A Quantitative Evaluation of Medication Histories and Reconciliation by Discipline

Background/Objective:

Medication reconciliation at transitions of care decreases medication errors, hospitalizations, and adverse drug events. We compared inpatient medication histories and reconciliation across disciplines and evaluated the nature of discrepancies.

Methods:

We conducted a prospective cohort study of patients admitted from the emergency department at our 760-bed hospital. Eligible patients had their medication histories conducted and reconciled in order by the admitting nurse (RN), certified pharmacy technician (CPhT), and pharmacist (RPh). Discharge medication reconciliation was not altered. Admission and discharge discrepancies were categorized by discipline, error type, and drug class and were assigned a criticality index score. A discrepancy rating system systematically measured discrepancies.

Results:

Of 175 consented patients, 153 were evaluated. Total admission and discharge discrepancies were 1,461 and 369, respectively. The average number of medications per participant at admission was 8.59 (1,314) with 9.41 (1,374) at discharge. Most discrepancies were committed by RNs: 53.2% (777) at admission and 56.1% (207) at discharge. The majority were omitted or incorrect. RNs had significantly higher admission discrepancy rates per medication (0.59) compared with CPhTs (0.36) and RPhs (0.16) (P < .001). RPhs corrected significantly more discrepancies per participant than RNs (6.39 vs 0.48; P < .001); average criticality index reduction was 79.0%. Estimated prevented adverse drug events (pADEs) cost savings were $589,744.

Conclusions:

RPhs committed the fewest discrepancies compared with RNs and CPhTs, resulting in more accurate medication histories and reconciliation. RPh involvement also prevented the greatest number of medication errors, contributing to considerable pADE-related cost savings.Key Words: admission, evaluation study, discharge, medication reconciliationObtaining medication histories and conducting medication reconciliation are challeng ing tasks with the advent of new molecular entities and orphan drugs.¹ As Franklin reported, “Patients who once came into the [physician] office carrying their medications in a purse, or pocket, now need a shopping bag.”² The importance of accurate medication histories cannot be overemphasized; nearly 27% of all hospital prescribing errors originate from incorrect admission medication histories, over 70% of drug-related problems are only discovered through patient interview, and more than 50% of discharge discrepancies are associated with admission discrepancies.^3–6In 2010, an Institute of Medicine report estimated that if hospitals prevented adverse drug events (pADEs) and redundant tests, the associated cost savings would be nearly $25 billion annually.⁷ One organization decreased inpatient care costs by 30% when no medication reconciliation errors were reported over 24 months. ⁷Multiple organizations have supported medication reconciliation to improve quality of care, reduce preventable hospital admissions and readmissions, and decrease the incidence of adverse health care- associated conditions.^8–11 Although The Joint Commission does not indicate the discipline to perform this role, evidence supports the role of registered pharmacists (RPhs), pharmacy students, and pharmacy technicians in collecting accurate medication histories. RPhs should be involved when high-risk medications are identified, more than 5 medications are reported, or patients are elderly.^6,8,11–40 Therefore, our primary study objective was to compare inpatient medication histories and reconciliation processes across disciplines and to evaluate the nature of discrepancies using a novel method.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

ISMP Medication Error Report Analysis

These medication errors have occurred in health care facilities at least once. They will happen again—perhaps where you work. Through education and alertness of personnel and procedural safeguards, they can be avoided. You should consider publishing accounts of errors in your newsletters and/or presenting them at your inservice training programs.Your assistance is required to continue this feature. The reports described here were received through the Institute for Safe Medication Practices (ISMP) Medication Errors Reporting Program. Any reports published by ISMP will be anonymous. Comments are also invited; the writers’ names will be published if desired. ISMP may be contacted at the address shown below.Errors, close calls, or hazardous conditions may be reported directly to ISMP through the ISMP Web site (www.ismp.org), by calling 800-FAIL-SAFE, or via e-mail at gro.pmsi@ofnipmsi. ISMP guarantees the confidentiality and security of the information received and respects reporters’ wishes as to the level of detail included in publications.

• Hosp Pharm. 2013 Jul-Aug; 48(7): 538–541.
»
• Tragedy in the Postanesthesia Care Unit

2013 Jul-Aug; 48(7): 538–541. Published online 2013 Jul 9. doi: 10.1310/hpj4807-538

Tragedy in the Postanesthesia Care Unit

Michael R. Cohen, RPh, MS, ScD^* and Judy L. Smetzer, RN, BSN^†

Michael R. Cohen

^*President, Institute for Safe Medication Practices, 200 Lakeside Drive, Suite 200, Horsham, PA 19044; phone: 215-947-7797; fax: 215-914-1492; e-mail: gro.pmsi@nehocm; Web site: gro.pmsi.www.Find articles by Michael R. Cohen

