Cardiac arrest in vascular surgical patients receiving anaesthetic care: an analysis from the 7th National Audit Project (NAP7) of the Royal College of Anaesthetists.

The 7th National Audit Project of the Royal College of Anaesthetists studied peri-operative cardiac arrest in the UK. We report the results of the vascular surgery cohort from the 12-month case registry, from 16 June 2021 to 15 June 2022. Anaesthesia for vascular surgery accounted for 2% of UK anaesthetic caseload and included 69 (8%) reported peri-operative cardiac arrests, giving an estimated incidence of 1 in 670 vascular anaesthetics (95%CI 1 in 520-830). The high-risk nature of the vascular population is reflected by the proportion of patients who were ASA physical status 4 (30, 43%) or 5 (19, 28%); the age of patients (80% aged > 65 y); and that most cardiac arrests (57, 83%) occurred during non-elective surgery. The most common vascular surgical procedures among patients who had a cardiac arrest were: aortic surgery (38, 55%); lower-limb revascularisation (13, 19%); and lower-limb amputation (8, 12%). Among patients having vascular surgery and who had a cardiac arrest, 28 (41%) presented with a ruptured abdominal aortic aneurysm. There were 48 (70%) patients who had died at the time of reporting to NAP7 and 11 (16%) were still in hospital, signifying poorer outcomes compared with the non-vascular surgical cohort. The most common cause of cardiac arrest was major haemorrhage (39, 57%), but multiple other causes reflected the critical illness of the patients and the complexity of surgery. This is the first analysis of the incidence, management and outcomes of peri-operative cardiac arrest during vascular anaesthesia in the UK.

The incidence of potentially serious complications during non-obstetric anaesthetic practice in the United Kingdom: an analysis from the 7th National Audit Project (NAP7) activity survey.

Complications and critical incidents arising during anaesthesia due to patient, surgical or anaesthetic factors, may cause harm themselves or progress to more severe events, including cardiac arrest or death. As part of the 7th National Audit Project of the Royal College of Anaesthetists, we studied a prospective national cohort of unselected patients. Anaesthetists recorded anonymous details of all cases undertaken over 4 days at their site through an online survey. Of 416 hospital sites invited to participate, 352 (85%) completed the survey. Among 24,172 cases, 1922 discrete potentially serious complications were reported during 1337 (6%) cases. Obstetric cases had a high reported major haemorrhage rate and were excluded from further analysis. Of 20,996 non-obstetric cases, 1705 complications were reported during 1150 (5%) cases. Circulatory events accounted for most complications (616, 36%), followed by airway (418, 25%), metabolic (264, 15%), breathing (259, 15%), and neurological (41, 2%) events. A single complication was reported in 851 (4%) cases, two complications in 166 (1%) cases and three or more complications in 133 (1%) cases. In non-obstetric elective surgery, all complications were 'uncommon' (10-100 per 10,000 cases). Emergency (urgent and immediate priority) surgery accounted for 3454 (16%) of non-obstetric cases but 714 (42%) of complications with severe hypotension, major haemorrhage, severe arrhythmias, septic shock, significant acidosis and electrolyte disturbances all being 'common' (100-1000 per 10,000 cases). Based on univariate analysis, complications were associated with: younger age; higher ASA physical status; male sex; increased frailty; urgency and extent of surgery; day of the week; and time of day. These data represent the rates of potentially serious complications during routine anaesthesia care and may be valuable for risk assessment and patient consent.

Peri-operative cardiac arrest: management and outcomes of patients analysed in the 7th National Audit Project of the Royal College of Anaesthetists.

