Alan W. Everett

Spatial and Temporal Distribution of Nerves, Ganglia, and Smooth Muscle during the Early Pseudoglandular Stage of Fetal Mouse Lung Development

Neural tissue and smooth muscle appear early in the developing fetal lung, but little is known of their origin and subsequent distribution. To investigate the spatial and temporal distribution of nerves, ganglia, and airway smooth muscle during the early pseudoglandular stage, fetal mouse lungs at embryonic days (E) 11 to 14 were immunostained as whole‐mounts and imaged by confocal microscopy. At E11, the primordial lung consisted of the future trachea and two budding epithelial tubules that were covered in smooth muscle to the base of the growing buds. The vagus and processes entering the lung were positive for the neural markers PGP 9.5 (protein gene product 9.5) and synapsin but no neurons were stained at this stage. An antibody to p75NTR revealed neural crest cells on the future trachea as well as in the vagus and in processes extending from the vagus to the lung. This finding indicates that even though neuronal precursors are already present at this stage, they are still migrating into the lung. By E12, neural tissue was abundant in the proximal part of the lung and nerves followed the smooth muscle‐covered tubules to the base of the growing buds. At E13 and E14, a neural network of interconnected ganglia, innervated by the vagus, covered the trachea. The postganglionic nerves mainly followed the smooth muscle‐covered tubules, but some extended out into the mesenchyme beyond the epithelial buds. Furthermore, we show in a model of cultured lung explants that neural tissue and smooth muscle persist and continue to grow and differentiate in vitro. By using fluorescent markers and confocal microscopy, we present the developing lung as a dynamic structure with smooth muscle and neural tissue in a prime position to influence growth and development.

The role of innervation in the establishment of the topographical distribution of primary myotube types during development

SummaryMany avian muscles contain a characteristic topographical distribution of fibre types. In order to study the role of nerves in the establishment and distribution of these fibre types, monoclonal antibodies (McAb) to the heavy chain subunit of myosin (MHC) were produced. The anti-fast McAb (2B12) bound to adult fast MHC and cross-reacted with the embryonic isoform of MHC. The anti-slow McAb (3D1) bound specifically to the heavy chain of slow myosin 2. By indirect immunofluorescence, anti-fast (2B12) stained all myotubes in the anterior latissimus dorsi and triceps and biceps muscles at stage 37 (11 days embryonic), whilst anti-slow (3D1) staining was largely restricted to the future slow fibres of these muscles. Brachial levels of the neural tube were surgically removed at stage 12 (2 days embryonic) so that muscles developed aneurally. Muscles at aneural stage 37 were smaller than normal, but the distribution of myotube types was not altered; all myotubes present still stained with anti-fast antibody while anti-slow staining was restricted to the anterior latissimus dorsi and myotubes in the deep parts of the triceps brachii pars scapularis, triceps brachii par humeralis and biceps brachii muscles (the future slow fibres of normal muscles). The results suggest that despite an overall reduction in MHC in aneural muscles, specialized fast and slow primary myotubes arise independently of the nerve in appropriate regions of the muscle.

Measuring the Assessment and Counseling Provided with the Supply of Nonprescription Asthma Reliever Medication: A Simulated Patient Study