Judy L. Smetzer

^†Vice President, Institute for Safe Medication Practices, Horsham, Pennsylvania.Find articles by Judy L. SmetzerAuthor information Copyright and License information Disclaimer^*President, Institute for Safe Medication Practices, 200 Lakeside Drive, Suite 200, Horsham, PA 19044; phone: 215-947-7797; fax: 215-914-1492; e-mail: gro.pmsi@nehocm; Web site: gro.pmsi.www.^†Vice President, Institute for Safe Medication Practices, Horsham, Pennsylvania.Copyright notice In April 2012, after receiving a dose of intravenous (IV) fentanyl in the postanesthesia care unit (PACU) at an outpatient ambulatory surgery center, a 17-year-old girl died following an uncomplicated tonsillectomy. The case made headline news recently when a civil lawsuit filed by the teen’s parents was resolved. Although it is too late to reverse the tragic outcome of this case, we call upon all hospitals and outpatient surgery centers to learn from the event and take action to prevent a similar tragedy in your facility.Following surgery, the teen arrived in the PACU where a nurse anesthetist administered a dose of fentanyl by slow IV push to the patient to help manage pain. The drug led to respiratory depression and eventual respiratory arrest. The patient had no pulse and was breathless 25 minutes after receiving the fentanyl. Resuscitation efforts were initiated, and the patient was transferred from the ambulatory surgery center to a hospital emergency department. Tragically, as a result of oxygen deprivation, the patient suffered profound, irreversible brain injury and died.After investigating the causes of this adverse event, the ambulatory surgery center staff identified several reasons why the PACU staff failed to notice the patient’s declining respiratory status.

Inadequate Monitoring

After receiving the IV fentanyl, the teen was not observed or assessed for 25 minutes. The attention of the teen’s PACU nurse was temporarily diverted to tend to a patient who had developed postoperative complications. An initial set of vital signs had been taken upon the patient’s arrival in the PACU, but no further assessment of the patient occurred until she was found in cardiac arrest 25 minutes later.

Muted Alarms

The alarms on the monitoring equipment used to alert health care professionals to changes in the patient’s cardiac and respiratory status were muted. In fact, all the alarms associated with monitoring equipment in the PACU were muted, most likely due to alarm fatigue. The purpose of a medical device alarm is to warn caregivers of potential problems with patients who may require immediate action. The cacophony of sounds from alarms that echo through a hospital or PACU, however, can be overwhelming.¹ The sheer number of alarms—up to 700 per patient per day²—along with a high rate of false or clinically insignificant alarms, can quickly desensitize staff and cause alarm fatigue, leading to missed alarms, ignored alarms, delayed responses to alarms, muted or low volume alarm settings, or adjustments to alarm limits outside a safe range.^1,2 Alarm fatigue has been described by those who experience it as follows³:

• When a nurse or other caregiver is overwhelmed with hundreds of alarm signals per patient per day
• When a patient can’t rest with the multitude of alarm signals going off
• When a true life-threatening event is lost in the noise because of the multitude of devices with competing alarm signals, all trying to capture attention, without clarity about what action is needed

Additionally, in a PACU where nurses are rarely far from the patient’s bedside, staff may have a mistaken belief that the risk associated with muting alarms is not significant.

Obstructed View of the Patient

A curtain had been drawn around the patient, obstructing the nurses’ view and preventing them from maintaining an ongoing visual assessment of the patient.Patients in the PACU are particularly vulnerable to adverse events and are more likely to encounter medical difficulties as they emerge from anesthesia versus later in their recovery.⁴ In the operating room, an entire team of practitioners is directly caring for the patient and monitoring the patient’s response. Once the patient moves into the PACU, highly trained PACU nurses are available to provide care, but they may be caring for more than one patient. Although PACU nurses may be experts in interpreting and responding to events during the brief but intense period immediately following a procedure requiring anesthesia, staffing patterns that do not support necessary monitoring, alarms that are inaudible, and a blocked line of sight for observing patients invite untoward clinical events. While airway obstruction may be the most common untoward event in the PACU, inadequate ventilation and oversedation from residual anesthesia-related medications and pain medications administered in the PACU are a close corollary.⁴Unexpected patient emergencies can quickly arise in the PACU and result in the diversion of staff attention, so all hospitals and ambulatory surgery centers must recognize the likelihood of these events their effects on patient care and consider the following recommendations to reduce the risks in the PACU (or a similar unit such as the emergency department).

Maintain a Direct Line of Sight to Patients

Although patient privacy is an important concern during the postoperative experience,⁵ a direct line of sight to PACU patients is vital to allow staff to observe all patients at all times.⁴ Privacy curtains should be used judiciously, and a PACU staff member who is assigned only to one patient may need to remain behind the curtain with that patient if close observation is required. In the ambulatory surgery center where this event occurred, it is now prohibited to draw curtains that would restrict the view of patients, according to the news.

