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Introduction: Pursuant to the COVID-19 pandemic, an executive order issued by the New York State (NYS) governor allowed pharmacists to act as laboratory directors for a limited-service laboratory (LSL) to order and perform Food and Drug Administration (FDA) and Emergency Use Authorization (EUA) Clinical Laboratory Improvement Amendment (CLIA)-waived COVID-19 point-of-care testing (POCT). Objectives: To (i) assess the status of NYS community pharmacists with POCT in the early stages of the COVID-19 pandemic, (ii) assess the readiness and willingness of community pharmacists to incorporate COVID-19 POCT into their workflow during a pandemic, and (iii) assess community pharmacists' perception of the barrier to initiating COVID-19 POCT. Methods: This is a prospective cross-sectional study conducted from February 4 to February 21, 2021. An electronic survey consisting of 66 Likert-type questions, select all that apply, and fill-in-style questions were emailed to 250 Community Pharmacy Enhanced Service Network (CPESN) NY pharmacies, with a follow-up email sent halfway into the data collection period. The data were analyzed using descriptive statistics. Results: The result indicated that most participants (median = 5) demonstrated readiness and willingness to offer COVID-19 testing. Barriers to COVID-19 POCT were identified: impact on pharmacy workflow (59%), lack of payment mechanism (55%) and lack of sufficient training (21%). Most participants expressed interest in continuing POCT beyond the pandemic (86.1%). Conclusion: Community pharmacists in NYS reported willingness to initiate COVID-19 POCT. Addressing the identified barriers, such as workflow disruption and reimbursement challenges, will enable pharmacies to be better prepared to provide patient care, including POCT., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Primary care pharmacists are uniquely positioned to improve care quality by intervening within care transitions in the postdischarge period. However, additional evidence is required to demonstrate that pharmacist-led interventions can reduce health care utilization in a cost-effective manner. The study's objective was to evaluate the clinical and economic effectiveness of a pharmacy-led transition of care (TOC) program within a primary care setting., Methods: This cluster randomized trial was conducted between 2019 and 2021 and included three primary care practices. Eligible patients were ≥18 years of age and at high risk of readmission. The multifaceted pharmacy intervention included medication reconciliation, comprehensive medication review, and patient and provider follow-up. The primary composite endpoint included hospital readmissions and emergency department (ED) visits within 30 days of discharge. Differences in outcomes were modeled using a generalized estimated equations approach and outcomes were assumed to be distributed as a Poisson random variable. A cost-benefit analysis was embedded within the study and estimated economic outcomes from a provider group/health system perspective. Cost measures included: net benefit, benefit to cost ratio (BCR), and return on investment (ROI)., Results: Of 300 eligible patients, 36 were in the intervention group and 264 in the control group. The intervention significantly reduced the primary composite outcome of all-cause readmissions and ED visits within 30 days (adjusted incidence rate ratio [aIRR], 0.54; 95% CI, 0.44-0.66; P < 0.001). There were significant reductions in both 30-day all-cause readmissions (aIRR, 0.64; 95% CI, 0.60-0.67; P < 0.001) and ED visits (aIRR, 0.25; 95% CI, 0.20, 0.31; P < 0.001) between groups. The net benefit of the intervention was $9,078, with a BCR of 2.11 and a ROI of 111%. Sensitivity analyses were robust to changes in economic inputs., Conclusion: This care transition program had positive clinical and economic benefits, providing further support for the essential role pharmacists demonstrate in providing TOC services., Competing Interests: Disclosure DMJ is supported by the National Institutes of Health/National Heart, Lung, and Blood Institute under award number K23HL153582. This research reported in this publication was supported by the National Center for Advancing Translational Sciences and ULTR001412 to the University at Buffalo. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH., (Copyright © 2023 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Transition from hospital to home is a vulnerable period for patients with COPD exacerbations, with a high risk for readmission and mortality. Twenty percent of patients with an initial hospitalization for a COPD exacerbation are readmitted to a hospital within 30 days, costing the health care system over $15 billion annually. While nebulizer therapy directed at some high-risk COPD patients may improve the transition from hospital to home, patient and social factors are likely to contribute to difficulties with their use. Current literature describing the COPD patient's experience with utilizing nebulizer therapy, particularly during care transitions, is limited. Therefore, the objective of this study was to explore underlying COPD patient and social factors contributing to practical difficulties with nebulizer use at the care transition from hospital to home., Methods: This was a qualitative study conducted between September 2020 and June 2022. Patients were included if they were ≥ 40 years old, had a current diagnosis of COPD, had an inpatient admission at a hospital, and were discharged directly to home with nebulizer therapy. Semi-structured, one-on-one interviews with patients were conducted covering a broad range of patient and social factors and their relationships with nebulizer use and readmission. Interviews were recorded and transcribed verbatim. A thematic analysis was performed using a mixed inductive and deductive approach., Results: Twenty-one interviews were conducted, and subjects had a mean age of 64 ± 8.4 years, 62% were female, and 76% were White. The predominant interview themes were health care system interactions and medication management. The interviews highlighted that discharge counseling methods and depth of counseling from hospitals were inconsistent and were not always patient-friendly. They also suggested that patients could appropriately identify, set up, and utilize their nebulizer treatment without difficulties, but additional patient education is required for nebulizer clean up and maintenance., Conclusions: Our interviews suggest that there is room for improvement within the health care system for providing consistent, effective discharge counseling. Also, COPD patients discharged from a hospital on nebulizer therapy can access and understand their treatment but require additional education for nebulizer clean up and maintenance., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Community pharmacies in the United States are beginning to serve as patient care service destinations addressing both clinical and health-related social needs (HRSN). Although there is support for integrating social determinant of health (SDoH) activities into community pharmacy practice, the literature remains sparse on optimal pharmacy roles and practice models., Objective: To assess the feasibility of a community pharmacy HRSN screening and referral program adapted from a community health worker (CHW) model and evaluate participant perceptions and attitudes toward the program., Methods: This feasibility study was conducted from January 2022 to April 2022 at an independent pharmacy in Buffalo, NY. Collaborative relationships were developed with 3 community-based organizations including one experienced in implementing CHW programs. An HRSN screening and referral intervention was developed and implemented applying a CHW practice model. Pharmacy staff screened subjects for social needs and referred to an embedded CHW, who assessed and referred subjects to community resources with as-needed follow-up. Post intervention, subjects completed a survey regarding their program experience. Descriptive statistics were used to report demographics, screening form, and survey responses., Results: Eighty-six subjects completed screening and 21 (24.4%) an intervention and referral. Most participants utilized Medicaid (57%) and lived within a ZIP Code associated with the lowest estimated quartile for median household income (66%). Eighty-seven social needs were identified among the intervention subjects, with neighborhood and built environment (31%) and economic stability challenges (30%) being the most common SDoH domains. The CHW spent an average of 33 minutes per patient from initial case review through follow-up. All respondents had a positive perception of the program, and the majority agreed that community pharmacies should help patients with their social needs (70%)., Conclusions: This feasibility study demonstrated that embedding a CHW into a community pharmacy setting can successfully address HRSN and that participants have a positive perception toward these activities., (Copyright © 2023 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Pharmacies belonging to the Community Pharmacy Enhanced Service Networks (CPESN) are transforming their practices with support of the Flip the Pharmacy initiative. These pharmacies are submitting eCare plans that describe care that they have provided to patients., Objectives: The objectives of this study were (1) To develop a taxonomy for services reported by community pharmacies participating in year 1 of the Flip the Pharmacy initiative and (2) to illustrate the use of the taxonomy for hypertension-related services., Methods: A retrospective observational study design was used. The analyzed data were extracted from eCare plans submitted by participating pharmacies during the first year of the Flip the Pharmacy initiative (October 1, 2019-September 30, 2020). Systematized Nomenclature of Medicine - Clinical Terms (SNOMED-CT) codes submitted for encounter reason and for procedures were sorted into categories based on similarity of terminology in the SNOMED-CT code labels. All SNOMED-CT codes in the encounter reasons that had blood pressure or hypertension in their labels were mapped to taxonomy categories. Descriptive statistics were calculated for all variables., Results: A total of 368,297 eCare plans reporting 1,049,061 SNOMED-CT procedures were submitted for 133,210 patients by 526 pharmacies. Seven categories of community pharmacy patient care services were identified: medication synchronization, medication review, monitoring, immunizations, patient education, adherence, and recommendations. Over half of the encounter reasons (63.5%) and procedures (56.2%) were for medication synchronization. Both medication review and monitoring accounted for about 10% of the encounters, and medication review made up over 30% of procedures. A total of 18,307 encounters were related to hypertension. Of these, monitoring was the most frequent, with 11,285 encounters (61.6%) encounters, followed by patient education, with 5173 encounters (28.3%)., Conclusion: CPESN pharmacies are delivering a wide range of patient care services. This taxonomy provides a concise way to organize and report services being delivered by community pharmacies., (Copyright © 2022 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: While community pharmacies are an ideal setting for social needs screening and referral programs, information on social risk assessment within pharmacy practice is limited., Objectives: Our primary objective was to describe 2 social determinant of health (SDOH) practice models implemented within community pharmacies. The secondary objective was to evaluate implementation practices utilizing the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework., Practice Description: Two pharmacy groups participated in a 3-month study, one in New York (9 pharmacies) and another in Missouri (1 pharmacy). The New York pharmacies implemented an SDOH specialist practice model, in which pharmacy staff members facilitate the program. The Missouri pharmacy implemented a community health worker (CHW) model by cross training their technicians. Each pharmacy developed their program using the Community Pharmacy Enhanced Services Network Care Model., Practice Innovation: Both programs expanded the technician role to take on additional responsibilities. The SDOH specialist model partnered with a local independent practice association to create a social needs referral program using a technology platform for closed-loop communication. All workflow steps of the self-contained CHW program were completed within the pharmacy, placing additional responsibility on the CHW and pharmacy staff., Evaluation Methods: RE-AIM framework dimensions of Reach, Effectiveness, and Adoption., Results: Social challenges were identified in 49 of 76 (65%) generated SDOH screenings. The most prevalent social needs reported were affordability of daily needs (33%) and health care system navigation (15%). While most pharmacy staff indicated that workflow steps were clearly defined, assessments and referral tools were identified as potential gaps. While approximately 50% of pharmacy staff were comfortable with their assigned roles and in addressing SDOH challenges, physical and mental health concerns required additional education for intervention., Conclusion: The successful implementation of community pharmacy SDOH programs connected patients with local resources. Community pharmacies are ideally positioned to expand their public health footprint through SDOH interactions that consequently improve patient care., (Copyright © 2022 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Early hospital readmissions remain common in patients with conditions targeted by the CMS Hospital Readmission Reduction Program (HRRP). There is still no consensus on whether readmission measures should be adjusted based on social factors, and there are few population studies within the U.S. examining how social characteristics influence readmissions for HRRP-targeted conditions. The objective of this study was to determine if specific socio-demographic and -economic factors are associated with 30-day readmissions in HRRP-targeted conditions: acute exacerbation of chronic obstructive pulmonary disease, pneumonia, acute myocardial infarction, and heart failure., Methods: The Nationwide Readmissions Database was used to identify patients admitted with HRRP-targeted conditions between January 1, 2010 and September 30, 2015. Stroke was included as a control condition because it is not included in the HRRP. Multivariate models were used to assess the relationship between three social and economic characteristics (gender, urban/rural hospital designation, and estimated median household income within the patient's zip code) and 30-day readmission rates using a hierarchical two-level logistic model. Age-adjusted models were used to assess relationship differences between Medicare vs. non-Medicare populations., Results: There were 19,253,997 weighted index hospital admissions for all diagnoses and 3,613,488 30-day readmissions between 2010 and 2015. Patients in the lowest income quartile (≤$37,999) had an increased odds of 30-day readmission across all conditions (P < 0.0001). Female gender and rural hospital designation were associated with a decreased odds of 30-day readmission for most targeted conditions (P < 0.05). Similar findings were also seen in patients ≥65 years old., Conclusions: Socio-demographic and -economic factors are associated with 30-day readmission rates and should be incorporated into tools or interventions to improve discharge planning and mitigate against readmission., (© 2021. The Author(s).)