The 7th National Audit Project of the Royal College of Anaesthetists studied peri-operative cardiac arrest in the UK, a topic of importance to patients, anaesthetists and surgeons. We report the results of the 12-month registry phase, from 16 June 2021 to 15 June 2022, focusing on management and outcomes. Among 881 cases of peri-operative cardiac arrest, the initial rhythm was non-shockable in 723 (82%) cases, most commonly pulseless electrical activity. There were 665 (75%) patients who survived the initial event and 384 (52%) who survived to hospital discharge. A favourable functional outcome (based on modified Rankin Scale score) was reported for 249 (88%) survivors. Outcomes varied according to arrest rhythm. The highest rates of survival were seen for bradycardic cardiac arrests with 111 (86%) patients surviving the initial event and 77 (60%) patients surviving the hospital episode. The lowest survival rates were seen for patients with pulseless electrical activity, with 312 (68%) surviving the initial episode and 156 (34%) surviving to hospital discharge. Survival to hospital discharge was worse in patients at the extremes of age with 76 (40%) patients aged > 75 y and 9 (45%) neonates surviving. Hospital survival was also associated with surgical priority, with 175 (88%) elective patients and 176 (37%) non-elective patients surviving to discharge. Outcomes varied with the cause of cardiac arrest, with lower initial survival rates for pulmonary embolism (5, 31%) and bone cement implantation syndrome (9, 45%), and hospital survival of < 25% for pulmonary embolism (0), septic shock (13, 24%) and significant hyperkalaemia (1, 20%). Overall care was rated good in 464 (53%) cases, and 18 (2%) cases had overall care rated as poor. Poor care elements were present in a further 245 (28%) cases. Care before cardiac arrest was the phase most frequently rated as poor (92, 11%) with elements of poor care identified in another 186 (21%) cases. These results describe the management and outcomes of peri-operative cardiac arrest in UK practice for the first time.

Peri-operative cardiac arrest: epidemiology and clinical features of patients analysed in the 7th National Audit Project of the Royal College of Anaesthetists.

The 7th National Audit Project of the Royal College of Anaesthetists studied peri-operative cardiac arrest in the UK, a topic of importance to patients, anaesthetists and surgeons. Here we report the results of the 12-month registry, from 16 June 2021 to 15 June 2022, focusing on epidemiology and clinical features. We reviewed 881 cases of peri-operative cardiac arrest, giving an incidence of 3 in 10,000 anaesthetics (95%CI 3.0-3.5 per 10,000). Incidence varied with patient and surgical factors. Compared with denominator survey activity, patients with cardiac arrest: included more males (56% vs. 42%); were older (median (IQR) age 60.5 (40.5-80.5) vs. 50.5 (30.5-70.5) y), although the age distribution was bimodal, with infants and patients aged > 66 y overrepresented; and were notably more comorbid (73% ASA physical status 3-5 vs. 27% ASA physical status 1-2). The surgical case-mix included more weekend (14% vs. 11%), out-of-hours (19% vs. 10%), non-elective (65% vs. 30%) and major/complex cases (60% vs. 28%). Cardiac arrest was most prevalent in orthopaedic trauma (12%), lower gastrointestinal surgery (10%), cardiac surgery (9%), vascular surgery (8%) and interventional cardiology (6%). Specialities with the highest proportion of cases relative to denominator activity were: cardiac surgery (9% vs. 1%); cardiology (8% vs. 1%); and vascular surgery (8% vs. 2%). The most common causes of cardiac arrest were: major haemorrhage (17%); bradyarrhythmia (9%); and cardiac ischaemia (7%). Patient factors were judged a key cause of cardiac arrest in 82% of cases, anaesthesia in 40% and surgery in 35%.

The 7th National Audit Project (NAP7) baseline survey of individual anaesthetists: preparedness for and experiences of peri-operative cardiac arrest.