Provide Staffing to Ensure Proper Monitoring

Review current staffing patterns and monitoring practices in the PACU to ensure patients are adequately observed and cared for during the immediate postanesthesia period, particularly when IV opioid analgesics are administered. Guidelines from applicable professional associations and national and state regulatory agencies should serve as a resource to ensure that staffing and monitoring practices comply with current standards of care. For example, the American Society of Anesthesiologists (ASA) Standards for PostAnesthesia Care note that a quantitative method of assessing oxygenation such as pulse oximetry should be used during the initial phase of recovery from anesthetics⁶ and that physiologic monitoring such as cardiac monitoring has become a de facto standard.⁴ The ASA also suggests that, during the initial 15 minutes in the PACU, one nurse should be caring exclusively for that patient.⁴ The American Society of PeriAnesthesia Nurses (ASPAN)—the professional association to which the ASA defers for issues of nursing care—has promulgated a standard requiring a 1:1 nurse-patient ratio from the time the patient is first admitted to the PACU until explicit critical elements are met and the patient is hemodynamically stable.^5,7Because of the cumulative effects of opioids given near the end of a surgical procedure and then again in the PACU, which may contribute to respiratory depression, the surgical center where the adverse event happened now requires one-on-one nursing care for patients who have received opioids in the PACU. Because patient emergencies can quickly arise in the PACU and require unexpected staff attention, the facility also established a charge nurse position in the PACU to monitor the patient flow and staffing and to redeploy resources as needed.

Manage Alarm Hazards

According to the ECRI Institute—a leading organization that evaluates medical technology—alarm hazards are once again number one on its list of the Top 10 Health Technology Hazards for 2013.¹ The potential for alarm-related harm exists every day in every health care facility. Given the ubiquitous nature of medical alarms, ECRI suggests that the potential for alarm-related events may always warrant inclusion on a list of the most pressing technology hazards.¹ Nonetheless, health care facilities must do more to improve the manner in which alarms are managed. Awareness of the problem is not at issue—the absence of meaningful action is.Driving this point home, The Joint Commission (TJC) has been evaluating a proposed 2014 National Patient Safety Goal (NPSG) related to alarm management,⁸ which would require accredited organizations to:

• Establish alarm safety as a priority.
• Prepare an annual inventory of device alarms and identify default settings.
• Identify the most important alarms to manage.
• Establish policies for managing important alarms, including clear guidelines regarding when alarms can be disabled, when alarm parameters can be changed, who has the authority to make these decisions, how to monitor and respond to alarms, and when to check alarms for accuracy.
• Educate staff about alarm policies and procedures.

The public comment period for the proposed NPSG has ended. However, TJC recently published a Sentinel Event Alert describing the extent of alarm-related events reported to TJC along with recommendations for improvement.⁹The ambulatory surgery center where the event happened no longer allows muted alarms with monitoring equipment in the PACU. While ISMP concurs with this action, the scope of alarm hazards is larger than just muted alarms and broader than can be addressed with a few bullet points in this article. Therefore, ISMP strongly encourages health care organizations to utilize external resources that are more appropriate to guide the assessment and improvement of alarm hazards. Two great resources are the upcoming TJC Sentinel Event Alert⁹ as well as an in-depth resource that describes priority issues and consensus recommendations from a 2011 medical device alarms summit convened in October 2011 by the Association for the Advancement of Medical Instrumentation (AAMI), the US Food and Drug Administration (FDA), the American College of Clinical Engineers (ACCE), TJC, and ECRI Institute.³ The report, A Siren Call to Action, identifies and prioritizes a range of issues with medical alarms and priority actions for addressing them, including, among others, the following³:

• Conduct clinical testing and analyze alarm data to optimize alarm limits and delays for self-correcting alarm conditions that will reduce clinically non-actionable alarms.
• Test the acoustics on clinical floors; environmental noise impacts patient and staff well-being and patient safety.
• Change single-use sensors frequently to reduce nuisance alarm conditions, except in pediatric units (eg, change ECG leads every 24 hours).

The Sentinel Event Alert and the summit report make it clear that clinical leadership support and interdisciplinary efforts are an absolute requirement to make headway with this challenging problem.     

Selective ligands for opioid receptors: β-Cyclopropylalanyl containing analogs of enkephalin

The synthesis and resolution of the amino acid β-cyclopropylalanine (Cpr) and its incorporation into four enkephalin analogs is reported. The analogs prepared were: Tyr - l - Cpr - Gly - Phe - Pen (des - COOH - Nle = n - pentylamide = Pen) (l -Cpr²-Pen⁵-ENK), Tyr-d -Cpr-Gly-Phe-Pen (d -Cpr²-Pen⁵-ENK), l -Cpr-Tyr-d -Ala-Gly-Phe-Pen (l -Cpr⁰-d -Ala²-Pen⁵-ENK) and d -Cpr-Tyr-d -Ala-Gly-Phe-Pen (d -Cpr⁰-d -Ala²-Pen⁵-ENK). Each was tested for its ability to inhibit the field stimulated guinea pig ileum (GPI) and rat vas deferens (RVD) and the results compared to the effect d -Ala²-d -Leu⁵-enkephalin (DADLE) has on the same preparations. The results show that at concentrations up to 10^-5 m all four analogs, as well as DADLE, are full agonists on the GPI preparation. The concentrations necessary to produce a 50% inhibition of the twitch response were, DADLE, 3.5 °× 10^-8 m ; l - Cpr⁰-d -Ala²-Pen⁵-ENK, 6.0 × 10^-8 m ; d -Cpr²-Pen⁵-ENK, 1.1 × 10^-7 m ; l -Cpr²-Pen⁵-ENK, 1.2 × 10^-6 m and d -Cpr⁰-d -Ala²-Pen⁵-ENK, > 10^-5 m . On RVD a different result was observed with only DADLE (1.3 × 10^-6 m ) and l -Cpr⁰-Pen⁵-enkephalin (1.8 × 10^-6 m ) showing full agonist activity. d -Cpr²-Pen⁵-ENK was a partial agonist (29 · 5% inhibition of the twitch at 10^-5 m ) while d -Cpr⁰-d -Ala²-Pen⁵-ENK and l -Cpr²-Pen⁵-ENK did not inhibit the twitch at concentrations up to 10^-5 m . These compounds which were inactive or of low potency on each preparation were also tested as antagonists. Only d -Cpr²-Pen⁵-ENK was an antagonist (pA₂ = 6.09) versus DADLE on RVD while d -Cpr⁰-d -Ala²-Pen⁵-ENK was inactive as an antagonist on both GPI and RVD. d -Cpr²-Pen⁵-ENK, therefore, represents the first enkephalin analog to be categorized as a mixed agonist-antagonist.     