Coasts are areas of vitality because they host numerous activities worldwide. Despite their major importance, the knowledge of the main characteristics of the majority of coastal areas (e.g., coastal bathymetry) is still very limited. This is mainly due to the scarcity and lack of accurate measurements or observations, and the sparsity of coastal waters. Moreover, the high cost of performing observations with conventional methods does not allow expansion of the monitoring chain in different coastal areas. In this study, we suggest that the advent of remote sensing data (e.g., Sentinel 2A/B) and high performance computing could open a new perspective to overcome the lack of coastal observations. Indeed, previous research has shown that it is possible to derive large-scale coastal bathymetry from S-2 images. The large S-2 coverage, however, leads to a high computational cost when post-processing the images. Thus, we develop a methodology implemented on a High-Performance cluster (HPC) to derive the bathymetry from S-2 over the globe. In this paper, we describe the conceptualization and implementation of this methodology. Moreover, we will give a general overview of the generated bathymetry map for NA compared with the reference GEBCO global bathymetric product. Finally, we will highlight some hotspots by looking closely to their outputs.

Background: Nonadherence to medications is a concern due to adverse outcomes and higher costs of care. The Centers for Medicare and Medicaid Services has made adherence a key measurement for Star ratings., Objective: To evaluate the impact of a collaborative pilot program between a third-party payer, local pharmacy organization, and academic institution focusing on improving medication adherence with community pharmacies., Methods: Twenty-five community pharmacies implemented adherence-based interventions in patients ≥65 years old, who were Medicare Advantage Plan members, taking targeted medications (statins, oral diabetic medications, angiotensin-converting enzyme inhibitors [ACE-Is] and angiotensin receptor blockers [ARBs]). Outcome measures were (1) pharmacy intervention completion rate, (2) type of adherence interventions, (3) change in the proportion of days covered (PDC) following pharmacist intervention based on adherence group, and (4) nonadherence barriers., Results: A total of 1263 interventions met the eligibility criteria, and common interventions included explaining the benefit of the medication (n = 453, 35.9%) and provider follow-up (n = 109, 8.6%). Among nonadherent subjects who became adherent, the mean PDC increased by 14% (74%-88%, P < .0001), with a 12% decrease in mean PDC score in the nonadherent who remained nonadherent group (71%-58%, P < .0001). Common patient barriers for nonadherence were forgetfulness (n = 451, 35.7%) and denial (n = 84, 6.7%). System and therapeutic barriers included complexity (n = 155, 12.3%) and adverse side effects (n = 42, 3.3%)., Conclusion: This collaborative effort successfully implemented a community pharmacist-led adherence intervention in 25 independent pharmacies. Our findings highlight increased interactions with patients and in some cases improved adherence measures. Future research must include implementation outcomes in order to effectively implement these interventions in the community pharmacy setting.

Social determinants of health (SDoH) account for up to 90% of health outcomes, whereas medical care accounts for only 10%-15%; despite this disparity, only 24% of hospitals and 16% of physician practices screen for the 5 social needs. Community-embedded and highly accessible, pharmacies are uniquely positioned to connect individuals to local community and social resources and thereby address SDoH. In this article, we explore novel community pharmacy practice models that address SDoH, provide real-world examples of these models, and discuss pathways for reimbursement and sustainability. A number of innovative community pharmacy practice models that focus on social issues are currently being explored. These include integrating community health workers (CHWs) or SDoH specialists, wherein CHWs are frontline public health workers who can effectively bridge the health care system and their community, whereas SDoH specialists are pharmacy team members trained with substantial SDoH knowledge and how to use it to connect pharmacy patients to community resources. Three community pharmacy networks have implemented pilot programs using either a CHW or SDoH specialist model. An essential component for program success in all cases has been partnership development and increased interdependence between the pharmacies, local community organizations, and the public health sector. New payment models and financial incentives will be necessary to expand and sustain these programs. A potential Approach may be the use of Z codes, a subset of ICD-10-CM codes specific to assessing SDoH. Although opportunities are developing for community pharmacies to play a major role in sustainably addressing SDoH, additional work is needed before there is a widespread acceptance of pharmacies becoming service referral destinations for patients with social needs. Evaluation of these models on a wider scale will be necessary to fully evaluate their effectiveness, costs, and implementation within different community pharmacy settings., (Copyright © 2021 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: The Hospital Readmissions Reduction Program (HRRP) was introduced to reduce readmission rates among Medicare beneficiaries, however little is known about readmissions and costs for HRRP-targeted conditions in younger populations. The primary objective of this study was to examine readmission trends and costs for targeted conditions during policy implementation among younger and older adults in the U.S., Methods: We analyzed the Nationwide Readmission Database from January 2010 to September 2015 in younger (18-64 years) and older (≥65 years) patients with acute myocardial infarction (AMI), heart failure (HF), pneumonia, and acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Pre- and post-HRRP periods were defined based on implementation of the policy for each condition. Readmission rates were evaluated using an interrupted time series with difference-in-difference analyses and hospital cost differences between early and late readmissions (≤30 vs. > 30 days) were evaluated using generalized linear models., Results: Overall, this study included 16,884,612 hospitalizations with 3,337,266 readmissions among all age groups and 5,977,177 hospitalizations with 1,104,940 readmissions in those aged 18-64 years. Readmission rates decreased in all conditions. In the HRRP announcement period, readmissions declined significantly for those aged 40-64 years for AMI (p < 0.0001) and HF (p = 0.003). Readmissions decreased significantly in the post-HRRP period for those aged 40-64 years at a slower rate for AMI (p = 0.003) and HF (p = 0.05). Readmission rates among younger patients (18-64 years) varied within all four targeted conditions in HRRP announcement and post-HRRP periods. Adjusted models showed a significantly higher readmission cost in those readmitted within 30 days among younger and older populations for AMI (p < 0.0001), HF (p < 0.0001), pneumonia (p < 0.0001), and AECOPD (p < 0.0001)., Conclusion: Readmissions for targeted conditions decreased in the U.S. during the enactment of the HRRP policy and younger age groups (< 65 years) not targeted by the policy saw a mixed effect. Healthcare expenditures in younger and older populations were significantly higher for early readmissions with all targeted conditions. Further research is necessary evaluating total healthcare utilization including emergency department visits, observation units, and hospital readmissions in order to better understand the extent of the HRRP on U.S. healthcare.