The Royal College of Anaesthetists' 7th National Audit Project baseline survey assessed knowledge, attitudes, practices and experiences of peri-operative cardiac arrests among UK anaesthetists and Anaesthesia Associates. We received 10,746 responses, representing a 71% response rate. In-date training in adult and paediatric advanced life support was reported by 9646 (90%) and 7125 (66%) anaesthetists, respectively. There were 8994 (84%) respondents who were confident in leading a peri-operative cardiac arrest, with males more confident than females, but only 5985 (56%) were confident in leading a debrief and 7340 (68%) communicating with next of kin. In the previous two years, 4806 (46%) respondents had managed at least one peri-operative cardiac arrest, of which 321 (7%) and 189 (4%) of these events involved a child or an obstetric patient, respectively. Respondents estimated the most common causes of peri-operative cardiac arrest to be hypovolaemia, hypoxaemia and cardiac ischaemia, with haemorrhage coming fifth. However, the most common reported causes for the most recently attended peri-operative cardiac arrest were haemorrhage; (927, 20%); anaphylaxis (474, 10%); and cardiac ischaemia (397, 9%). Operating lists or shifts were paused or stopped after 1330 (39%) cardiac arrests and 1693 (38%) respondents attended a debrief, with 'hot' debriefs most common. Informal wellbeing support was relatively common (2458, 56%) and formal support was uncommon (472, 11%). An impact on future care delivery was reported by 196 (4%) anaesthetists, most commonly a negative psychological impact. Management of a peri-operative cardiac arrest during their career was reported by 8654 (85%) respondents. The overall impact on professional life was more often judged positive (2630, 30%) than negative (1961, 23%), but impact on personal life was more often negative.

Organisation of UK hospitals and anaesthetic departments in the treatment of peri-operative cardiac arrest: an analysis from the 7th National Audit Project (NAP7) local co-ordinator baseline survey.

We report the results of the Royal College of Anaesthetists' 7th National Audit Project organisational baseline survey sent to every NHS anaesthetic department in the UK to assess preparedness for treating peri-operative cardiac arrest. We received 199 responses from 277 UK anaesthetic departments, representing a 72% response rate. Adult and paediatric anaesthetic care was provided by 188 (95%) and 165 (84%) hospitals, respectively. There was no paediatric intensive care unit on-site in 144 (87%) hospitals caring for children, meaning transfer of critically ill children is required. Remote site anaesthesia is provided in 182 (92%) departments. There was a departmental resuscitation lead in 113 (58%) departments, wellbeing lead in 106 (54%) and departmental staff wellbeing policy in 81 (42%). A defibrillator was present in every operating theatre suite and in all paediatric anaesthesia locations in 193 (99%) and 149 (97%) departments, respectively. Advanced airway equipment was not available in: every theatre suite in 13 (7%) departments; all remote locations in 103 (57%) departments; and all paediatric anaesthesia locations in 23 (15%) departments. Anaesthetic rooms were the default location for induction of anaesthesia in adults and children in 148 (79%) and 121 (79%) departments, respectively. Annual updates in chest compressions and in defibrillation were available in 149 (76%) and 130 (67%) departments, respectively. Following a peri-operative cardiac arrest, debriefing and peer support programmes were available in 154 (79%) and 57 (29%) departments, respectively. While it is likely many UK hospitals are very well prepared to treat anaesthetic emergencies including cardiac arrest, the survey suggests this is not universal.

Patient characteristics, anaesthetic workload and techniques in the UK: an analysis from the 7th National Audit Project (NAP7) activity survey.

Detailed contemporary knowledge of the characteristics of the surgical population, national anaesthetic workload, anaesthetic techniques and behaviours are essential to monitor productivity, inform policy and direct research themes. Every 3-4 years, the Royal College of Anaesthetists, as part of its National Audit Projects (NAP), performs a snapshot activity survey in all UK hospitals delivering anaesthesia, collecting patient-level encounter data from all cases under the care of an anaesthetist. During November 2021, as part of NAP7, anaesthetists recorded details of all cases undertaken over 4 days at their site through an online survey capturing anonymous patient characteristics and anaesthetic details. Of 416 hospital sites invited to participate, 352 (85%) completed the activity survey. From these, 24,177 reports were returned, of which 24,172 (99%) were included in the final dataset. The work patterns by day of the week, time of day and surgical specialty were similar to previous NAP activity surveys. However, in non-obstetric patients, between NAP5 (2013) and NAP7 (2021) activity surveys, the estimated median age of patients increased by 2.3 years from median (IQR) of 50.5 (28.4-69.1) to 52.8 (32.1-69.2) years. The median (IQR) BMI increased from 24.9 (21.5-29.5) to 26.7 (22.3-31.7) kg.m-2 . The proportion of patients who scored as ASA physical status 1 decreased from 37% in NAP5 to 24% in NAP7. The use of total intravenous anaesthesia increased from 8% of general anaesthesia cases to 26% between NAP5 and NAP7. Some changes may reflect the impact of the COVID-19 pandemic on the anaesthetic population, though patients with confirmed COVID-19 accounted for only 149 (1%) cases. These data show a rising burden of age, obesity and comorbidity in patients requiring anaesthesia care, likely to impact UK peri-operative services significantly.