A higher frequency of IL‐10‐producing B cells in Hepatitis B virus‐associated membranous nephropathy

The purpose of this study is to elucidate the potential role of interleukin (IL)‐10⁺ regulatory B cells and other B cell subsets in the development of hepatitis B virus‐associated membranous nephropathy (HBV‐MN). A total of 14 patients with new onset HBV‐MN, 12 individuals with immune‐tolerant HBV infection (HBV‐IT), and 12 healthy controls (HC) were examined for the percentages of CD38⁺, CD86⁺, CD27⁺, CD95⁺ and IL‐10⁺ B cells by flow cytometry. Serum IL‐10 concentration was examined by enzyme‐linked immunosorbent assay (ELISA). The percentages of CD38⁺CD19⁺, CD86⁺CD19⁺, CD38⁺CD86⁺CD19⁺, and CD95⁺CD19⁺ B cells were significantly higher in HBV‐MN patients than the HBV‐IT and HC. The percentages of CD5⁺CD19⁺, IL‐10⁺CD19⁺ B cells and serum IL‐10 level in HBV‐MN patients were significantly higher than the HC, and lower than the HBV‐IT. Percentages of CD38⁺CD19⁺, and CD86⁺CD19⁺ B cells were reduced after treatment, while the percentages of CD5⁺CD1d⁺CD19⁺, CD5⁺CD1d⁺IL‐10⁺CD19⁺, and IL‐10⁺CD19⁺ B cells were increased. The 24 h urinary protein concentration was positively correlated with the percentage of CD38⁺CD19⁺, and negatively correlated with the percentage of IL‐10⁺CD19⁺ B cells and serum IL‐10 level. Similarly, the value of eGFR was negatively correlated with the percentage of CD38⁺CD19⁺, and positively correlated with the percentage of IL‐10⁺CD19⁺ B cells and serum IL‐10 level. Serum IL‐10 level and the percentage of IL‐10⁺CD19⁺ were negatively correlated with the percentages of CD38⁺CD19⁺, and CD86⁺CD19⁺ B cells. These results suggest that CD86⁺CD19⁺, CD38⁺CD86⁺CD19⁺, CD95⁺CD19⁺, and especially CD38⁺CD19⁺ and IL‐10⁺CD19⁺ cells may participate in the pathogenesis of HBV‐MN.     

Manganese Ions Induce Tonic Contraction after Relaxation in a High-K+ Medium in Ileal Longitudinal Smooth Muscle of Guinea-pig

Abstract— In ileal longitudinal muscle 5Mm Mn²⁺ inhibited completely the K⁺ (60 Mm )-induced tonic tension to the base line; however, the tension progressively increased to above the level of original tonic response evoked by K⁺ after 3 h in the presence of Mn²⁺. Tetrodotoxin (5 × 10^?5 m ) had no influence on the tension development in the presence of Mn²⁺ in the high-K⁺ medium. Mn²⁺ also increased the tension in a high-K⁺, Ca²⁺-free medium. The Ca²⁺ antagonist, gallopamil (10^?6 m ) inhibited the development of tension in the presence of Mn²⁺ in the high-K⁺ medium. The ⁴⁵Ca uptake determined by the lanthanum method remained unchanged from control levels after 3 h of the 5 Mm Mn²⁺ application in the high-K⁺ medium in spite of the development of the tension. The manganese uptake in the high-K⁺ medium, increased in accordance with the increase of duration of 5 Mm Mn²⁺ application. Gallopamil inhibited manganese uptake in the high-K⁺ medium. These results suggest that Mn²⁺ firstly reduces K⁺-induced tension by inhibition of Ca²⁺ influx; subsequently, Mn²⁺ ions accumulate in the intracellular compartments through voltage-operated Ca²⁺ channels and may activate contractile proteins in the ileal muscle.     