Background: Over one quarter of asthma reliever medications are provided without prescription by community pharmacies in Australia. Evidence that community pharmacies provide these medications with sufficient patient assessment and medication counseling to ensure compliance with the governments Quality Use of Medicines principles is currently tacking. Objective: To assess current practice when asthma reliever medication is provided in the community pharmacy setting and to identify factors that correlate with assessment of asthma control. Methods: Researchers posing as patients visited a sample of Perth metropolitan community pharmacies in May 2007. During the visit, the simulated patient enacted a standardized scenario of someone with moderately controlled asthma who wished to purchase a salbutamol (albuterol) inhaler without prescription. Results of the encounter were recorded immediately after the visit. Regression analysis was performed, with medication use frequency (a marker of asthma control) as the dependent variable. Results: One hundred sixty community pharmacies in the Perth metropolitan area were visited in May 2007. Pharmacists and/or pharmacy assistants provided some form of assessment in 84% of the visits. Counseling was provided to the simulated patients in 24% of the visits. Only 4 pharmacy staff members asked whether the simulated patient knew how to use the inhaler. Significant correlation was found between assessment and/or counseling of reliever use frequency and 3 independent variables: visit length (p < 0.001), number of assessment questions asked (p < 0.001), and the simulated patient who conducted the visit (p < 0.02). Conclusions: Both patient assessment and medication counseling were suboptimal compared with recommended practice when nonprescription asthma reliever medication was supplied in the community pharmacy setting. Pharmacy and pharmacist demographic variables do not appear to affect assessment of asthma control. This research indicates the need for substantial improvements in practice in order to provide reliever medication in line with Quality Use of Medication principles of ensuring safe and effective use of medication.

Cellular localization of voltage-gated calcium channels and synaptic vesicle-associated proteins in the guinea pig cochlea.

The cellular localization of voltage-gated calcium channels (VGCCs) and synaptic vesicle-associated proteins, SV2, synapsin I, and vesicle-associated membrane protein (VAMP) (synaptobrevin), was investigated in the guinea pig cochlea using immunocytochemistry and confocal laser scanning microscopy. Reactivity, in guinea pig, of antibodies to the α1 subunits of L-type, α1C [Cav1.2] and α1D [Cav1.3]; P/Q-type, α1A [Cav2.1]; and R-type, α1E [Cav2.3] high voltage-activated calcium channels, was determined by Western blotting and immunolabeling of cerebellum. In the cochlea the sensory inner hair cells of the organ of Corti displayed strong intracellular staining, predominantly localized to their basolateral poles, with an antibody directed against the α1C subunit. Some α1C labeling was also observed in the inner pillar cells, in cell bodies of afferent neurons in the spiral ganglion, and in the inferior region of the spiral ligament. The supporting pillar cells were strongly immunoreactive throughout for α1D, but no α1D labeling of the inner hair cells was seen. The α1A subunit showed a cytoplasmic distribution in all three rows of outer hair cells. α1E labeling localized to the outer hair cells, predominantly in the subcuticular plate region, and also to nerve fiber bundles beneath these hair cells. Strong immunoreactivity was consistently seen with antibodies directed against SV2 and synapsin I in neuronal structures surrounding the basolateral surfaces of both the inner and outer hair cells but was absent from the sensory cells themselves. VAMP labeling was found throughout the cytoplasm of the inner hair cells and in neuronal structures beneath the hair cells. These results reveal a differential distribution of VGCC-types in the sensory and nonsensory elements of the guinea pig cochlea, with the inner hair cells expressing α1C L-type channels and VAMP but not synapsin I or SV2.

Isomyosin expression in human heart in early pre- and post-natal life.

Monoclonal antibodies to the fast (high ATPase) alpha-type myosin heavy chain and to the slow (low ATPase) beta-type myosin heavy chain of rabbit heart were used to investigate myosin expression in human fetal, infant and adult heart by immunocytochemical procedures. With the anti-HC alpha antibody human atria always showed homogeneous staining from 14 weeks gestation through to the adult. No staining of ventricles was observed with this antibody between 14 and 18 weeks gestation but in infant ventricle a wide range of staining patterns was found; most hearts (44 out of 53) displayed none or a scattering of stained cells, five hearts showed fairly widespread but mostly weak staining while only two hearts showed strong staining of the majority of the ventricular myocytes. Normal adult ventricles more closely resembled the majority of infant hearts in showing none or a handful of stained cells scattered throughout the left ventricular wall. With the anti-HC beta antibody, all ventricular cells of all hearts stained strongly while a minority of atrial cells also showed staining from 14 weeks gestation. It is concluded that the alpha-type myosin heavy chain only appears in the ventricles around the time of birth but most commonly only ever constitutes a minor proportion of total ventricular myosin.

