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Infrared beak treatment (IRBT) results in a change in beak shape; however, it is unclear what effect variations in post-treatment beak shape have on young pullets. Additionally, the impact of sloughing of the treated beak tissue is not fully understood. Two experiments were conducted to determine the effects of beak tissue sloughing and post-treatment beak shape on the productivity of infrared beak-treated Lohmann Brown (LB) and Lohmann LSL-Lite (LW) pullets and hens. Birds were treated on day of hatch and IRBT equipment settings were adjusted to create 4 specific beak shapes: shovel (SHV), step (STP), standard (STAN), and an untreated sham control (C). Experiment 1 pullets (n = 160) were housed in cages from 1 to 29 d of age and had access to water through chick founts or 360° nipple drinkers (2 replicate cages per treatment). Data collected included body weight (BW), feed intake (FI), feed efficiency (FE), and water disappearance (WD). Experiment 2 pullets (n = 640) were housed in floor pens from 1 d to 18 wk of age (2 replicate pens per treatment) then conventional cages during the laying period (6 replicate cages per treatment). Data collected included BW, FI, egg production, and egg quality. Data were analyzed using PROC MIXED (SAS® 9.4) and differences were significant when P ≤ 0.05. During early life, the IRBT treatments and sloughing had minor effects on FI, FE, and BW. At 4 wk of age, STAN pullets were lighter than C pullets; however, differences were no longer apparent after this age. Pullets with STP or STAN beak shapes had lower WD than C pullets when allowed access to water via nipple drinkers but this did not result in reduced growth. Throughout the laying period, SHV hens laid more saleable eggs than C hens, with no other effects on production. Overall, variations in beak shape and sloughing of the beak tissue had minimal impacts on the productivity of LW and LB pullets and hens.

Despite previous research on the impacts of beak treatment on laying hens, little information exists regarding how variation in beak morphology that can occur following beak treatment affects production, behavior, and welfare. Following infrared beak treatment (IRBT), variations in beak shape, such as a shovel beak (bottom beak longer than top), cracks (Cr), or bubbles (B) may occur if the IRBT equipment is damaged or if a quality control program is not followed at the hatchery. This study aimed to determine if variations in beak morphology post-IRBT impacted laying hen production or welfare. Infrared beak-treated Lohmann LSL-Lite hens (n = 80) were selected from a 56-wk-old flock and randomly assigned into 1 of 8 treatments: flush beak (control), shovel beak extending 0–1 mm (SB0-1), 1–2 mm (SB1-2), 2–3 mm (SB2-3), 3–4 mm (SB3-4), or >4 mm (SB > 4), Cr, or B. Hens were housed in individual cages for 4 wk and production (body weight, feed intake, egg production, and egg quality), and welfare (behavior and histology) parameters were evaluated. Consumption of different particle sizes was assessed by measuring feed particle size of refused feed. Data were analyzed as a one-way ANOVA, in a completely randomized design using PROC GLM (SAS 9.4). The results indicated that the beak morphologies examined had minimal effects on the production or welfare of the hens. Histological assessment did not show the presence of neuromas in the beak tissue, suggesting that the hens were not experiencing chronic pain from the IRBT procedure.

This research examined how infrared beak treatment (IRBT), sloughing of the treated beak tissue, and the variations in beak shape that can occur post-IRBT impact the welfare and mortality of Lohmann LSL-Lite (LW) and Lohmann Brown (LB) pullets and hens. Two experiments were conducted and birds for both experiments were treated on the day of hatch. IRBT equipment settings were adjusted to create 4 specific beak shapes: shovel (SHV), step (STP), standard (STAN), and an untreated sham control (C). Experiment 1 pullets (n = 80 per strain) were reared in bioassay cages from 1 to 29 D of age (4 replicates per treatment). Data collected included time and presence of beak sloughing, pecking force, behavioral expression, and mortality. Experiment 2 pullets (n = 320 per strain) were reared in floor pens from 1 D to 18 wk of age (2 replicates per treatment) and then conventional cages from 18 to 60 wk of age (6 replicates per treatment). Data collected for Experiment 2 included behavioral expression, feather cover, comb damage, and mortality. Data were analyzed using PROC MIXED (SAS® 9.4) with Tukey's test to separate means. Differences were significant when P ≤ 0.05. IRBT and sloughing had no effect on pecking force or mortality throughout rearing. The variations in post-IRBT beak shape had minor effects on behavior. During rearing, STAN pullets were more active than C pullets but STP and STAN pullets performed less exploratory pecking. During the laying period, SHV and STP hens preened more than C hens. The IRBT treatments, regardless of beak shape, reduced feather loss, comb damage, and cannibalism-related mortality during the laying period. Overall, the results indicate that LW and LB pullets and hens can cope with the change in beak shape that occurs with IRBT, and that welfare is not negatively impacted if some variation in beak shape occurs.