Methods of the 7th National Audit Project (NAP7) of the Royal College of Anaesthetists: peri-operative cardiac arrest.

Cardiac arrest in the peri-operative period is rare but associated with significant morbidity and mortality. Current reporting systems do not capture many such events, so there is an incomplete understanding of incidence and outcomes. As peri-operative cardiac arrest is rare, many hospitals may only see a small number of cases over long periods, and anaesthetists may not be involved in such cases for years. Therefore, a large-scale prospective cohort is needed to gain a deep understanding of events leading up to cardiac arrest, management of the arrest itself and patient outcomes. Consequently, the Royal College of Anaesthetists chose peri-operative cardiac arrest as the 7th National Audit Project topic. The study was open to all UK hospitals offering anaesthetic services and had a three-part design. First, baseline surveys of all anaesthetic departments and anaesthetists in the UK, examining respondents' prior peri-operative cardiac arrest experience, resuscitation training and local departmental preparedness. Second, an activity survey to record anonymised details of all anaesthetic activity in each site over 4 days, enabling national estimates of annual anaesthetic activity, complexity and complication rates. Third, a case registry of all instances of peri-operative cardiac arrest in the UK, reported confidentially and anonymously, over 1 year starting 16 June 2021, followed by expert review using a structured process to minimise bias. The definition of peri-operative cardiac arrest was the delivery of five or more chest compressions and/or defibrillation in a patient having a procedure under the care of an anaesthetist. The peri-operative period began with the World Health Organization 'sign-in' checklist or first hands-on contact with the patient and ended either 24 h after the patient handover (e.g. to the recovery room or intensive care unit) or at discharge if this occured earlier than 24 h. These components described the epidemiology of peri-operative cardiac arrest in the UK and provide a basis for developing guidelines and interventional studies.

Validation of PAS Kinase, a Regulator of Hepatic Fatty Acid and Triglyceride Synthesis, as a Therapeutic Target for Nonalcoholic Steatohepatitis.

Hyperactivation of sterol regulatory element binding protein 1c (SREBP-1c), which transcriptionally induces expression of enzymes responsible for de novo lipogenesis and triglyceride (TG) formation, is implicated in nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) pathogenesis. Posttranslational SREBP-1c maturation and activation is stimulated by the protein per-arnt-sim kinase (PASK). PASK-knockout mice are phenotypically normal on a conventional diet but exhibit decreased hypertriglyceridemia, insulin resistance, and hepatic steatosis on a high-fat diet. We investigated the effects of pharmacologic PASK inhibition using BioE-1115, a selective and potent oral PASK inhibitor, in Zucker fatty (fa)/fa) rats, a genetic model of obesity, dyslipidemia, and insulin resistance, and in a dietary murine model of NAFLD/NASH. Female Zucker (fa/fa) rats and lean littermate (fa/+) controls received BioE-1115 (3-100 mg/kg/day) and/or omega-3 fatty acids, and blood glucose, hemoglobin A1c, glucose tolerance, insulin, and serum TG were measured. C57BL/6J mice fed a high-fat/high-fructose diet (HF-HFrD) were treated with BioE-1115 (100 mg/kg/day) or vehicle. Body weight and fasting glucose were measured regularly; serum TG, body and organ weights, and liver TG and histology were assessed at sacrifice. Messenger RNA (mRNA) abundance of SREBP-1c target genes was measured in both models. In Zucker rats, BioE-1115 treatment produced significant dose-dependent reductions in blood glucose, insulin, and TG (all greater than omega-3 fatty acids) and dose dependently restored insulin sensitivity assessed by glucose tolerance testing. In HF-HFrD mice, BioE-1115 reduced body weight, liver weight, fasting blood glucose, serum TGs, hepatic TG, hepatic fibrosis, hepatocyte vacuolization, and bile duct hyperplasia. BioE-1115 reduced SREBP-1c target mRNA transcripts in both models. Conclusion: PASK inhibition mitigates many adverse metabolic consequences associated with an HF-HFrD and reduces hepatic fat content and fibrosis. This suggests that inhibition of PASK is an attractive therapeutic strategy for NAFLD/NASH treatment.