Effects of monovalent cations on cardiac Na+, K+-ATPase activity and on contractile force

Summary The relationship between Na⁺, K⁺-ATPase inhibition by monovalent cations and their inotropic effect was studied in guinea pig hearts. The activity of partially purified cardiac enzyme was assayed in the presence of 5.8 mM KCl and either 20 or 150 mM NaCl. Rb⁺ and Tl⁺ inhibited Na⁺, K⁺-ATPase activity, the magnitude of the inhibition by these cations being greater in the assay media containing lower Na⁺ concentrations. Tl⁺ produced a dose-dependent inhibition of Na⁺, K⁺-ATPase activity in the presence of 20 mM Na⁺ and 75 mM K⁺, a cationic condition similar to that of intracellular fluid. Other monovalent cations such as K⁺, Cs⁺, NH₄ ⁺, Na⁺ or Li⁺ produced essentially no effect on the Na⁺, K⁺-ATPase activity or slightly stimulated it. In left atrial strips stimulated with field electrodes and bathed in Krebs-Henseleit solution (5.8 mM K⁺ and 145 mM Na⁺), addition of Cs⁺ failed to alter the isometric contractile force significantly. NH₄ ⁺ and K⁺ caused a transient positive inotropic effect which was partially blocked by propranolol. The positive inotropic response to K⁺ was followed by a negative inotropic response. Rb⁺ produced a sustained, dose-dependent inotropic response reaching a plateau at 1–2 min, whereas Tl⁺ produced a dose-dependent positive inotropic effect which developed slowly over a 30-min period. The positive inotropic effects produced by Rb⁺ and Tl⁺ were insensitive to propranolol pretreatment. Concentrations of Tl⁺ and cardiac glycosides which produce similar inotropic effects appear to cause the same degree of Na⁺-pump inhibition. The onset of the positive inotropic response to Rb⁺ or Tl⁺ was not dependent on the number of contractions which is in contrast to the cardiac glycoside-induced inotropic response. Substitution of 20 mM LiCl for an equimolar amount of NaCl in Krebs-Henseleit solution produced a significantly greater inotropic response than that observed when sucrose was substituted for NaCl. It appears that, among monovalent cations, only sodium pump inhibitors produce a sustained positive inotropic response.     

Synthetic glucagon antagonists and partial agonists

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys^17.18,Glu²¹]glucagon,¹ a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [d -Phe⁴,Tyr⁵,Arg¹²]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His¹ by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe⁶ residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip¹, Lys^17.18,Glu²¹]glucagon (2); [Tip¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹ glucagon (3); [dHis¹,d -Phe⁴,Tyr⁵,Arg¹², Lys^17.18,Glu²¹ glucagon (4); [dHis¹,Asp³,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹]glucagon (5); des-Phe⁶-[Tip¹,D-Phe⁴,Tyr⁵Arg¹²,Glu²¹ glucagon (6); des-Phe⁶-[Asp³,d -Phe⁴,Tyr⁵,Arg¹²,Glu²¹]glucagon (7) The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10^?5m . In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve. Hence these compounds are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase system.     

Kinetics of catecholamine sensitive Na+-K+ ATPase activity in mouse brain synaptosomes.

The effects of several neurotransmitters on mouse brain synaptosomal ATPase activities were determined in vitro. Both dopamine and norepinephrine activated Na⁺-K⁺ and Mg²⁺ ATPase activities in a dose-dependent manner. Na⁺-K⁺ ATPase was more sensitive to the catecholamines than was Mg²⁺ ATPase activity. Acetylcholine, γ-aminobutyric acid, L-glutamic acid and serotonin were without effect up to 10^?3 M concentration. Chlorpromazine, an antipsychotic agent, which has been shown to block the dopamine-receptor site, totally inhibited the dopamine-stimulated Na⁺-K⁺ and Mg²⁺ ATPase activities in mouse brain synaptosomes. Further, catecholamine-sensitive ATPase activities from mouse brain synaptosomal preparation were determined in relation to the substrate, pH and ionic concentrations in the reaction medium. The results indicate that Na⁺-K⁺ and Mg²⁺ ATPases were activated by dopamine (DA) and norepinephrine (NE) at different concentrations of ATP. Lineweaver-Burk plots reveal that Na⁺-K⁺ ATPase was activated non-competitively to ATP with a K_m value of 5.5 × 10^?4 M, whereas Mg²⁺ ATPase exhibited a mixed type of activation in that K_m was decreased and V_max increased in the presence of DA or NE. A maximum stimulation occurred by catecholamines at the optimum pH of 7.5 for Na⁺-K⁺ and 8.0 for Mg²⁺ ATPase activities. Both catecholamines increased the Na⁺-K⁺ ATPase activity in the presence of Na⁺ and K⁺ in the reaction medium. However, in the absence of Na⁺ ions the K⁺ -ATPase activity was stimulated by DA and NE but in the absence of K⁺ ions the Na⁺ ATPase, was not activated by DA or NE, indicating that the ATPase activity was more sensitive to catecholamines in the presence of K⁺ than Na⁺.     