Background/objectives: To examine the prevalence of potentially inappropriate medication (PIM) prescribing and its association with healthcare utilization and related expenditures utilizing nationally representative data from the United States., Design: Retrospective cohort study., Setting: The 2011-2015 Medical Expenditure Panel Survey (MEPS)., Participants: Community-dwelling sample of U.S. adults aged 65 and older during the first round of each MEPS cycle., Measurements: A qualified definition operationalized from the 2019 American Geriatrics Society Beers Criteria® was used to estimate the prevalence of PIM prescribing over the study period. Negative binomial models were assembled to examine associations between PIM exposure and healthcare utilization including hospitalizations, emergency department (ED) visits, and outpatient provider visits. Generalized linear models with the log link function and gamma distribution were used to analyze associations between PIM exposure and healthcare expenditures. Sensitivity analyses were conducted utilizing inverse probability treatment weighting using propensity scores for being prescribed a PIM., Results: The period prevalence of PIM prescribing over the 5-year sample was 34.4%. PIM prescribing was positively associated with hospitalizations (adjusted incidence rate ratio [aIRR] = 1.17; 95 confidence interval [CI] = 1.08-1.26; P < .001), ED visits (aIRR = 1.26; 95% CI = 1.17-1.35; P < .001), and outpatient provider visits (aIRR = 1.18; 95% CI = 1.14-1.21; P < .001). PIM exposure was associated with higher marginal costs within outpatient visits ($116; 95% CI = $105-$243; P < .001), prescription medications ($128; 95% CI = $72-$199; P < .001), and total healthcare expenditures ($458; 95% CI = $295-$664; P < .001). Similar results were found in our propensity score analyses., Conclusion: PIMs continue to be prescribed at a high rate among older adults in the United States. Our results suggest that receipt of PIMs is associated with higher rates of healthcare utilization and increased costs across the healthcare continuum. Further work is needed to implement evidence-based deprescribing interventions that may in turn reduce unnecessary healthcare utilization., (© 2020 The American Geriatrics Society.)

Background: Pharmacists are positioned as an accessible source of patient care services (PCS). Despite the adversity community pharmacies continue to face, the expanding opportunity of offering PCS continues to be a pathway forward., Objective: To identify community pharmacists' perceptions to deliver PCS within an enhanced service network., Methods: One-on-one semi-structured phone interviews were conducted as part of a mixed-methods approach. Interview transcripts were analyzed using a consensus codebook to draft thematic findings. Participants were recruited from an electronic survey targeting community pharmacists from the New York chapters of the Community Pharmacy Enhanced Services Network (CPESN)., Results: Twelve pharmacists were interviewed with four main themes identified. The majority of study participants were pharmacy owners (92%) devoting an average of 15 h/week to PCS and 8 h/week addressing social barriers. The main themes identified include: (1) perceptions of pharmacy profession, (2) reimbursement models and sustainability of PCS, (3) provision of patient care services, and (4) how PCS address social determinants of health., Conclusions: Offering PCS opportunities for patients is a direction many community pharmacists have embraced and are working to succeed. Ongoing research is needed focusing on community pharmacists' self-perceptions of the clinical impact and role they hold in an evolving healthcare system.

Objective: To initiate a call to action for ambulatory care pharmacists to play a more active role in transitional care management within primary care settings by discussing relevant opportunities, experiences, and challenges., Summary: With the shift to value-based health care, greater emphasis is being placed on improving patient care quality at the lowest cost. This represents an opportunity for pharmacist integration into primary care teams to address medication management challenges in the postdischarge period. Primary care pharmacists are uniquely positioned to close gaps in care not typically addressed by hospital-based programs. These pharmacists can provide complex transition interventions tailored toward individual patients, including comprehensive medication review, patient counseling, and direct collaboration with providers. For broad acceptance of these services, current challenges include identifying and prioritizing high-risk patients, establishing the cost-effectiveness of these strategies, and ultimately applying dissemination and implementation methodologies to increase the potential impact of these interventions., Conclusion: Opportunities are expanding for primary care pharmacists to play a more substantial role in transitional care management in sustainable ways. For widespread implementation of these strategies, additional research is necessary to determine their clinical effectiveness as well as cost-effectiveness and to understand better the barriers and facilitators to adopting these interventions., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Sphingosine kinase [(SK), isoforms SK1 and SK2] catalyzes the formation of the bioactive lipid, sphingosine 1-phosphate (S1P). This can be exported from cells and bind to S1P receptors to modulate vascular function. We investigated the effect of short-term hypoxia on SK1 expression and the response of arteries to S1P. SK1 expression in rat aortic and coronary artery endothelial cells was studied using immunofluorescence and confocal microscopy. Responses of rat aortic rings were studied using wire myography and reversible hypoxia induced by bubbling myography chambers with 95% N 2 :5% CO 2 Inhibitors were added 30 minutes before induction of hypoxia. S1P induced endothelium-dependent vasodilation via activation of S1P 3 receptors and generation of nitric oxide. Hypoxia significantly increased relaxation to S1P and this was attenuated by (2 R )-1-[[(4-[[3-methyl-5-[(phenylsulfonyl)methyl] phenoxy]methyl]phenyl]methyl]-2-pyrrolidinemethanol [(PF-543), SK1 inhibitor] but not ( R )-FTY720 methyl ether [(ROMe), SK2 inhibitor]. Hypoxia also increased vessel contractility to the thromboxane mimetic, 9,11-dideoxy-11 α ,9 α -epoxymethanoprostaglandin F2 α , which was further increased by PF-543 and ROMe. Hypoxia upregulated SK1 expression in aortic and coronary artery endothelial cells and this was blocked by PF-543 and 2-( p -hydroxyanilino)-4-( p -chlorophenyl)thiazole [(SKi), SK1/2 inhibitor]. The effects of PF-543 and SKi were associated with increased proteasomal/lysosomal degradation of SK1. A short period of hypoxia increases the expression of SK1, which may generate S1P to oppose vessel contraction. Under hypoxic conditions, upregulation of SK1 is likely to lead to increased export of S1P from the cell and vasodilation via activation of endothelial S1P 3 receptors. These data have significance for perfusion of tissue during episodes of ischemia., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Quantitative outcomes assessment remains a persistent challenge in orthopaedic trauma. Although patient-reported outcome measures (PROMs) and radiographic assessments such as Radiographic Union Scale for Tibial Fractures (RUST) scores are frequently used, very little evidence has been presented to support their validity for measuring structural bone formation or biomechanical integrity., Methods: In this pilot study, a sequential cohort of patients with a tibial shaft fracture were prospectively recruited for observation following standard reamed intramedullary nailing in a level-I trauma center. Follow-up at 6, 12, 18, and 24 weeks included radiographs and completion of PROMs (EuroQol 5-Dimension [EQ-5D] and pain scores). Low-dose computed tomography (CT) scans were also performed at 12 weeks. Scans were reconstructed in 3 dimensions (3D) and subjected to virtual mechanical testing via the finite element method to assess torsional rigidity in the fractured limb relative to that in the intact bone., Results: Patients reported progressive longitudinal improvement in mobility, self-care, activity, and health over time, but the PROMs were not correlated with structural bone healing. RUST scoring showed moderate intrarater agreement (intraclass coefficient [ICC] = 0.727), but the scores at 12 weeks were not correlated with the time to union (R = 0.104, p = 0.193) and were only moderately correlated with callus structural integrity (R = 0.347, p = 0.010). In contrast, patient-specific virtual torsional rigidity (VTR) was significantly correlated with the time to union (R = 0.383, p = 0.005) and clearly differentiated 1 case of delayed union (VTR = 10%, union at 36 weeks) from the cases in the normally healing cohort (VTR > 60%; median union time, 19 weeks) on the basis of CT data alone., Conclusions: PROMs provide insight into the natural history of the patient experience after tibial fracture but have limited utility as a measure of structural bone healing. RUST scoring, although reproducible, may not reliably predict time to union. In contrast, virtual mechanical testing with low-dose CT scans provides a quantitative and objective structural callus assessment that reliably predicts time to union and may enable early diagnosis of compromised healing., Level of Evidence: Therapeutic Level IV. Please see Instructions for Authors for a complete description of levels of evidence.