Renal adenylate cyclase and the interrelationship between parathyroid hormone and vitamin D in the regulation of urinary phosphate and adenosine cyclic 3',5'-monophosphate excretion.

This study examined the role of cyclic AMP in the phosphaturic response to parathyroid hormone in vitamin D-deficient rats. Infusion of purified bovine parathyroid hormone (13.3 mug/h) into control, D-fed, or D-deficient, thyroparathyroidectomized rats produced a sixfold increase in renal phosphate and cyclic AMP excretion in D-fed rats, but only a two- to threefold increase in both parameters in D-deficient animals. Intravenous injection of parathyroid hormone over the dosage range from 1-50 mug/kg resulted in a dose-dependent increase in phosphate and cyclic AMP excretion with both D-fed and D-deficient thyroparathyroidectomized rats. However, the D-deficient rats responded to these injections of parathyroid hormone with a two- to threefold increase in both renal phosphate and cyclic AMP excretion at the highest dose of 50 mug/kg, whereas the D-fed animals' response was 35-fold and 11-fold over control excretion levels of phosphate and cyclic AMP, respectively. To directly examine the role of the renal cortical adenylate cyclase system in the blunted phosphaturic and urinary cyclic AMP responses to parathyroid hormone in D-deficient rats, we prepared a plasma membrane fraction enriched in this enzyme activity from the renal cortex of D-fed and D-deficient thyroparathyroidectomized rats. The renal cortical adenylate cyclase of D-deficient rats showed significantly (P less than 0.001) less activation by parathyroid hormone over the hormone concentration range from 0.3 to 7.0 mug/ml than was observed with the enzyme prepared from D-fed animals. Basal adenylate cyclase activity and the fluoride-stimulated enzyme activity were not altered by the state of D-deficiency. These experiments demonstrate that the blunted phosphaturic response to parathyroid hormone observed in D-deficient rats is associated with the reduced responsiveness of the renal cortical adenylate cyclase to the hormone. Moreover, the defect in the renal membrane adenylate cyclase system appears to be localized at the level of PTH binding to membrane receptors or, alternatively, at the level of transmission of the hormone-receptor binding signal to the catalytic moiety of this membrane enzyme.

A peptidomimetic antagonist of the alpha(v)beta3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.

Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.

A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy.

The integrin alpha(v)beta3 interacts with the arginine-glycine-aspartic acid (RGD) tripeptide recognition sequence of a variety of extracellular matrix proteins. Recent studies show that alpha(v)beta3 plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha(v)beta3 inhibit angiogenic processes that include endothelial cell adhesion and migration. Consequently, we reasoned that an RGD-based peptidomimetic antagonist of alpha(v)beta3 might inhibit tumor angiogenesis and tumor growth in vivo. An RGD-peptidomimetic library was screened to identify antagonists of vitronectin binding to alpha(v)beta3, and the compounds chosen were modified to produce selective and potent inhibitors of alpha(v)beta3. One of these compounds, beta-[[2-2-[[[3-[(aminoiminomethyl)amino]-phenyl]carbonyl]amino]ac etyl]amino]-3,5-dichlorobenzenepropanoic acid (SC-68448), inhibited vitronectin binding to both alpha(v)beta3 and the closely related platelet receptor, alpha(IIb)beta3, in a dose-responsive manner. SC-68448 inhibited vitronectin binding to alpha(v)beta3 (IC50, 1 nM) and fibrinogen binding to the platelet receptor alpha(IIb)beta3 (IC50, >100 nM), demonstrating that SC-68448 was 100-fold more potent as an inhibitor of alpha(v)beta3 versus alpha(IIb)beta3. In cell-based studies, SC-68448 inhibited alpha(v)beta3-mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. In accord with these results, SC-68448 inhibited angiogenesis in vivo in a basic fibroblast growth factor-induced rat corneal neovascularization model. A xenogeneic severe combined immune deficiency mouse/rat Leydig cell tumor model was developed for testing SC-68448 as an inhibitor of tumor growth in vivo. Rat Leydig cell tumors grew rapidly in severe combined immune deficiency mice and produced humoral hypercalcemia of malignancy. SC-68448 inhibited the growth of the tumors in mice by up to 80% and completely blocked the development of hypercalcemia. Together, these results demonstrate the feasibility of antitumor therapies based upon the development of nontoxic small molecule pharmacological antagonists of integrin alpha(v)beta3.

Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding.

Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1-34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1-34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1-34) bound rapidly to both rat and dog renal membranes. At 22 degrees C reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely greater than 90% for rat kidney and greater than 95% for dog kidney. Similar results were obtained at 4 degrees C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.

Radioiodinated rat parathyroid hormone-(1-34) binds to its receptor on rat osteosarcoma cells in a manner consistent with two classes of binding sites.

Binding of 125I-labeled rat (r) PTH-(1-34) to ROS 17/2.8 osteoblastic bone cells and to membranes from these cells was examined. Competitive binding inhibition experiments were performed using unlabeled rPTH-(1-34) with particular emphasis on concentrations of peptide below 1 nM. In intact cells, binding of labeled rPTH-(1-34) was highly specific, and inhibition of binding by unlabeled ligand suggested the presence of two classes of binding sites, one with high affinity and low capacity (KD = 40 pM, approximately 20% of total binding sites) and the other with lower affinity and high capacity (KD = 2 nM, approximately 80% of the sites). Membranes prepared from ROS cells also exhibited a pattern of binding from competitive inhibition curves consistent with two distinct binding sites (KD = 30 pM and 6 nM). In intact ROS cells, cellular cAMP levels increased over the range of 10(-11)-10(-9) M rPTH-(1-34) with an ED50 intermediate between the two KD values (0.25 nM). These data suggest that osteoblastic bone cells possess two distinct classes of membrane receptors for PTH. Since the KD of the higher affinity site more closely approximates circulating concentrations of PTH, binding to this site may have physiologic relevance.

Parathyroid hormone and calcium: interactions in the control of renin secretion in the isolated, nonfiltering rat kidney.

The present study was designed to characterize the interaction of calcium and PTH in the control of renin release in isolated rat kidneys perfused in a closed circuit at constant flow. Kidneys were rendered nonfiltering using low perfusion pressures (70 mm Hg) and a hyperoncotic perfusate (100 g/liter BSA). Under these conditions, differences in perfusion pressure were less than 9 mm Hg between control and PTH-treated kidneys over the 50 min of perfusion. In the absence of PTH, renin release was inversely correlated with ionized calcium (Ca2+) concentration, with the highest release of renin noted with 1 mM EGTA and no added calcium. Also, verapamil treatment markedly elevated renin release, even in the presence of 2 mM Ca2+. In contrast, renin secretion was strongly depressed by 20 nM BAY-K8644 in the perfusate. In medium containing normal calcium concentrations (1 mM Ca2+), rat PTH(1-34) induced a 2-fold greater renin accumulation than in the control, non-PTH-treated kidneys. Isoproterenol induced a 5-fold stimulation under the same conditions. In the 0 Ca2+/1 mM EGTA perfusion, PTH did not elevate renin secretion. Renin release in response to PTH in 2 mM Ca2+ was similar to that observed in the 1 mM Ca2+ perfusion. PTH also reversed the effects of BAY-K8644 to suppress renin release. In verapamil-treated kidneys, PTH failed to stimulate renin release. These results indicate that PTH stimulates renin release by a process independent of the baroreceptors and macula densa. The Ca2+ modulation of PTH-induced renin release is consistent with the reported ability of PTH to block calcium channels and relax vascular smooth muscle.