Early changes in myocardial protein synthesis in vivo in response to right ventricular pressure overload in the dog

Pulmonary artery stenosis was produced by inflation of a cuff positioned around the artery in dogs. Right ventricular pressures averaged 40 ± 2 (s.e.) mmHg before inflation and 72 ± 3 mmHg 1 day after inflation of the cuff. After 1 day stenosis the mass of the right ventricle averaged 12% above that in control dogs in which the cuff was not inflated. The protein synthesis rate was measured in both ventricles at this time by a continuous intravenous infusion of [14C]-tyrosine. Stenosis resulted in an increase in the specific radioactivity of tyrosine in protein while that of the tissue free tyrosine was unchanged. The rate of mixed protein synthesis in the right ventricle increased from 7.2%/day to 13.9%/day after 1 day of stenosis. Myofibrillar and sarcoplasmic protein fractions showed similar increases.

Provision of Primary Care to Patients with Chronic Cough in the Community Pharmacy Setting

Background: Community pharmacies are at the forefront of primary care providers and have an important role in the referral of patients to a medical practitioner for review when necessary. Chronic cough is a common disorder in the community and requires medical assessment. The proficiency of community pharmacy staff to refer patients with chronic cough is currently unknown. Objective: To assess the ability of community pharmacy staff to recognize and medically refer patients with a chronic nonproductive cough. Methods: Following ethics approval, a simulated patient study of 156 community pharmacies in Perth, Western Australia, was conducted over a 3-month period. Simulated patients presented to the pharmacy requesting treatment for a cough. The simulated patient required a referral based on a designated scenario. Demographic details, assessment questions, and advice provided were recorded by the simulated patient immediately postvisit. A logistic regression analysis was performed, with referral for medical assessment as the dependent variable. Results: Of the 155 community pharmacies included in the analysis, 38% provided appropriate medical referral. Cough suppressants were provided as therapy in 72% of all visits. Predictors of medical referral were assessment of symptom duration, medical history, current medications being taken, frequency of reliever use, and the position of the pharmacy staff member conducting the consultation. A third of community pharmacies provided appropriate primary care by recommending medical referral advice to patients with chronic cough. The majority of pharmacy staff members acquired information from the patient that suggested a need for medical referral, yet did not provide referral advice. Conclusions: Appropriate medical referral is more likely when adequate assessment is undertaken and when a pharmacist is directly involved in the consultation. This highlights the need for pharmacies to ensure that processes are in place for patients to access the pharmacist.

Postsynaptic production of nitric oxide implicated in long-term depression at the mature amphibian (Bufo marinus) neuromuscular junction

We report here evidence for endogenous NO signalling in long‐term (> 1 h) synaptic depression at the neuromuscular junction induced by 20 min of 1 Hz nerve stimulation. Synaptic depression was characterized by a 46% reduction in the end‐plate potential (EPP) amplitude and a 21% decrease in miniature EPP (MEPP) frequency, but no change to MEPP amplitude, indicating a reduction in evoked quantal release. Both the membrane‐impermeant NO scavenger cPTIO and the NOS inhibitor+ release from the sarcoplasmic reticulum and muscle contraction were blocked with dantrolene. These data suggest that the depression depends on transmission, but not muscle contraction. The calcineurin inhibitors cyclosporin A and FK506, as well as ODQ, an inhibitor of NO‐sensitive soluble guanylyl cyclase, Rp‐8‐pCPT‐cGMPS, an inhibitor of cGMP‐dependent protein kinase, and the calmodulin antagonist phenoxybenzamine also blocked depression. We propose that low frequency synaptic transmission leads to production of NO at the synapse and depression of transmitter release via a cGMP‐dependent mechanism. The NO could be generated either directly from the muscle, or possibly from the Schwann cell in response to an unidentified muscle‐derived messenger. We showed that the long‐lasting depression of transmitter release was due to sustained activity of the NO signalling pathway, and suggest dephosphorylation of NOS by calcineurin as the basis for continued NO production.