Membrane fusion in all eukaryotic cells is regulated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. The molecular mechanisms that control this process are conserved through evolution and require several protein families, including Sec1p/Munc18 (SM) proteins. Here, we demonstrate that the mammalian SNARE protein syntaxin 16 (Sx16, also known as Syn16) is a functional homologue of the yeast SNARE Tlg2p, in that its expression fully complements the mutant phenotypes of tlg2Δ mutant yeast. We have used this functional homology to demonstrate that, as observed for Tlg2p, the function of Sx16 is regulated by the SM protein Vps45p. Furthermore, in vitro SNARE-complex assembly studies demonstrate that the N-terminal domain of Tlg2p is inhibitory to the formation of SNARE complexes, and that this inhibition can be lifted by the addition of purified Vps45p. By combining these cell-biological and biochemical analyses, we propose an evolutionarily conserved regulatory mechanism for Vps45p function. Our data support a model in which the SM protein is required to facilitate a switch of Tlg2p and Sx16 from a closed to an open conformation, thus allowing SNARE-complex assembly and membrane fusion to proceed.

Sec1p/Munc18 (SM) proteins play a key role in the regulation of soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptor (SNARE)-mediated intracellular membrane trafficking events in all eukaryotic cells. Understanding the molecular mechanisms by which SM proteins function has not been straight forward as SM proteins bind to their cognate SNARE proteins by at least two distinct mechanisms, suggesting that they provide more than one function. We have previously characterised two binding modes used by the yeast SM protein Vps45p to interact with its SNARE proteins. In one of these modes, the N terminus of the syntaxin Tlg2p inserts into a hydrophobic pocket in the SM protein. We now report that disruption of this high-affinity binding between Vps45p and Tlg2p leads to downregulation of Tlg2p, and propose that this pocket-mode of binding of SM proteins to their cognate syntaxins serves to regulate cellular levels of the syntaxin.

Increasing numbers of cougars (Puma concolor) have been documented in Great Plains and Midwestern states in recent years. This includes numerous reports of cougars in Michigan; however, evidence of a natural population has not been obtained. We conducted track surveys and operated camera stations from November 2004 through April 2005 in Sleeping Bear Dunes National Lakeshore (SBDNL), Michigan. We obtained no images of cougars during 863 camera nights and did not observe cougar tracks or associated sign (e.g., scats, hair, kill sites) during 493 km of track surveys. Presence of 10 carnivore species, including bobcat (Lynx rufus), coyote (Canis latrans), and domestic dog (Canis familiaris) was documented. We also investigated four cougar sightings and one report of cougar tracks within and adjacent to SBDNL. We were unable to locate any cougar sign at the sighting locations but did observe sign of coyote and bobcat. A domestic dog made the reported cougar tracks. We found no evidence supporting the...

Severe head injury in childhood is associated with considerable mortality and morbidity. In this study we determined age-related differences in the relationship between outcome and intracranial pressure (ICP) and cerebral perfusion pressure (CPP) in the first 6 h of monitoring in a large cohort of head-injured children. Two hundred and thirty-five head-injured children (admitted to five UK hospitals over a 15-year period) in whom intracranial pressure monitoring was clinically indicated were studied. Patients were divided into three age groups (2–6, 7–10 and 11–16 years). The sensitivity of ICP and CPP were similar. Differences were found in the specificity of ICP and CPP for each group and these were more marked for CPP. For a specificity of 50% the pressures were 53, 63 and 66 mmHg for the three age groups. There are age-related differences in the specificity of intracranial pressure and cerebral perfusion pressure in relation to outcome. These differences may be important in the clinical management of head-injured children. Thus cerebral perfusion pressures of 53, 63 and 66 mmHg should be the minimum to strive for in these three age groups respectively.