Cardiovascular responsiveness to parathyroid hormone (PTH) and PTH-related protein in genetic hypertension.

Hypertension is often accompanied by abnormalities of calcium homeostasis, including hyperparathyroidism with reduced target organ responses to PTH in kidney and bone. Due to this association between PTH and hypertension and since PTH and the paracrine factor PTH-related protein (PTHrp) have both been shown to exert marked changes in cardiovascular activity, these actions of PTH and PTHrp were examined in spontaneously hypertensive rats (SHR) and in control normotensive Wistar-Kyoto rats (WKY). Fourteen-week-old SHR [systolic blood pressure (SBP), 201 +/- 4.4 mm Hg] and WKY (SBP, 141 +/- 2.5 mm Hg) were studied. Renal cortical membranes were prepared and assayed for radioligand binding with [125I]PTH-(1-34) and [125I]PTHrp-(1-34). There was no apparent alteration in the affinity of the binding sites to either peptide in the SHR, but specific binding in SHR renal tissue was only 60% of that observed in WKY tissue for both peptides. Serum immunoreactive PTH levels were 4-fold higher in SHR than WKY, while serum total calcium and 1,25-dihydroxyvitamin D3 levels were not different. The iv administration of both PTH and PTHrp produced dose-dependent reductions in SBP and increases in heart rate in conscious unrestrained SHR and WKY. Both peptides caused greater absolute reductions in blood pressure in SHR than in WKY. However, when the hypotensive response was normalized for the higher baseline pressure in the SHR, the blood pressure reductions caused by PTH and PTHrp were not different in SHR and WKY. Conversely, the chronotropic responses to PTH and PTHrp were lower in SHR compared to WKY. These findings indicate that the SHR exhibits elevated PTH levels, with a reduced number of renal PTH/PTHrp receptors and a depressed chronotropic response to either PTH or PTHrp. In contrast, the hypotensive response to PTH or PTHrp was not altered, indicating possible tissue-specific receptor subclasses or tissue-specific regulation of PTH and PTHrp receptors.

Binding of parathyroid hormone and parathyroid hormone-related protein to vascular smooth muscle of rabbit renal microvessels.

The humoral hypercalcemia of malignancy factor (also called PTH-related protein or PTHrp) has been shown to produce effects similar to PTH in the kidney, bone, and cardiovascular system. Binding of PTHrp and PTH has been characterized in renal and osseous tissues, but not in vascular tissue. We have attempted to characterize the interaction of both human PTHrp and rat PTH to renal microvessels as a model of vascular smooth muscle and in a renal tubule preparation from the same rabbit kidneys. Previous studies have shown the microvessel and tubule preparations to be distinct based upon morphological examination, differential enzyme markers, calcitonin and vasopressin-sensitive adenylate cyclase distribution, and different characteristics of guanine nucleotide and of oxidized PTH activation of the adenylate cyclases associated with the preparations. Human PTHrp and rat PTH were iodinated by standard techniques and purified by HPLC. Both ligands bound to microvessels and tubules in a saturable, specific manner, Maximal specific binding of either ligand was 65-75% in microvessels and 80-90% in renal tubules. The time courses of binding of both ligands were identical with steady state achieved within 20 min in the smooth muscle of microvessels and 15 min in the tubules at 22 C. In equilibrium competition binding experiments, bound 125I-PTHrp was displaced by both PTHrp and PTH in microvessels and tubules. Rat PTH displayed slightly higher affinity in microvessels and tubules than PTHrp. Identical results were obtained with 125I-PTH as ligand. Specificity of binding of PTHrp and PTH to both microvessels and tubules was excellent, with competition observed between the radioactive ligand and bovine and rat PTH, PTHrp, and the antagonists, [Nle8,18, Tyr34]bovine PTH and [Nle8,18, Tyr34]bovine PTH but not with several other peptides of unrelated structure. The only major difference in binding between microvessels and tubules was a smaller number of binding sites in microvessels compared to tubules. These results indicate that vascular tissue contains receptor sites for PTH and PTHrp as identified by radioligand binding techniques. These receptors are similar in characteristics to the receptors of renal tubular tissue. Both PTH and PTHrp appear to interact with the receptors of rabbit kidney microvessels and tubules.