The structure of the blood vessel wall has historically been studied using thin cut sections using standard histological stains. In the mid-80s laser scanning confocal microscopes became available and offered investigators the chance to examine the 3D structure of thicker sections (i.e. ~60 μm depth penetration for a typical vascular wall). Unfortunately, desktop computers lagged far behind in their capacity to process and display large 3D (confocal) data sets. Even extremely highly priced graphics workstations of the early to mid-90s offered little in the way of flexible 3D viewing. Today's gaming PCs provide the kind of processing power that 3D confocal users have been waiting for. Coupled with high end animation software, virtual reality and game design software, we now have the capacity to exploit the huge data sets that modern microscopes can produce. In this chapter, the vascular wall will be used as an example of a biological tissue that can benefit from these developments in imaging hardware and software.

The mostly widely used bronchodilators in asthma therapy are β 2 -adrenoreceptor (β 2 AR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that β 2 AR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that β 2 AR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models. Our data show that inhibition of β 2 AR signaling with an aerosolized antagonist attenuates airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus-production responses to IL-13, whereas treatment with an aerosolized agonist worsens these phenotypes, suggesting that β 2 AR signaling on resident lung cells modulates the asthma phenotype. Labeling with a fluorescent β 2 AR ligand shows the receptors are highly expressed in airway epithelium. In β 2 AR -/- mice, transgenic expression of β 2 ARs only in airway epithelium is sufficient to rescue IL-13-induced AHR, inflammation, and mucus production, and transgenic overexpression in WT mice exacerbates these phenotypes. Knockout of β-arrestin-2 (βarr-2 -/- ) attenuates the asthma phenotype as in β 2 AR -/- mice. In contrast to eosinophilic inflammation, neutrophilic inflammation was not promoted by β 2 AR signaling. Together, these results suggest β 2 ARs on airway epithelial cells promote the asthma phenotype and that the proinflammatory pathway downstream of the β 2 AR involves βarr-2. These results identify β 2 AR signaling in the airway epithelium as capable of controlling integrated responses to IL-13 and affecting the function of other cell types such as airway smooth muscle cells., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

Objectives. To characterize pharmacy/MBA professionals during their entry-level and current positions and to describe their attitudes and perceptions toward their combined degree. Methods. A cross-sectional survey of University at Buffalo (UB) alumni who obtained both pharmacy and MBA degrees was used. An electronic survey was developed through collaboration with the UB School of Management and administered in winter 2015. Results. A total of 68/115 (59% response rate) pharmacy/MBA professionals responded to the survey. Post-graduate training was completed by 24% of respondents, and most commonly it was a residency program. After adjusting for inflation to 2014 dollars, the median entry-level salary for pharmacy/MBA professionals was $140,123 (mean = $144,327) and this increased to $179,947 (mean = $205,623) for those in their current position. Practice settings for entry-level professionals included pharmaceutical industry (25%) and chain pharmacies (18%). Most respondents believed that a combined degree helped in career advancement (85%) and made them more competitive in the job market (90%). Conclusion. Pharmacy/MBA professionals are well-compensated, work in a wide-range of professional settings, and have a high-level of satisfaction with their combined degree.

Objectives. To understand pharmacy students' reasons for pursuing a dual PharmD/MBA degree and their perceptions of the impact a dual degree will have on their careers. Methods. This was a cross-sectional survey of registered students in the University at Buffalo PharmD/MBA program. An electronic survey was developed through collaboration with the UB School of Management and administered in January 2015. Results. A total of 23/24 (96% response rate) students who were enrolled in the PharmD/MBA curriculum responded to the survey. Respondents identified employment opportunities following graduation and career advancement as the most influential determinants in deciding to pursue an MBA degree. All respondents (100%) felt the job marketplace for pharmacy graduates is becoming increasingly difficult, 96% believe they will earn a higher midpoint salary with a PharmD/MBA, and 82% would recommend a PharmD/MBA to first year pharmacy students. Conclusion. Students are increasingly perceptive of the challenges in the current job marketplace and are taking advantage of a dual PharmD/MBA degree.

Background: For tibial fractures, the decision to fix a concomitant fibular fracture is undertaken on a case-by-case basis. To aid in this clinical decision-making process, we investigated whether loss of integrity of the fibula significantly destabilises midshaft tibial fractures, whether fixation of the fibula restores stability to the tibia, and whether removal of the fibula and interosseous membrane for expediency in biomechanical testing significantly influences tibial interfragmentary mechanics., Methods: Tibia/fibula pairs were harvested from six cadaveric donors with the interosseous membrane intact. A tibial osteotomy fracture was fixed by reamed intramedullary (IM) nailing. Axial, torsion, bending, and shear tests were completed for four models of fibular involvement: intact fibula, osteotomy fracture, fibular plating, and resected fibula and interosseous membrane., Findings: Overall construct stiffness decreased slightly with fibular osteotomy compared to intact bone, but this change was not statistically significant. Under low loads, the influence of the fibula on construct stability was only statistically significant in torsion (large effect size). Fibular plating stiffened the construct slightly, but this change was not statistically significant compared to the fibular osteotomy case. Complete resection of the fibula and interosseous membrane significantly decreased construct torsional stiffness only (large effect size)., Interpretation: These results suggest that fixation of the fibula may not contribute significantly to the stability of diaphyseal tibial fractures and should not be undertaken unless otherwise clinically indicated. For testing purposes, load-sharing through the interosseous membrane contributes significantly to overall construct mechanics, especially in torsion, and we recommend preservation of these structures when possible., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sophisticated three-dimensional animation and video compositing software enables the creation of complex multimedia instructional movies. However, if the design of such presentations does not take account of cognitive load and multimedia theories, then their effectiveness as learning aids will be compromised. We investigated the use of animated images versus still images by creating two versions of a 4-min multimedia presentation on vascular neuroeffector transmission. One version comprised narration and animations, whereas the other animation comprised narration and still images. Fifty-four undergraduate students from level 3 pharmacology and physiology undergraduate degrees participated. Half of the students watched the full animation, and the other half watched the stills only. Students watched the presentation once and then answered a short essay question. Answers were coded and marked blind. The "animation" group scored 3.7 (SE: 0.4; out of 11), whereas the "stills" group scored 3.2 (SE: 0.5). The difference was not statistically significant. Further analysis of bonus marks, awarded for appropriate terminology use, detected a significant difference in one class (pharmacology) who scored 0.6 (SE: 0.2) versus 0.1 (SE: 0.1) for the animation versus stills group, respectively (P = 0.04). However, when combined with the physiology group, the significance disappeared. Feedback from students was extremely positive and identified four main themes of interest. In conclusion, while increasing student satisfaction, we do not find strong evidence in favor of animated images over still images in this particular format. We also discuss the study design and offer suggestions for further investigations of this type., (Copyright © 2016 The American Physiological Society.)