The development of topographical maps and fibre types in toad (Bufo marinus) glutaeus muscle during synapse elimination.

1. The toad glutaeus muscle consists of two muscle compartments. A study has been made of the topographical distribution of motor units in these compartments, in relation to the fibre types which arise during different stages of development. 2. Monoclonal antibodies to myosin allowed the distribution of fibre types to be determined. In mature muscles (from toads of greater than 30 g body weight) clusters of type 5 (tonic) fibres were found exclusively at the dorsal surface of the muscle, surrounded by a layer of type 3 (slow‐twitch) fibres. A homogeneous layer of type 2 (fast‐twitch red) fibres was found beneath this dorsal rind of slow and tonic fibres. The rest of the muscle, including the ventral surface, consisted of a mosaic of type 1 (fast‐twitch white) and type 2 fibres. 3. Glycogen‐depletion methods, together with the myosin antibodies, allowed the distribution of single motor units and their fibre types to be determined. In mature muscles, axons originating from rostral spinal cord possessed muscle units located in a band extending from the ventral surface to beyond the middle of the muscle; these units consisted of 78% type 1 and 22% type 2 fibres found amongst the mosaic of type 1 and type 2 fibres. Intermediate axons possessed muscle units located primarily in the middle and dorsal half of the muscle. These units consisted mostly of type 2 fibres (29% type 1, 71% type 2) also found amongst the mosaic of type 1 and type 2 fibres. Thus rostral and intermediate units were of mixed fibre type, with type 1 fibres predominating in the former units and type 2 in the latter. Caudal axons possessed muscle units located mostly in the homogeneous layer of type 2 fibres, beneath the dorsal rind of tonic fibres; these units were almost always composed entirely of type 2 fibres. 4. The distribution of single motor units and their fibre types were determined for the caudal axons during development. In juvenile animals (toads of about 10 g body weight) the dorsal rind of tonic and slow fibres, together with the underlying homogeneous layer of type 2 fibres, were still present, but the rest of the muscle to the ventral surface consisted almost entirely of type 1 fibres. Caudal axons innervated the type 2 fibre layer at the dorsal surface as they do in mature animals. 5. The glutaeus in post‐metamorphic toads (0.15 g body weight) had only a small number of tonic and slow‐twitch fibres in the very dorsal layer of cells; the muscle was largely type 1.(ABSTRACT TRUNCATED AT 400 WORDS)

A review of the information-gathering process for the provision of medicines for self-medication via community pharmacies in developing countries

BACKGROUND Currently, no review has been completed regarding the information-gathering process for the provision of medicines for self-medication in community pharmacies in developing countries. OBJECTIVE To review the rate of information gathering and the types of information gathered when patients present for self-medication requests. METHODS Six databases were searched for studies that described the rate of information gathering and/or the types of information gathered in the provision of medicines for self-medication in community pharmacies in developing countries. The types of information reported were classified as: signs and symptoms, patient identity, action taken, medications, medical history, and others. RESULTS Twenty-two studies met the inclusion criteria. Variations in the study populations, types of scenarios, research methods, and data reporting were observed. The reported rate of information gathering varied from 18% to 97%, depending on the research methods used. Information on signs and symptoms and patient identity was more frequently reported to be gathered compared with information on action taken, medications, and medical history. CONCLUSION Evidence showed that the information-gathering process for the provision of medicines for self-medication via community pharmacies in developing countries is inconsistent. There is a need to determine the barriers to appropriate information-gathering practice as well as to develop strategies to implement effective information-gathering processes. It is also recommended that international and national pharmacy organizations, including pharmacy academics and pharmacy researchers, develop a consensus on the types of information that should be reported in the original studies. This will facilitate comparison across studies so that areas that need improvement can be identified.