Gonadotropin-releasing hormone (GnRH) is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2) which is produced from a precursor polypeptide in hypothalamic neurons and secreted in a pulsatile fashion to stimulate the secretion of LH and FSH via its interaction with a cognate receptor on gonadotropes. Low doses of the native peptide delivered in a pulsatile manner to mimic that found in the hypothalamic portal vessels restore fertility in hypogonadal patients, and are also effective in treating cryptorchidism and delayed puberty. Administration of high doses of GnRH, or agonist analogues, causes desensitization of the gonadotrope with consequent decline in gonadal gametogenesis and steroid and peptide hormone synthesis. This phenomenon finds extensive therapeutic application in clinical medicine in a wide spectrum of disease (Table 1). In addition, GnRH analogues have promise as new generation male and female contraceptives in conjunction with steroid hormone replacement. GnRH antagonists inhibit the reproductive system through competition with endogenous GnRH for the receptor and, in view of their rapid effects, are being increasingly used for the above mentioned applications. The peptide agonists and antagonists currently available require parenteral administration, typically in the form of long-acting depots. A new generation of non-peptide GnRH antagonists are beginning to emerge which should allow oral administration and, therefore, may provide greater flexibility of dosing, lower costs and increased patient acceptance.

Aims The BTS Asthma Guidelines and NICE advise that patients discharged from hospital, following an exacerbation of their asthma, should have a personalised discharge plan. This forms part of patient education and self management. There is no standard action plan available for children currently. The lack of national guidance on designing discharge plans results in a wide variation in practice between hospitals. The BTS National Asthma Audit in 2014 identified that paediatric asthma patients at Royal Hampshire County Hospital were not routinely given an asthma discharge plan. Basingstoke Hospital had a discharge plan that needed updating. It was agreed that a HHFT-wide asthma discharge plan should be designed, agreed, implemented and re-audited. Methods Discharge plans around the region were reviewed and compared with other NHS hospitals and international examples. An asthma discharge plan was then designed and developed by a working group and sent to a wider paediatric group for comment and modification. Whilst seeming straightforward, the process was slow and required repeated reviews and discussion. The discharge plan has a traffic light system, enabling parents to wean their child’s bronchodilators, recognise deterioration, and take appropriate action. The plans are individual and can be tailored to meet patient’s needs. Results The asthma discharge plan was implemented in July 2015 and audited in November 2015, both locally and as part of the national asthma audit. User feedback was positive, stating that the plans are effective and simple to use. The use of th plans improves communication at discharge, whilst reducing time spent on typing individual discharge plans within the discharge summary. Learning points It is important to follow national best practice and important for merged organisations to follow the same guidelines. Currently there are no national guidelines on designing individualised asthma discharge plans, resulting in wide variation in guidelines both locally and between hospitals. Gaining consensus across the two hospitals with different initial practice was difficult, but was achieved with persistence and using Delphi consensus strategies. A good result was achieved and is currently being implemented. A national asthma plan template would help standardise practice across UK hospitals.

Parents often report that young children have "smelly urine" or a particular urinary odour. There is little evidence that these observations are relevant to the diagnosis of urinary tract infection (UTI).To determine whether parental reporting of smelly urine is of any relevance to the diagnosis of UTI in children less than 6 years of age.Parents whose children were having urine collected as part of their admission to a large district hospital were given a simple questionnaire to complete regarding the current smell of their child's urine. Parents were asked whether their child's urine smelled different from usual or had a particular smell. Microscopy and culture results of the child's urine were compared to their parent's questionnaire answers to see if there was a association between parental reporting of a different or particular urine smell and a diagnosis of UTI.One hundred and ten questionnaires and urine samples were obtained. Fifty two per cent of parents thought that their child's urine smelled different from usual or had a particular smell. Only 6.4% of children were diagnosed as having a UTI. There was no statistically significant association between parental reporting of abnormal urine smell and diagnosis of UTI.In determining whether a young child has a UTI, asking parents about urine smell is unlikely to be of benefit.