The evidence describing the autonomic innervation of body fat is reviewed with a particular focus on the role of the sympathetic neurotransmitters. In compiling the evidence, a strong case emerges for the interaction between autonomic nerves and perivascular adipose tissue (PVAT). Adipocytes have been shown to express receptors for neurotransmitters released from nearby sympathetic varicosities such as adrenoceptors (ARs), purinoceptors and receptors for neuropeptide Y (NPY). Noradrenaline can modulate both lipolysis (via α2- and β3-ARs) and lipogenesis (via α1- and β3-ARs). ATP can inhibit lipolysis (via P1 purinoceptors) or stimulate lipolysis (via P2y purinoceptors). NPY, which can be produced by adipocytes and sympathetic nerves, inhibits lipolysis. Thus the sympathetic triad of transmitters can influence adipocyte free fatty acid (FFA) content. Substance P (SP) released from sensory nerves has also been shown to promote lipolysis. Therefore, we propose a mechanism whereby sympathetic neurotransmission can simultaneously activate smooth muscle cells in the tunica media to cause vasoconstriction and alter FFA content and release from adjacent adipocytes in PVAT. The released FFA can influence endothelial function. Adipocytes also release a range of vasoactive substances, both relaxing and contractile factors, including adiponectin and reactive oxygen species. The action of adipokines (such as adiponectin) and reactive oxygen species (ROS) on cells of the vascular adventitia and nerves has yet to be fully elucidated. We hypothesise a strong link between PVAT and autonomic fibres and suggest that this poorly understood relationship is extremely important for normal vascular function and warrants a detailed study., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Recent advances in intramedullary (IM) nailing have focused on removing free play at the nail-screw interface to provide enhanced construct torsional stiffness. These changes also increase axial construct stiffness and reduce axial interfragmentary movement, which is required for optimal secondary fracture healing. This study tested whether a novel intramedullary nail, the Flexible Axial Stimulation (FAST) nail, can simultaneously provide controlled axial interfragmentary motion with enhanced torsional stiffness., Methods: Novel tibial nails and matched controls (N=6 per group) were tested in a cadaveric osteotomy fracture model and in explanted bench testing. In cadaver and bench tests, nails were tested in axial tension/compression, torsion, bending, and shear. Overall construct stiffness values were calculated in each loading mode and axial and torsional low-load micromotion plateaus were quantified., Findings: The novel nails produced 1 mm of controlled axial interfragmentary motion, which was associated with a 22% reduction in axial stiffness compared to standard controls (P=0.026, effect size 2.5). The novel constructs also allowed less low-load torsional movement compared to the controls (3.8 deg vs. 7.1 deg, P=0.010, effect size 1.9), which was associated with a 14% increase in overall construct torsional stiffness (P=0.003, effect size 1.3). There were no observable differences in performance between the novel and control nails in anteroposterior/mediolateral bending or shear., Interpretation: These results suggest that an IM nailing construct can provide axial interfragmentary motion while retaining high torsional stiffness, a combination which may potentially enhance healing., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Animal studies and clinical trials have suggested that early application of controlled axial micromotion can accelerate healing of long bone fractures compared to rigid fixation. However, experimental investigations of micromotion constructs have been limited to external fixators, which have a higher incidence of complications than intramedullary nails. The purpose of this study was to assess whether a novel intramedullary nail design can generate stimulatory micromotion under minimal weight-bearing loads typical of the early healing period., Methods: Eight cadaver tibiae were reamed, osteotomised, and implanted with commercially-available IM nails fitted with a custom insert that allowed 1mm of axial micromotion after proximal/distal interlocking. Specimens were mounted in a materials testing machine and subjected to cyclic axial loading while interfragmentary motion was measured using an extensometer. Implants were also tested in standard statically-locked mode., Findings: The average force required to cause distraction of the fracture gap in micromotion mode was 37.0 (SD 21.7) N. The mean construct stiffness was 1046.8 (SD 193.6) N/mm in static locking mode and 512.4 (SD 99.6) N/mm in micromotion mode (significantly different, P<0.001)., Interpretation: These results support the development of a micromotion-enabled IM nail because the forces required to cause interfragmentary movements are very low, less than the weight of the hanging shank and foot. In contrast to rigid-fixation nails, which require significant weight-bearing to induce interfragmentary motion, the micromotion-enabled nail may allow movement in non-weight-bearing patients during the early healing period when the benefits of mechanical stimulation are most critical., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The use of fluorescent ligands to analyze receptor distribution is increasing in popularity. This is due to the ever growing number of fluorescent ligands and the increased sensitivity of microscope-based technologies. Image-analysis methods have advanced to a stage where quantification of fluorescent signals is relatively simple (if used appropriately). In this chapter we describe a method of analyzing the 2D and 3D distribution of fluorescent ligands in segments of blood vessels. In addition, we introduce the issues surrounding the accurate analysis of colocalization of two different fluorescent ligands.

The discovery of β-adrenoceptors in previously unsuspected cell types is contributing to the rethinking of new drug targets. Recent developments in β-adrenoceptor pharmacology might have excited and surprised James Black, given his interest in developing drugs based on the selective manipulation of receptors to alter physiological responses. β-adrenoceptors continue to generate surprises at molecular and pharmacological levels that often require knowledge of receptor location to interpret. In this review, we emphasize the use of fluorescent ligands as the most selective means of demonstrating receptor localization. Fluorescent ligand binding in live tissues can provide quantitative pharmacological data, under carefully controlled conditions, relevant to other signalling parameters. Consideration of the role of β-adrenoceptors in many cell types (previously ignored) is needed to understand the actions of drugs at β-adrenoceptors throughout the body, particularly in the lung epithelium, vascular endothelium, immune cells and other 'structural' and 'restorative' cell types., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Blood vessels are capable of structural changes in a dynamic process called 'vascular remodelling', which involves cell growth, death, phenotypic change and migration, as well as extracellular matrix synthesis and degradation. An integrated view of the interrelationships of the different elements of the arterial wall is made possible by fluorescence confocal microscopy which enables collection of serial optical sections of relatively thick specimens without the need to cut them as with conventional histology. With the aid of image analysis software, these serial sections can be further reconstructed to obtain 3-D images, where the structures of interest are localized and quantified. Confocal microscopy can be combined with pressure myography to obtain, simultaneously, information on vascular function and 3-D structure at near-to-physiological conditions. There are a vast number of fluorescent compounds useful for imaging vessel structure and function. Nuclear dyes allow the identification of the different types of vascular cells and the quantification of their number, shape and orientation. The speed of confocal image acquisition and processing makes it possible to scan entire intact arteries stained with fluorescent kits or antibodies to locate infrequent events such as cell apoptosis, proliferation or migration. Confocal microscopy is not only useful for imaging vascular wall structure, but also to visualize and quantify, by the intensity of fluorescence, the generation of vascular cell factors such as nitric oxide or superoxide anion. In conclusion, confocal microscopy and image analysis software provide insight into vascular wall structure and function and the active process of vascular remodelling in physiological and pathological situations.

Since pyrethroids are involved in reactive oxygen species production and no investigations have yet been performed on smooth muscle cell integrity, we studied the influence of permethrin- and cypermethrin-treatment on rabbit aorta using confocal laser scanning fluorescence microscopy, which allows cell viability to be assessed within the wall of living rabbit aorta. The data obtained show that the pyrethroid-treatment (10-100μM) impairs the smooth muscle cell viability. A double-labeling protocol allowed us to distinguish cytotoxic effects of permethrin- and cypermethrin-treatment in aortic rings. In conclusion, permethrin seems to induce more oxidative stress on the aorta wall than that cypermethrin does., (Copyright © 2006 Elsevier B.V. All rights reserved.)

