned to the three conditions. Women donors assigned to the AMT condition reported significantly fewer donation-related symptoms, required less chair reclining for reactions, and were more likely to produce a full unit of blood than women in both the no-treatment and placebo control conditions. Women in the AMT condition also said they would be more likely to recommend it to a friend who was going to give blood, but there were no significant effects of AMT on the rated probability of giving blood again or blood pressure change. CONCLUSIONS: Although it was not universally effective and the mechanisms of its effects are unclear, AMT is a simple behavioral technique that may be useful in reducing reactions to blood donation.

On-site training in applied muscle tension to reduce vasovagal reactions to blood donation.

Vasovagal reactions significantly complicate the blood collection process and, more importantly, discourage people who might otherwise donate blood many times from returning. Applied muscle tension is a simple behavioral technique that may reduce vasovagal reactions by maintaining blood pressure. It has been successfully used to treat patients with blood and injury phobias, but has not been applied in the more general, time-limited context of blood collection clinics. Thirty-seven inexperienced blood donors (maximum number of prior donations = 2) attending mobile blood collection clinics were asked to practice applied tension after watching a 2-min instructional video presented on a notebook computer. They were compared with 94 untreated donors with similar donation experience and 47 more experienced blood donors. Treatment reduced the number of symptoms reported on a postdonation questionnaire. It also significantly reduced the amount of medical treatment required (chair reclining) among those who practiced applied tension for the entire period they were in the donation chair.