Surgical sterilization is the mainstay of dog and cat population control, but its use is still often limited by the costs and effort involved, especially in developing countries. An ideal non-surgical sterilant that is safe, effective, permanent, administered as a single injection and capable of being manufactured inexpensively could have a significant impact on the world-wide dog and cat overpopulation problem. One approach towards developing such an agent is the targeting of pituitary gonadotrophic cells with cytotoxic agents using gonadotropin-releasing hormone (GnRH). GnRH is a peptide that binds to high-affinity receptors selectively expressed on gonadotrophs and some types of cancers. Both small molecules and proteins have been conjugated to GnRH analogues to generate targeted cytotoxic and imaging agents. Although most of these efforts have focused on development of human cancer therapeutics, available reproductive studies in rats and dogs suggest that current compounds do not have sufficient therapeutic windows for complete gonadotroph ablation, in part owing to poor stability of peptide targeting sequences. The only reported longer-term study of gonadotroph ablation in dogs reported suppression of serum testosterone for 8 months, but endocrine function then recovered, raising important questions about the mechanism of reproductive suppression and its recovery. Although studies to date suggest that this is a potentially attractive approach to non-surgical sterilization, ideal agents are yet to be developed, and important mechanistic questions remain to be answered.

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.

A wild-type T7 virion ejects about 850 bp of the 40 kb genome into the bacterial cell by a transcription-independent process. Internalization of the remainder of the genome normally requires transcription. Inhibition of transcription-independent DNA translocation beyond the leading 850 bp is not absolute but the time taken by a population of phage genomes in overcoming the block averages about 20 minutes at 30 degrees C. There are additional blocks to transcription-independent translocation and less than 20 % of infecting DNA molecules completely penetrate the cell cytoplasm after four hours of infection. Mutant virions containing an altered gene 16 protein either prevent the blocks to transcription-independent DNA translocation or effect rapid release from blocking sites and allow the entire phage DNA molecule to enter the cell at a constant rate of about 75 bp per second. This rate is likely the same at which the leading 850 bp is ejected into the cell from a wild-type virion. All mutations fall into two clusters contained within 380 bp of the 4 kb gene 16, suggesting that a 127 residue segment of gp16 controls DNA ejection from the phage particle. We suggest that this segment of gp16 acts as a clamp to prevent transcription-independent DNA translocation.

In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K(i)0.5 nM) for the rat GnRH receptor (rGnRHR). The most favored cycles bridged the side chains of residues (4-10),(1,2) (5-8),(2) (4-10/5-8),(2) (1-3),(3) and (1-3/4-10).(3) Our goal was to identify a consensus model of bioactive conformations of GnRH antagonists, yet these biocompatible constraints did not sufficiently restrain the spatial location of the N-terminal tripeptide with respect to the C-terminal heptapeptide, due largely to the rotational freedom about the bonds connecting these regions. Examination of models derived from NMR studies of cyclo(4-10) analogues suggested a large number of possible cyclic constraints such as cyclo (0-8), (1-8), or (2-8). All analogues tested with these substitutions were inactive as antiovulatory agents at 1 mg/rat (5-9) and had low affinity for rGnRHR. On the other hand, bridging positions 3 and 8 with a [DAsp(3)] to [Dbu(8)] (12, K(i) = 13 nM) or [Orn(8)] (13, K(i) = 14 nM) in the parent compound cyclo(3-8)[Ac-DNal(1),DCpa(2),DXaa(3), Arg(5),DNal(6),Xbb(8),DAla(10)]GnRH yielded analogues that blocked ovulation at 250 microgram/rat. Analogue 14 (K(i) = 2.3 nM), with a [DAsp(3), Lys(8)] bridge, was fully active at 50 microgram/rat. Loss of potency (20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K(i) = 23 nM). Dicyclic analogues possessing the (4-10) cycle and selected (1-6), (2-6), and (2-8) cycles led to analogues that were inactive at doses of 500 microgram/rat or larger. Two analogues with (1-8/4-10) cycles (16, K(i) = 1.1 nM) or (3-8/4-10) cycles (22, K(i) = 17 nM) showed full antiovulatory potency at 250 microgram/rat. None of these substitutions yielded analogues potent enough (80% inhibition of ovulation at 5 microgram/rat or less and K(i)0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1, 1'-5/4-10) analogues met this criterion: dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (32, K(i) = 0.22 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DNal(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (34, K(i) = 0.38 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(betaAla),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (40, K(i) = 0.15 nM), and dicyclo(1, 1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (41, K(i) = 0.24 nM). Since they differed slightly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.