Purpose: In nongenital arteries a sex difference has been postulated in the dominant endothelium-derived relaxant factor(s), eg nitric oxide, prostacyclin or endothelial-derived hyperpolarizing factor. Knowledge of endothelium-derived relaxant factor mechanisms in genital tissues could influence the development of novel treatments for sexual dysfunction. We compared nitric oxide and endothelial-derived hyperpolarizing factor contributions to acetylcholine induced relaxation in the genital arteries of the 2 sexes., Materials and Methods: Male dorsal and cavernous penile arteries, and female extravaginal and intravaginal arteries from New Zealand White rabbits were studied. Acetylcholine concentration-vasodilator response curves were constructed in the presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester, K(+) channel blockers (apamin and charybdotoxin) or a combination. Indomethacin was present throughout to exclude prostacyclins., Results: Extravaginal artery relaxation was predominantly endothelial-derived hyperpolarizing factor induced. Apamin plus charybdotoxin decreased maximal relaxations from a mean +/- SEM of 77% +/- 4% to 23% +/- 3% in 6 preparations (p <0.01). However, nitric oxide and endothelial-derived hyperpolarizing factor contributed to overall function. Dorsal artery relaxation was largely nitric oxide induced. Nomega-nitro-L-arginine methyl ester decreased maximal relaxations from 90% +/- 3% to 41% +/- 9% (p <0.001) with no endothelial-derived hyperpolarizing factor involvement (p >0.05). In cavernous and intravaginal arteries nitric oxide and endothelial-derived hyperpolarizing factor contributed to acetylcholine induced relaxation, while nitric oxide predominated. Blocking nitric oxide synthase or K(+) channels indicated that myogenic tone and constitutive activity of endothelium-derived relaxant factors were present. Vasodilator nerve mediated responses were influenced by each with the former more effective., Conclusions: Vaginal inflow arteries showed a dominance of endothelial-derived hyperpolarizing factor, contrasting with nitric oxide in penile arteries. Penile arteries followed the trend that endothelial-derived hyperpolarizing factor involvement increased with decreasing vessel caliber, while the reverse was demonstrated in female arteries.

Background and Purpose: Maintained penile erection depends on the absence of alpha-adrenoceptor (alpha-AR) activation and so can be facilitated by alpha-blockers. This study seeks the alpha(1)-AR subtypes involved in order to inform the pro-erectile consequences of subtype selective blockade., Experimental Approach: Wire myography was used with dorsal (nutritional supply) and cavernous (erectile inflow) penile arteries; standard alpha-AR-selective agonists and antagonists were employed to classify responses., Key Results: In both penile arteries noradrenaline (NA) and phenylephrine (PE, alpha(1)-AR agonist) caused concentration-dependent contractions. Sensitivity to NA was increased by NA uptake blockers, cocaine (3 microM) and corticosterone (30 microM). PE responses were antagonised by phentolamine (non-selective alpha-AR: dorsal pK(B) 8.00, cavernous 8.33), prazosin (non-subtype-selective alpha(1)-AR: dorsal 8.60, cavernous 8.41) and RS100329 (alpha(1A)-AR selective: dorsal 9.03, cavernous 8.80) but not by BMY7378 (alpha(1D)-AR selective: no effect at 1-100 nM) or Rec15/2615 (alpha(1B)-AR selective: no effect at 1-100 nM). Schild analysis was straightforward in cavernous artery, indicating that PE activates only alpha(1A)-AR. In dorsal artery Schild slopes were low, though alpha(1A)-AR was still indicated. Analysis using UK 14,304 and rauwolscine indicated an alpha(2)-AR component in dorsal artery that may account for low slopes to alpha(1)-AR antagonists., Conclusions and Implications: Penile arteries have a predominant, functional alpha(1A)-AR population with little evidence of other alpha(1)-AR subtypes. Dorsal arteries (nutritional supply) also have alpha(2)-ARs. Thus, alpha-AR blockers with affinity for alpha(1A)-AR or alpha(2)-AR would potentially have pro-erectile properties; the combination of these perhaps being most effective. This should inform the design of drugs to assist/avoid penile erection.

Hypertension is associated with vascular structural alterations known as "vascular remodelling", which initially are adaptive but in the long run, lead to vascular damage and loss of function. Despite decades of study, there is still modest information on the 3-dimensional (3D) arrangement of vascular cells and extracellular matrix (ECM) and how they change under pathological situations. To address this problem we developed a technique which combines fluorescence confocal microscopy, pressure myography and image analysis, "confocal myography", which permits the study of intact resistance-sized vessels at cellular level and at physiological pressure. With the aid of this method, we have identified, in arteries from hypertensive rats, abnormal orientation of endothelial, smooth muscle cells (SMC) and elastic fibres; elongation and denudation of endothelial cells, and adventitial hypercellularity. Confocal myography offers a new approach to the study of vascular remodelling in intact small arteries from a 3D point of view.

Resistance artery narrowing and stiffening are key elements in the pathogenesis of essential hypertension, but their origin is not completely understood. In mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR), we have shown that inward remodeling is associated with abnormal elastic fiber organization, leading to smaller fenestrae in the internal elastic lamina. Our current aim is to determine whether this alteration is an early event that precedes vessel narrowing, or if elastic fiber reorganization in SHR arteries occurs because of the remodeling process itself. Using MRA from 10-day-old, 30-day-old, and 6-mo-old SHR and normotensive Wistar Kyoto rats, we investigated the time course of the development of structural and mechanical alterations (pressure myography), elastic fiber organization (confocal microscopy), and amount of elastin (radioimmunoassay for desmosine) and collagen (picrosirius red). SHR MRA had an impairment of fenestrae enlargement during the first month of life. In 30-day-old SHR, smaller fenestrae and more packed elastic fibers in the internal elastic lamina were paralleled by increased wall stiffness. Collagen and elastin levels were unaltered at this age. MRA from 6-mo-old SHR also had smaller fenestrae and a denser network of adventitial elastic fibers, accompanied by increased collagen content and vessel narrowing. At this age, elastase digestion was less effective in SHR MRA, suggesting a lower susceptibility of elastic fibers to enzymatic degradation. These data suggest that abnormal elastic fiber deposition in SHR increases resistance artery stiffness at an early age, which might participate in vessel narrowing later in life.

The role of alpha(1D)-adrenoceptors in vasoconstrictor responses to noradrenaline in mouse femoral resistance arteries was investigated using wire myography in alpha(1D)-adrenoceptor knockout (alpha(1D)-KO) and wild-type (WT) mice of the same genetic background.alpha(1D)-KO mice were 2.5-fold less sensitive than WTs to exogenous noradrenaline and BMY 7378 was significantly less potent against noradrenaline in alpha(1D)-KO mice than in WTs, showing a minor contribution of alpha(1D)-adrenoceptors in response to noradrenaline. Prazosin and 5-methyl-urapidil were equally effective against noradrenaline in alpha(1D)-KO and WT mice. Chloroethylclonidine produced a significantly greater attenuation of the response to noradrenaline in alpha(1D)-KO mice than in WTs. Responses to electrical field stimulation (EFS), at 2-20 Hz for 10 s and 0.09 ms pulse width were significantly smaller overall in alpha(1D)-KOs than in WTs although no significant differences were seen at the different frequencies.BMY 7378 produced significantly greater inhibition of responses at 2 and 5 Hz than at higher frequencies in WTs. In alpha(1D)-KOs, this greater sensitivity to BMY 7378 at lower frequencies was not apparent, confirming that the effect of BMY 7378 was due to blockade of alpha(1D)-adrenoceptors. Prazosin and 5-methyl-urapidil had similar inhibitory effects on responses to EFS in alpha(1D)-KO and WT mice. Chloroethylclonidine inhibited responses to EFS to a significantly greater extent in alpha(1D)-KO mice. The present study with alpha(1D)-KO mice shows that alpha(1D)-adrenoceptors contribute to vasoconstrictor responses to exogenous and neurally released noradrenaline in femoral resistance arteries.

1 Recent evidence supports additional subtypes of vasodilator beta-adrenoceptor (beta-AR) besides the 'classical' beta(2). The aim of this study was to investigate the distribution of beta-ARs in the wall of rat mesenteric resistance artery (MRA), to establish the relative roles of beta-ARs in smooth muscle and other cell types in mediating vasodilatation and to analyse this in relation to the functional pharmacology. 2 We first examined the vasodilator beta-AR subtype using 'subtype-selective' agonists against the, commonly employed, phenylephrine-induced tone. Concentration-related relaxation was produced by isoprenaline (pEC(50): 7.70+/-0.1) (beta(1) and beta(2)). Salbutamol (beta(2)), BRL 37344 (beta(3)) and CGP 12177 (atypical beta) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the 'beta(3)-adrenoceptor agonist' CL 316243 was ineffective. 3 In arteries precontracted with 5-HT or U 46619, isoprenaline produced concentration-related relaxation but salbutamol, BRL 37344, CGP 12177 and CL 316243 did not. SR 59230A, CGP 12177 and BRL 37344 caused a parallel rightward shift in the concentration-response curve to phenylephrine indicating competitive alpha(1)-AR antagonism, explaining the false-positive 'vasodilator' action against phenylephrine-induced tone. Endothelial denudation but not L-NAME slightly attenuated isoprenaline-mediated vasodilatation in phenylephrine and U 46619 precontracted MRA. 4 The beta-AR fluorescent ligand BODIPY TMR-CGP 12177 behaved as an irreversible beta(1)-AR antagonist in MRA and bound to the surface and inside vascular smooth muscle cells in intact vascular wall. Beta-ARs in smooth muscle cells were observed in a perinuclear location, consistent with the location of Golgi and endoplasmic reticulum. 5 Binding of BODIPY TMR-CGP 12177 was inhibited by BAAM (1 microM) in all three vascular tunics, confirming the presence of beta-ARs in adventitia, media and intima. Binding in adventitia was observed in both neuronal and non-neuronal cell types. Lack of co-localisation with a fluorescent ligand for alpha-ARs confirms the selectivity of BODIPY TMR-CGP 12177 for beta-ARs over alpha-ARs. 6 Our results support the presence of functional vasodilator beta(1)-ARs and show that they are mainly located in smooth muscle cells. Furthermore, we have demonstrated, for the first time, the usefulness of BODIPY TMR-CGP 12177 for identifying beta-AR distribution in the 'living' vascular wall.

UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine]-mediated vasodilator responses were studied on wire myograph-mounted mouse aorta to determine the cells involved, mechanisms of action, and subtypes of alpha(2)-adrenoceptors. In the presence of induced tone, UK-14,304 produced concentration-related vasodilatation that was abolished by rauwolscine, N(omega)-nitro-L-arginine methyl ester (L-NAME), or endothelium removal, indicating that endothelial alpha(2)-adrenoceptors can release nitric oxide. In the alpha(2A)-adrenoceptor knockout mouse and the D79N mouse, a functional knockout of the alpha(2A)-adrenoceptor, these relaxant effects of UK-14,304 were lost, indicating the involvement of the alpha(2A)-adrenoceptor. UK-14,304 could also contract aorta: a small contraction occurred at high concentrations, was enhanced by L-NAME, and was absent in the alpha(1D)-adrenoceptor knockout mouse, indicating activation of the alpha(1D)-adrenoceptor. There was no evidence for a contractile alpha(2)-adrenoceptor-mediated response. A fluorescent ligand, quinazoline piperazine bodipy, antagonized the relaxant action of UK-14,304. This compound could be visualized on aortic endothelial cells, and its binding could be prevented by rauwolscine, providing direct evidence for the presence of alpha(2)-adrenoceptors on the endothelium. Norepinephrine reduced tone in the alpha(1D)-adrenoceptor knockout and controls, an effect blocked by rauwolscine and L-NAME but not by prazosin. This suggests that norepinephrine activates endothelial alpha(2)-adrenoceptors. In conclusion, the endothelium of mouse aorta has an alpha(2A)-adrenoceptor that responds to norepinephrine; promotes the release of nitric oxide, causing smooth muscle relaxation; and that can be directly visualized. Knockout or genetic malfunction of this receptor should increase arterial stiffness, exacerbated by raised catecholamines, and contribute to heart failure.

Conventionally, the architecture of arteries is based around the close-packed smooth muscle cells and extracellular matrix. However, the adventitia and endothelium are now viewed as key players in vascular growth and repair. A new dynamic picture has emerged of blood vessels in a constant state of self-maintenance. Recent work raises fundamental questions about the cellular heterogeneity of arteries and the time course and triggering of normal and pathological remodelling. A common denominator emerging in hypertensive remodelling is an early increase in adventitial cell density suggesting that adventitial cells drive remodelling and may initiate subsequent changes such as re-arrangement of smooth muscle cells and extracellular matrix. The organization of vascular smooth muscle cells follows regular arrangements that can be modelled mathematically. In hypertension, new patterns can be quantified in these terms and give insights to how structure affects function. As with smooth muscle, little is known about the organization of the vascular endothelium, or its role in vascular remodelling. Current observations suggest that there may be a close relationship between the helical organization of smooth muscle cells and the underlying pattern of endothelial cells. The function of myoendothelial connections is a topic of great current interest and may relate to the structure of the internal elastic lamina through which the connections must pass. In hypertensive remodelling this must present an organizational challenge. The objective of this paper is to show how the functions of blood vessels depend on their architecture and a continuous interaction of different cell types and extracellular proteins.

Conventional techniques to visualise microvascular structure often involve fixed tissue slices that provide two-dimensional images. A previous study using diffusive labelling of fresh, dissected tissue samples with fluorescently-tagged endothelial markers demonstrated the possibility of examining the three-dimensional architecture of the microvasculature using confocal microscopy. The present study extends the use of this quick and simple method of diffusive labelling to examine the possibility of repeatedly measuring changes in the morphology of intact microvessel in response to pharmacological stimuli. Initially, three-dimensional surface-rendered images of the same microvessel derived from the placenta and subcutaneous biopsies demonstrated morphological and topological changes in response to temperature and increasing potassium changes of physiological salt solutions, respectively. Furthermore, a dose-response study was performed with subcutaneous microvessels using the potent vasodilator, adrenomedullin. Analysis of a series of z-stack, superimposed to form a single maximum brightness image, demonstrated an inverse dose-response relationship, with responses to increasing adrenomedullin concentrations (10(-12) to 10(-8) M). In vessels that had constricted in response to noradrenaline (diameters: 22.4 to 58.0 microm), physiological concentrations of 10(-12) M increased vessel diameter by 108% above baseline conditions. Control treatment using physiological salt solution did not demonstrate any changes. The technique described suggest that diffusive labelling with vascular endothelial markers such as ulex europeaus agglutinin I in live tissue samples may be used in conjunction with confocal microscopy to demonstrate heterogeneous morphological and topological changes in intact segments of the microvasculature.

The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine alpha(1a)-adrenoceptors (ARs). Data indicate that the receptor-ligand complex is spontaneously internalized by beta-arrestin-dependent endocytosis. Internalization of the ligand did not occur in beta-arrestin-deficient cells; was blocked or reversed by another alpha(1) ligand, phentolamine, indicating it to reflect binding to the orthosteric recognition site; and was prevented by blocking clathrin-mediated endocytosis. The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50 to 60 min and was reversible on ligand removal, indicating a dynamic equilibrium. In cells expressing a human alpha(1a)-AR-enhanced green fluorescent protein (EGFP) 2 fusion protein, BODIPY-R-558/568-prazosin (RQAPB) colocalized with the fusion, indicating that the ligand gained access to all compartments containing the receptor, and, conversely, that the receptor has affinity for the ligand at all of these sites. The distribution of QAPB binding sites was similar for receptors with or without EGFP2, validating the fusion protein as an indicator of receptor location. The ligand partially colocalized with beta-arrestin in recycling and late endosomes, indicating receptor transit without destruction. Organelles containing receptors showed considerable movement consistent with a transportation function. This was absent in beta-arrestin-deficient cells, indicating that both constitutive receptor internalization and subsequent intracellular transportation are beta-arrestin-dependent. Calculations of relative receptor number indicate that at steady state, less than 30% of receptors reside on the cell surface and that recycling is rapid. We conclude that alpha(1a)-ARs recycle rapidly by an agonist-independent, constitutive, beta-arrestin-dependent process and that this can transport "alpha-blockers" into cells carrying these receptors.

1. alpha(1)-Adrenoceptor (AR) subtypes in mouse carotid arteries were characterised using a combination of agonist/antagonist pharmacology and knockout (KO) mice. 2. Phenylephrine (PE) was most potent in the alpha(1B)-KO (pEC(50)=6.9+/-0.2) followed by control (pEC(50)=6.3+/-0.06) and alpha(1D)-KO (pEC(50)=5.5+/-0.07). Both N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulphonamide hydrobromide (A-61603) and 5-hydroxytryptamine (5-HT) were more potent in the alpha(1D)-KO (pEC(50)=7.4+/-0.27 and 7.4+/-0.05, respectively) than the control (pEC(50)=6.9+/-0.09 and 6.9+/-0.08, respectively) and equipotent with the control in the alpha(1B)-KO (pEC(50)=6.7+/-0.07 and 6.8+/-0.04). Maximum responses to PE and A-61603 were reduced in the alpha(1D)-KO compared to control; there was no difference in maximum responses to 5-HT. 3. In control arteries, prazosin and 5-methylurapidil acted competitively with pA(2) of 9.6 and 7.5, respectively. BMY7378 produced antagonism only at the highest concentration used (100 nM; pK(B) 8.3). 4. Prazosin, 5-methylurapidil and BMY7378 acted competitively in alpha(1B)-KO carotid arteries with pA(2) of 10.3, 7.6 and 9.6, respectively. 5. In the alpha(1D)-KO, against PE, 5-methylurapidil produced a pA(2) of 8.1. pK(B) values were calculated for prazosin (10.6) and BMY7378 (7.0). Against A-61603, 5-methylurapidil had a pA(2) of 8.5, prazosin 8.6, while BMY7378 had no effect. 6. In conclusion, the alpha(1B)-KO mediates contraction solely through alpha(1D)-ARs and the alpha(1D)-KO through alpha(1A)-ARs. Extrapolating back to the control from the knockout data suggests that all three subtypes could be involved in the responses, but we propose that the alpha(1D)-AR causes the contractile response and that the role of the alpha(1B)-AR is mainly regulatory.