Endothelium-independent linkage of parathyroid hormone receptors of rat vascular tissue with increased adenosine 3',5'-monophosphate and relaxation of vascular smooth muscle.

We examined the mechanisms involved in the relaxation of rat vascular smooth muscle by PTH. PTH increased intracellular cAMP 10-fold in cultured vascular smooth muscle cells from rat aorta. Forskolin, methylisobutylxanthine, and papaverine all potentiated PTH action. The cAMP responses to PTH were not altered by concurrent addition of propranolol, phentolamine, atropine, or [Sar1,Ile8]angiotensin II. Only the synthetic PTH antagonist analog [Nle8,Nle18,Tyr34] bovine PTH-(3-34) inhibited the cAMP and vascular relaxation responses to PTH. Isoproterenol produced increases in intracellular cAMP and adenylate cyclase activity which were additive to those produced by PTH. In contracted rat aortic strips, PTH caused a dose-dependent relaxation which was not altered by removal of the vessel intima or treatment with nordihydroguaiaretic acid. Also, membrane preparations from intact aortas or aortas with the endothelium or adventitia removed displayed identical PTH-stimulated adenylate cyclase activities. These findings indicate that the relaxant action of PTH in rat aorta does not require an intact endothelium and results from a direct effect on the vessel medial layer. Relaxation appears to be mediated by a receptor unique for PTH which is linked to the adenylate cyclase of vascular smooth muscle cells.

Synthetic alphaVbeta3 antagonists inhibit insulin-like growth factor-I-stimulated smooth muscle cell migration and replication.

Porcine aortic smooth cells respond to insulin-like growth factor-I (IGF-I) with increases in DNA synthesis and cell migration. Because ligand occupancy of the alphaVbeta3 integrin has been shown to be necessary for IGF-I to stimulate maximal increases in both processes, we determined whether synthetic alphaVbeta3 antagonists could inhibit IGF-I-stimulated actions on this cell type. Low-molecular-weight compounds that had been selected based on their ability to compete with vitronectin for binding to purified human alphaVbeta3 in vitro were analyzed for their ability to compete with 125I-kistrin (a known ligand for porcine alphaVbeta3) for binding to porcine alphaVbeta3. Nine compounds were screened, and five were found to be potent competitive inhibitors. The most potent compound, SC-69000, resulted in 88% competition at 10(-7) M and was nearly equipotent with echistatin. The compounds that were the most potent inhibitors of kistrin binding were tested for their capacity to inhibit the cell migration response to IGF-I. Three compounds caused between 81-88% inhibition of IGF-I-stimulated migration at 10(-7) M. To determine whether these compounds could inhibit other IGF-I-stimulated actions, their ability to inhibit IGF-I-stimulated [3H]-thymidine incorporation into DNA was analyzed. The four compounds that were the most potent inhibitors of cell migration also inhibited IGF-I-stimulated DNA replication. IGF-I stimulates the synthesis of IGF binding protein-5 by these cells. Preincubation with the four most active compounds also resulted in significant inhibition of the ability of IGF-I to stimulate IGF binding protein-5 synthesis. AlphaVbeta3 occupancy by the ligand vitronectin has been shown to enhance the capacity of IGF-I to activate its receptor tyrosine kinase. The four most active compounds were shown to inhibit IGF-I-stimulated IGF-I receptor autophosphorylation. These findings suggest that blockade of ligand occupancy of the alphaVbeta3 integrin globally inhibits several IGF-I-stimulated biologic actions and that synthetic inhibitors are very active in this regard. Because these compounds can be administered to whole animals, they should be very useful in determining whether blocking alphaVbeta3 occupancy in vivo results in alteration in responsiveness to IGF-I.