Background and Aims Neonatal intensive care units (NICU) across the UK use different guidelines for the treatment of neonates at risk of sepsis. However, unless specific symptoms/risk factors for sepsis are present, antibiotics are usually stopped at 48 hours if blood culture (BC) results are negative. We aimed to determine whether it would be safe to stop antibiotics at 36 hours. Methods We conducted a retrospective audit of all blood cultures over a two-year-period (2009–2011) from neonates at risk of or with suspected sepsis admitted to Winchester NICU - a medium-sized level 2 neonatal unit (3000 deliveries/year). BC were analysed with the automated BacT ALERT ® 3D Signature system, (Biomerieux, Durham, UK), using paediatric blood culture bottles (BacT/ALERT ® PF; incubated for a total of 5 days. Results A total of 402 BC were identified and included in the analysis. Eighteen were positive (4.4%). The median time to BC positivity was 14.5 hours (25 th –75 th percentile:11.5–21.5 hours). There was no significant difference between the proportion of positive BC results at 36 and 48 hours (p=0.4857; odds ratio 0.178 (95% CI: 0.008–3.995)). Only two BC were positive after 36 hours; both were considered to be contaminants and did not changed management. Conclusions Our data suggest that it is safe, in similar units using similar methods, to stop antibiotics after 36 hours if BC are negative. This would result in a substantial reduction in antibiotic use, invasive procedures and admission time, and thereby has significant implications for neonatal care.

To describe the pathophysiology and nursing care of patients with Lambert-Eaton myasthenic syndrome (LEMS), a little-known paraneoplastic syndrome.Published articles, books and book chapters, and clinical trial experience.LEMS most commonly is associated with small cell lung cancer. Clinical presentation is characterized by weakness of the proximal muscles of the pelvis, thighs, shoulders, and arms and a weakening or absence of deep tendon reflexes. These symptoms are the manifestations of an abnormality at the neuromuscular junction and may precede the diagnosis of lung cancer.The oncology nurse knowledgeable about the pathophysiology, clinical features, diagnostic evidence, and treatment of LEMS is better able to care for and teach patients with this disorder.Nurses must assist patients in adjusting not only to a diagnosis of cancer and its treatment effects but also to the symptoms of LEMS, including impaired mobility, self-care deficits, and fatigue.

Activin A is a potent growth and differentiation factor related to transforming growth factor beta. In somatotrophs, activin suppresses the biosynthesis and secretion of growth hormone (GH) and cellular proliferation. We report here that, in MtTW15 somatotrophic tumor cells, activin decreased GH mRNA levels and inhibited expression of transfected GH promoter--chloramphenicol acetyltransferase fusion genes. Deletion mapping of nucleotide sequences mediating this inhibition led to the identification of a region that has previously been characterized as binding the pituitary-specific transcription factor Pit-1/GHF-1. Characterization of nuclear factor binding to this region demonstrated that binding of Pit-1 to the GH promoter is lost on activin treatment. These results indicate that activin-induced repression of GH biosynthesis is mediated by the loss of tissue-specific transcription factor binding to the GH promoter and suggest a possible general mechanism for other activin responses, whereby activin regulates the function of other POU- or homeodomain-containing transcription factors.