Fluorescent ligands provide the means of studying receptors in whole tissues using confocal laser scanning microscopy and have advantages over antibody- or non-fluorescence-based method. Confocal microscopy provides large volumes of images to be measured. Histogram analysis of 3-D image volumes is proposed as a method of graphically displaying large amounts of volumetric image data to be quickly analyzed and compared. The fluorescent ligand BODIFY FL-prazosin (QAPB) was used in mouse aorta. Histogram analysis reports the amount of ligand-receptor binding under different conditions and the technique is sensitive enough to detect changes in receptor availability after antagonist incubation or generic manipulations. QAPB binding was concentration dependent, causing concentration-related rightward shifts in histogram. In the presence of 10 microM phenoxybenzamine (blocking agent), the QAPB (50 nM) histogram overlaps the autofluorescence curve. The histogram obtained for the 1D knockout aorta lay to the left of that control and 1B knockout aorta, indicating a reduction in 1D receptors. We have shown, for the first time, that it is possible to graphically display binding of a fluorescent drug to a biological tissue. Although our application is specific to adrenergic receptors, the general method could be applied to any volumetric, fluorescence-image-based assay.

Fluorescent molecules bound to receptors can show their location and, if binding is reversible, can provide pharmacological information such as affinity and proximity between interacting molecules. The spatial precision offered by visualisation transcends the diverse localisation and low molecular concentration of receptor molecules. Consequently, the relationships between receptor location and function and life cycles of receptors have become better understood as a result of fluorescent labeling. Each of these aspects contributes new insights to drug action and potential new targets. The relationships between spatial distribution of receptor and function are largely unknown. This is particularly apparent for native receptors expressed in their normal host tissues where communication between heterogeneous cell types influences receptor distribution and function. In cultured cell systems, particularly for G-protein-coupled receptors (GPCR), fluorescence-based methods have enabled the visualisation of the cycle of agonist-stimulated receptor clustering, endocytic internalisation to the perinuclear region, degradation of the receptor-ligand complex, and recycling back to the surface membrane. Using variant forms of green fluorescent protein (GFP), antibodies, or fluorescent ligands, it is possible to detect or visualise the formation of oligomeric receptor complexes. Careful selection of fluorescent molecules based on their spectral properties enables resonance energy transfer and multilabel visualisation with colocalisation studies. Fluorescent agonist and antagonist ligands are now being used in parallel with GFP to study receptor cycling in live cells. This review covers how labeling and visualisation technologies have been applied to the study of major pharmacologically important receptors and illustrates this by giving examples of recent techniques that have relied on GFP, antibodies, or fluorescent ligands alone or in combination for the purpose of studying GPCR.

Chronic hypertension is associated with resistance artery remodelling and mechanical alterations. However, the contribution of elastin has not been thoroughly studied. Our objective was to evaluate the role of elastin in vascular remodelling of mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR). MRA segments from Wistar Kyoto rats (WKY) and SHR were pressurised under passive conditions at a range of physiological pressures with pressure myography. Confocal microscopy was used to determine differences in the quantity and organisation of elastin in intact pressure-fixed arteries. To assess the contribution of elastin to MRA structure and mechanics, myograph-mounted vessels were studied before and after elastase incubation. When compared with WKY, MRA from SHR showed: (1) a smaller lumen, (2) decreased distensibility at low pressures, (3) a leftward shift of the stress-strain relationship, (4) redistribution of elastin within the internal elastic lamina (IEL) leading to smaller fenestrae but no change in fenestrae number or elastin amount. Elastase incubation (1) fragmented the structure of IEL in a concentration-dependent fashion, (2) abolished all the structural and mechanical differences between strains, and (3) decreased distensibility at low pressures. The study shows the overriding role of elastin in determining vascular dimensions and mechanical properties in a resistance artery. In addition, it informs hypertensive remodelling. MRA remodelling and increased stiffness are accompanied by elastin restructuring within the IEL and elastin degradation reverses structural and mechanical alterations of SHR MRA. Differences in elastin organisation are, therefore, a central element in small artery remodelling in hypertension.

Objective: The purpose of this study was to examine the relationship of performance on the Developmental Test of Visual-Motor Integration (VMI; Beery, 1997) to handwriting legibility in children attending kindergarten. The relationship of using lined versus unlined paper on letter legibility, based on a modified version of the Scale of Children's Readiness in PrinTing (Modified SCRIPT; Weil & Cunningham Amundson, 1994) was also investigated., Method: Fifty-four typically developing kindergarten students were administered the VMI; 30 students completed the Modified SCRIPT with unlined paper, 24 students completed the Modified SCRIPT with lined paper. Students were assessed in the first quarter of the kindergarten school year and scores were analyzed using correlational and nonparametric statistical measures., Results: Strong positive relationships were found between VMI assessment scores and student's ability to legibly copy letterforms. Students who could copy the first nine forms on the VMI performed significantly better than students who could not correctly copy the first nine VMI forms on both versions of the Modified SCRIPT., Conclusion: Visual-motor integration skills were shown to be related to the ability to copy letters legibly. These findings support the research of Weil and Cunningham Amundson. Findings from this study also support the conclusion that there is no significant difference in letter writing legibility between students who use paper with or without lines.

Pharmacological analysis alone has failed to clarify the role of the three alpha(1)-adrenoceptor subtypes in modulating vascular tone, due to a lack of sufficiently selective antagonists, particularly for the alpha (1B)-adrenoceptor, and the complexity when three receptor subtypes are potentially activated by the same agonist. We adopted a combined genetics/ pharmacology strategy based on the alpha(1B)-adrenoceptor knockout (KO) mouse. The potency of three alpha(1)-adrenoceptor antagonists vs. phenylephrine was tested in aorta, carotid, mesenteric, and caudal isolated arteries from KO and wild-type (WT) mice. In the KO mouse the pharmacology became straightforward, showing alpha(1D) in two major conducting arteries (aorta and carotid) and alpha(1A) in two distributing arteries (mesenteric and caudal). By combining antagonist pharmacology and genetics, we provide a simplified analysis of alpha(1)-mediated vasoconstriction, demonstrating that alpha(1D) and alpha(1A) are the major subtypes involved in vasoconstriction, with a minor but definite contribution from alpha(1B) in every vessel.

This paper presents a theoretical analysis of the spatially averaged free-field responses of phase-sensitive and phase-insensitive receivers centered in the beam of a harmonically excited piston transmitter. The responses of unfocused circular plane piston receivers are analyzed, and both unfocused and spherically focused piston transmitters are considered. A set of closed-form expressions figures prominently in the analysis. The expressions are based on the Lommel diffraction formulation which is, in turn, based on the Fresnel approximation. Although approximate, the expressions allow for quick and easy estimation of phase-sensitive or phase-insensitive unfocused piston receiver responses. It is shown that the spatial averaging effects associated with phase-sensitive and phase-insensitive receivers are virtually identical when gamma < or = 0.1, where gamma = b/a is the ratio of receiver radius b to transmitter radius a. In addition, numerical results obtained from the closed-form expressions are compared with previously reported results. The comparisons indicate that the approximate results are valid from the m = 3 maxima forward under the assumption of linear propagation when ka > 58, where k is the circular wave number. Finally, it is pointed out that the closed-form expressions may prove useful in the estimation of the potential for bioeffects associated with diagnostic ultrasound.

alpha(1)-Adrenergic receptors (alpha(1A), alpha(1B), and alpha(1D)) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the alpha(1A) and alpha(1D) subtypes is thought to be mainly responsible for this activity, the role of the alpha(1B)-adrenergic receptor (alpha(1B)AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active alpha(1B)ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by alpha(1B)AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of alpha(1)ARs that is important in systemic regulation of blood pressure.