Effects of altered availability of Na+ on guinea-pig airway smooth muscle contractility

The effects of altering the availability of sodium ions (Na⁺) on contractility of the guinea-pig isolated trachealis was examined using regimens which are reported to inhibit Na⁺/K⁺ ATPase activity (ouabain), Na⁺/H⁺ exchange (amiloride, ammonium ion (NH₄⁺)) or Na⁺/Ca²⁺ exchange (reduced extracellular Na⁺). Inhibition of Na⁺/K⁺ ATPase and reversal of Na⁺/Ca²⁺ exchange resulted in increased ⁴⁵Ca uptake and contraction of the trachealis by voltage-sensitive and voltage-insensitive mechanisms respectively. When Na⁺/H⁺ exchange was inhibited by amiloride the tissues relaxed to below their baseline tension. The relaxation was not due to reduced Ca²⁺ influx. Treatment with NH₄⁺ produced a contractile response. Reduced extracellular Na⁺ caused a transient contraction as a result of reversal of the normal Na⁺/Ca²⁺exchange process leading to accumulation of Ca²⁺ within the cell. Since the effects of amiloride and reduced extracellular sodium were different, it is unlikely that amiloride is acting primarily by inhibiting Na⁺/Ca²⁺ exchange. Amiloride reduced tissue sensitivity to methacholine and KCI without affecting Ca²⁺ influx. This may involve a secondary stimulation of Na⁺/Ca²⁺ exchange following changes in [Na'];. Ouabain also reduced tissue sensitivity to methacholine and KCl.These findings suggest that Na⁺ are important in determining smooth muscle contractility. If NH₄⁺ is altering pH then, at the concentrations used, the changes in (H⁺] were not sufficient to alter responses to the spasmogens.     

Reaction mechanism underlying the in vitro transformation of thioarsenicals

Thioarsenicals have been paid much attention due to the toxicity of arsenic, since some of them are highly toxic and commonly found in the urine of mammals. We previously reported that thioarsenicals might be produced in red blood cells (RBCs). Here, we further characterized the mechanism underlying the production and metabolism of thioarsenicals in RBCs using ³⁴S-labeled dimethylmonothioarsinic acid (³⁴S-DMMTA^V) and purified rat hemoglobin (Hb) or a rat RBC lysate. ³⁴S-DMMTA^V did not bind to Hb on incubation with purified rat Hb, remaining in its original form. However, when ³⁴S-DMMTA^V was incubated with a rat RBC lysate, only arsenic, i.e., not sulfur (³⁴S), was detected in a form bound to Hb (As-Hb). In addition, another arsenic product containing sulfur (³⁴S) in the molar ratio of ³⁴S/As = 2 was detected, which was assigned as dimethyldithioarsinic acid (DMDTA^V), suggesting that arsenic does not bind to Hb in the form of ³⁴S-DMMTA^V but does so in the form of dimethylarsinous acid (DMA^III). Namely, DMMTA^V appeared to be hydrolyzed into dimethylarsinic acid (DMA^V) and H³⁴S^-, and the released H³⁴S^- reacted with DMMTA^V to produce DMDTA^V. Thus, DMMTA^V was transformed into DMDTA^V and DMA^V (2DMMTA^V - > DMDTA^V + DMA^V), the latter product being reduced to DMA^III in the presence of GSH and bound to Hb. In a separate experiment, ³⁴S-DMMTA^V was incubated with sulfide (Na₂S) and GSH. Although DMMTA^V was not transformed into DMDTA^V in the presence of only Na₂S or GSH, it was transformed into DMDTA^V in the presence of both Na₂S and GSH. Our results suggest that DMMTA^V is hydrolyzed enzymatically into DMA^V and sulfide, the former being reduced to DMA^III and bound to Hb, and the latter reacting with DMMTA^V to yield DMDTA^V. Thus, DMMTA^V is transformed into DMDTA^V and DMA^V through a hydrolytic reaction in a manner similar to a disproportionation reaction, DMA^V being reduced and bound to Hb (As-Hb), and DMDTA^V being produced more in the presence of sulfides in the medium.     

Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS

Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of ³⁴S labeled dimethylthioarsinic acid (³⁴S-DMTA^V) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched ³⁴S-DMTA^V made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS^V). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, ³⁴S-DMTA^V underwent several transformations. Labile ³⁴S was exchanged with more abundant ³²S to produce ³²S-DMTA^V, a thiol group was added to yield DMDTA^V, and a methyl group was added to yield ³⁴S-TMAS^V. Because incubation of ³⁴S-DMTA^V resulted in the formation of ³⁴S-TMAS^V, the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS^V from dimethylarsinic acid (DMA^V) would proceed via a dimethylarsinous acid (DMA^III) intermediate and would necessitate the loss of ³⁴S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA^V to TMAS^V. Additionally, the detection of isotopically pure ³⁴S-TMAS^V raises questions about the sulfur exchange properties of TMAS^V in the cecum material. Therefore, ³⁴S-TMAS^V was incubated and the exchange was monitored with respect to time. The data suggest that the As-S bond associated with TMAS^V is less labile than the As-S bond associated with DMTA^V.     