In order to improve the biological potency of cyclic gonadotropin releasing hormone (GnRH) antagonists, we have synthesized analogues, the conformations of which were restrained through internal side chain/side chain amide bridges linking aspartic acid or glutamic acid and L-2,3-diaminopropionic acid or L-ornithine. A disulfide bridge linking L-cysteine residues was also introduced. Residues belonging to the bridge spanned from position 4 to positions 9 or 10. Two series of analogues were synthesized and are characterized by residues at positions 1 [Ac-D-3-(2'-naphthyl)alanine], 2 [D-(4-chlorophenyl)alanine or D-(4-fluorophenyl)alanine], 3 [D-3-(3'-pyridyl)alanine or D-tryptophan], 5 (arginine or tyrosine), and 6 [D-3-(3'-pyridyl)alanine or D-arginine], respectively. These substitutions were selected in an effort to optimize high biopotency for inhibition of luteinizing hormone secretion, minimization of histamine release activity, and high (relative) hydrophilicity. The most potent analogues in the antiovulatory assay were cyclo(4-10) [Ac-DNal1,DCpa2,DPal3,(Asp4 or Glu4),Arg5,DPal]6,Dpr10]GnRH (compounds 5 and 7), which were fully active at ca. 12.5 micrograms/rat in the first series, and cyclo(4-10)[Ac-DNal1,DFpa2,DTrp3,Asp4,DArg6++ +,Dpr10]GnRH (compound 12), which was fully active at 2.5 micrograms/rat in the second.

Characterization, a word often used in the chemical/biochemical and biological sciences, refers in each of these disciplines to distinct but certainly complementary analytical approaches to defining intrinsic properties of a particular compound or activity (including bio-, immuno-, radio- etc. activities). Here we describe some aspects of our research efforts at characterizing the decapeptide GnRH (gonadotropin hormone releasing hormone) using analogs as probes for the study of receptor binding and activation.

We have used a smooth muscle cell line isolated from rabbit carotid artery (RCA) as a model system with which to study the expression of atrial natriuretic peptide (ANP) receptors and, in addition, the receptor-mediated degradation of ANP. RCA cells bind rat alpha ANP-(1-28) reversibly at 37 C, apparently to a single class of high affinity (Kd approximately equal to 50 pM) binding sites (approximately equal to 20,000 sites per cell). Binding of rat alpha ANP-(1-28) elicits rapid accumulation of intracellular cGMP. However, the concentrations of rat alpha ANP-(1-28) and related peptides, abbreviated at the N- and C-terminals, required to stimulate cGMP synthesis are substantially greater than those required for binding. Analysis by HPLC of 125I-labeled rat alpha ANP-(1-28) bound to RCA cells at 37 C illustrates the rapid and continuous degradation of the radioiodinated rat alpha ANP-(1-28) to two radiolabeled products, one of which, 125I-labeled tyrosine is the major radiolabeled component that dissociates from the cells. Measurement of rat alpha ANP-(1-28) interaction with RCA cells by radioligand binding techniques therefore subsumes several processes. One of these processes is the rapid and continuous degradation of specifically bound ANP by these cells and perhaps also other target cells that respond to ANP.

We have used a smooth muscle cell line isolated from rabbit carotid artery (RCA) as a model system with which to study the ligand-mediated regulation of the atrial natriuretic peptide (ANP) receptor. RCA cells bind rat alpha-ANP-(1-28) semi-irreversibly at 4 degrees C, apparently to a single class of high-affinity binding sites (Kd approximately equal to 5 pM). In contrast, ANP initially bound to RCA cells at 37 degrees C is fully reversible after the cells have been cooled to 4 degrees C. Although the measured dissociation constant for binding rat alpha-ANP-(1-28) is 10-fold lower at 4 degrees C than at 37 degrees C, the maximum amount of specifically bound radioligand is equivalent at both temperatures. We infer this to be suggestive of ANP receptor recycling occurring at 37 degrees C. This hypothesis is substantiated by experiments demonstrating the rapid disappearance of ANP receptors (measured at 4 degrees C) after exposure of RCA cells to ANP at 37 degrees C. We believe this reflects a loss of cell-surface receptors that is reversible on removal of ANP from the incubation medium at 37 degrees C. The concentration dependence and rank order of potency of ANP in eliciting receptor redistribution resembles that for binding to the ANP receptor and not for eliciting cyclic GMP formation. In contrast to this striking loss of presumed cell-surface receptors, incubation of RCA cells with ANP elicits only limited desensitization of the cyclic GMP response.(ABSTRACT TRUNCATED AT 250 WORDS)