Calmodulin-mediated cadmium inhibition of phosphodiesterase activity,in vitro

Ion-stripped bovine brain calmodulin (CaM) binds 4 moles Cd²⁺ as well as 4 moles Ca²⁺ per mole protein, with similar affinity; in the presence of 1 mM Mg²⁺ the molar binding ratio of CaM for Ca²⁺ decreased to 3, the apparent K_0.5 for Ca²⁺ nearly doubled, but the binding characteristics of CaM for Cd²⁺ were not changed. Saturating concentrations Ca²⁺ did not affect the molar binding ratio of CaM for Cd²⁺, but increased the apparent K_0.5 for Cd²⁺; vice versa, saturating concentrations Cd²⁺ decreased the molar binding ratio for Ca²⁺ to 2 without affecting the apparent K_0.5 for Ca²⁺.CaM-independent phosphodiesterase (PDE) activity was inhibited at [Cd²⁺]>10^–5 M. Cd²⁺-CaM as well as Ca²⁺-CaM activated PDE. However, the Cd²⁺-CaM complex is less effective than the Ca²⁺-CaM complex in stimulating CaM-dependent enzyme activities. Cd²⁺ inhibits Ca²⁺- and CaM-dependent PDE in a competitive way. Introduction of Cd²⁺ in a medium containing Ca²⁺ and CaM may, therefore, result in a reduction of CaM-dependent enzyme stimulation.By its interference with Ca²⁺- and CaM- dependent PDE activity, Cd²⁺ could upset the catabolic pathway of cellular cyclic nucleotide metabolism.     

Conformation-function relationships in LHRH analogs

A systematic conformational build-up procedure was performed for the LHRH molecule, pGlu¹-His²-Trp³-Ser⁴-Tyr⁵-Gly⁶-Leu⁷-Arg⁸-Pro⁹-Gly¹⁰-NH₂. The results showed a very high flexibility of the LHRH backbone, with 300 conformers being regarded as having low energy. At the same time, the conformational flexibility of LHRH differs among the fragments of the molecule. The most rigid fragments of LHRH are the Ser⁴-Tyr⁵-Gly⁶-Leu⁷ and Tyr⁵-Gly⁶-Leu⁷-Arg⁸ central tetrapeptides, the latter possessing only eight different types of low-energy backbone conformers. These eight conformer types belong to different kinds of chain reversals which are stabilized by different systems of intramolecular hydrogen bonds. Some of them resemble the β-II′ turn, which was derived as the LHRH structure from energy calculations by others. The results obtained are in good agreement with the experimental data on LHRH flexibility in solution.     

Co‐expression of LAG3 and TIM3 identifies a potent Treg population that suppresses macrophage functions in colorectal cancer patients

Regulatory T (Treg) cells are critical suppressors of inflammation and are thought to exert mainly deleterious effects in cancers. In colorectal cancer (CRC), Foxp3⁺ Treg accumulation in tumors was associated with poor prognosis. Hence, we examined the circulating Treg cells in CRC patients. Compared to controls, CRC patients presented mild upregulations in CD4⁺CD25^+/hi T cells and in the more canonical CD4⁺CD25^+/hiFoxp3⁺ Treg cells in peripheral blood mononuclear cells. Both of these Treg populations could be roughly divided into lymphocyte activation gene 3 negative T cell immunoglobulin and mucin‐domain containing‐3 negative (LAG3^?TIM3^?) and LAG3⁺TIM3⁺ subsets. In CRC patients, the LAG3⁺TIM3⁺ subset represented approximately half of CD4⁺CD25^+/hi T cells and greater than 60% of CD4⁺CD25^+/hiFoxp3⁺ Treg cells, which was significantly more frequent than in healthy controls. Compared to the LAG3^?TIM3^? CD4⁺CD25^+/hi T cells, the LAG3⁺TIM3⁺ CD4⁺CD25^+/hi T cells presented considerably higher transforming growth factor‐β and slightly higher interleukin (IL)‐10 secretion, together with higher cytotoxic T‐lymphocyte associated protein 4 and Foxp3 expression levels. Notably, macrophages following incubation with LAG3^?TIM3^? CD4⁺CD25^+/hi T cells and LAG3⁺TIM3⁺ CD4⁺CD25^+/hi T cells displayed different characteristics. Macrophages incubated with LAG3⁺TIM3⁺ CD4⁺CD25^+/hi T cells presented lower expression of major histocompatibility complex class II, CD80, CD86, and tumor necrosis factor‐α but higher expression of IL‐10, than macrophages incubated with LAG3^?TIM3^? CD4⁺CD25^+/hi T cells. Together, our investigations demonstrated that CRC patients presented an enrichment of circulating Treg cells, in which the LAG3⁺TIM3⁺ subset exhibited more potent expression of inhibitory molecules, and furthermore, the LAG3⁺TIM3⁺ Treg cells could suppress the proinflammatory activation of macrophages more potently than the LAG3^?TIM3^? Treg cells.     

NMR studies on the structure of some cyclic and linear antagonists of luteinizing hormone-releasing hormone (LHRH)

The structure of cyclic antagonists of luteinizing hormone-releasing hormone (LHRH), Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-c(Asp⁵-D-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( I ), Ac-D-Phe(p-Cl⁾l-D-Phe(p-Cl)²-D-Trp³-Ser⁴-c(Glu⁵-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( II ) and their linear analogues, Ac-D-Phe(p-Cl)¹-Phe(p-C1)²-Trp³-Ser⁴-Asp⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( III ) and Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-Glu⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( IV ), have been studied by NMR spectroscopy. The cyclic peptides I and II are more potent antagonists than the corresponding linear peptides in an in vivo assay. All the peptides show propensity of an unusual type II′β-turn involving residues 3–6. Cyclic analogues also show some additional structure around residues 7 and 8 which is absent in the linear peptides. This additional structure in the cyclic peptides may be due to a minor conformation with a β-turn between residues 5 and 8. © Munksgaurd 1995.     

New antagonists of LHRH II. Inhibition and potentiation of LHRH by closely related analogues

Modifications of the previously described LHRH antagonists, [Ac-d -Nal(2)¹, d -Phe(4Cl)², d -Trp³, d -Cit⁶, d -Ala¹⁰]LHRH and the corresponding d -Hci⁶ analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of d -Trp³ with the less hydrophobic d -Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the d -Cit/d -Hci⁶ analogues, but it appeared to further improve the toxicity lowering effect of d -Cit/d -Hci⁶ substitution. Antagonists containing d -Pal(3)³ and d -Cit/d -Hci⁶ residues, i.e. [Ac-d -Nal(2)¹, d -Phe(4Cl)², d -Pal(3)³d -Cit⁶, d -Ala¹⁰]LHRH (SB-75) and [Ac-d -Nal(2)¹, d -Phe(4Cl)², d -Pal(3)³, d -Hci⁶, d -Ala¹⁰]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the d -Trp³, d -Arg⁶ antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-d -Nal(2)¹, d -Phe(4Cl)², d -Trp³, d -Cit⁶, d -Ala¹⁰]LHRH and [Boc-d -Phe¹, d -Phe(4Cl)², d -Pal(3)³, d -Cit⁶, d -Ala¹⁰]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.     

Frequency-Dependence of the Metabolism of 3H-Noradrenaline Released from Rabbit Isolated Aorta: Effects of a Combination of Cocaine and Corticosterone or Hydrocortisone

Abstract The effect of cocaine (neuronal uptake inhibitor) in combination with either hydrocortisone or corticosterone (extraneuronal uptake inhibitors) on the metabolism of ³H-noradrenaline (³H-NA) released spontaneously or by electrical-field stimulation was studied on the isolated rabbit aorta preloaded with ³H-NA. In the spontaneous outflow ³H-O-methylated and deaminated metabolites (³H-OMDA) accounted for 41% of the total radioactivity whereas ³H-NA represented only 22%. Cocaine (3 × 10^-5M) + hydrocortisone (10^-4 M) neither changed the spontaneous outflow of total tritium nor the distribution of the ³H-outflow on ³H-NA and its ³H-metabolites. However, cocaine (3 × 100^-5M) + corticosterone (4 × 10^-5M) enhanced the passive ³H-outflow, increased the percentage recovered as ³H-3,4-dihydroxyphenylglycol (³H-DOPEG), and reduced the percentage of ³H-OMDA + ³H-NMN. The effect of field-stimulation was investigated during and ofter stimulation at two frequencies: 3 and 10 Hz. The stimulation-induced ³H-overflow consisted mainly of unmetabolized ³H-NA and ³H-OMDA in the sample collected during stimulation. The ratio between ³H-NA and ³H-OMDA was strongly dependent on the frequency of stimulation, Thus it was 1:2 at 3 Hz, but 3:1 at 10 Hz. These ratios were about the same during the poststimulation period. On the other hand, whereas the formation of ³H-DOPEG from ³H-NA released during stimulation at both frequencies was small, it increased rapidly in the poststimulation sample. At 3 Hz, inhibition of neuronal and extraneuronal uptake by cocaine + steroids did not change the stimulation-induced ³H-overflow, but the distribution on ³H-NA and ³H-metabolites was markedly altered. Thus the percentage of ³H-N A was doubled, ³H-OMDA was halved, and ³H-DOPEG was almost abolished. At 10 Hz, however, cocaine + corticosterone decreased the stimulation-induced ³H-overflow, but did not change the proportions of ³H-NA and ³H-OMDA. Only the formation of ³H-DOPEG was markedly reduced. It is concluded that the distribution of stimulation-induced ³H-overflow on ³H-NA and its ³H-metabolites and the effect of neuronal and extraneuronal uptake inhibition on this is strongly influenced by the frequency of stimulation. Furthermore, inhibition of both neuronal and extraneuronal uptake does not fully prevent metabolism of ³H-NA released by electrical-field stimulation.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis¹,Va1⁹,11e^11,16] glucagon amide, (3) [desHis¹,Val^9,11,16]glucagon amide, (4) [desHis¹,Va1⁹,Leu^11,16]glucagon amide, (5) [desHis¹,Nle⁹,11e^11,16]glucagon amide, (6) [desHis¹,Nle⁹,Val^11,16]glucagon amide, (7) [desHis¹,Nle⁹,Leu^11,16]glucagon amide, (8) [desHis¹,Val⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide and (9) [desHis¹,Nle⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC₅₀= 1.5 nM), analogues 2-9 were found to have IC₅₀ values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with PA₂, values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively. © Munksgaard 1997.