Evaluation of medical prescriptions and off-label use on board ships to improve healthcare quality.

OBJECTIVE: This study analyzed the appropriateness of drug therapies prescribed for a particular category of patients: the seafarers. We investigated an important problem of this area: the off-label use of drugs, which resulted to be a consequence of major shortcomings in the on-board pharmacies of ships. The off-label use of drugs is allowed, but can lead to some not negligible ethical and health problems, compromising the quality of provided healthcare. MATERIALS AND METHODS: The analysis was performed on electronic health records of patients onboard ships without physicians, and assisted by the CIRM from 2011 to 2015. This work is divided into two phases: in the first one, we classified the diagnoses registered onboard on the basis of the ICD-10 classification proposed by the WHO. In the second phase, we evaluated the congruence of the pharmacological therapies prescribed by CIRM physicians, according to the MICROMEDEX Database, which provides comprehensive information about drugs and their use. RESULTS: From the analysis emerged that prescribed drugs were not always corresponded to their primary indication of use. In particular, in 2011 off-label drug use was widely spread (more than 30%) in some ICD-10 classes. In the following years (2012-2015) a decrease of off-label use of drugs was noticed. CONCLUSIONS: The results suggest that a standardization of onboard pharmacies is crucial, in order to have a complete on-board pharmacy that will allow preventing and counteracting any situation of health danger, which may occur onboard, ensuring high quality healthcare to seafarers all over the world.

Knowledge and Attitudes on Food Hygiene among Food Services Staff on Board Ships.

BACKGROUND: Ships have long been sites for outbreaks of infectious diseases, particularly gastrointestinal diseases. The ship environment has the potential to facilitate the spread of such diseases, infecting susceptible cohorts of embarked passengers and crew. Gastrointestinal disorders among seafarers are fairly common and usually represent the first or second cause of requests for medical assistance aboard ships in international waters. STUDY DESIGN: The purpose of this study was to evaluate food safety and the level of knowledge among food service personnel on board merchant ships, where food handlers could be a cause of health problems for all crew members. METHODS: An anonymous self-administered questionnaire containing specific questions about food hygiene and safety knowledge was administered. RESULTS: The overall score of correct answers for the food safety aspects tested was 51.77 (SD 3.87) out of 76 points, corresponding to 68.12% of questions answered correctly. Food workers who followed a training course showed higher mean knowledge score (p < 0.05) compared to not-trained workers. The most relevant significant differences (p < 0.05) were noticeable in the personal hygiene, cross-contamination, safe storage, and knowledge of foodborne pathogens sections. CONCLUSION: Food services staff on board cargo ships should be adequately trained and should understand basic and fundamental aspects of food hygiene, related pathologies and sanitation, as they are responsible for the health and wellbeing of many seafarers.

Choline-Containing Phospholipids: Structure-Activity Relationships Versus Therapeutic Applications.

Choline is a quaternary ammonium salt, and being an essential component of different membrane phospholipids (PLs) contributes to the structural integrity of cell membranes. Choline-containing phospholipids (CCPLs) include phosphatidylcholine (PC), sphingomyelin (SM), and choline alphoscerate (GPC). PC is the major phospholipid in most eukaryotic cells. It is involved in SM synthesis, choline/choline metabolite re-generation, and fatty acid/GPC formation. This paper has reviewed chemical, biological and therapeutic features of CCPLs by analyzing: a) effects of exogenous CCPLs, b) influence of GPC treatment on brain cholinergic neurotransmission, and c) neuroprotective effects of GPC alone or in association with acetylcholinesterase inhibitors in animal models of brain vascular injury, d) synthesis of the choline analogs, containing a short alkyl chain instead of a methyl group. Cytidine-diphosphocholine and GPC, protect cell membranes and could be helpful in the sequelae of cerebrovascular accident treatment. Moreover, cellular membrane breakdown is suggested as a feature of neurodegeneration both in acute (stroke) and in chronic (Alzheimer and vascular dementia) brain disorders. Published data were focused to a larger extent on the biosynthesis, relevant role in cell life, and crucial involvement of CCPLs in cholinergic neurotransmission. The possibility of their use in the treatment of cerebrovascular and neurodegenerative disorders is suggested by published clinical studies. In line with these potential practical applications in pharmacotherapy, the need of further research in the field of the synthesis of new choline derivatives with possible activity in nervous system diseases characterized by cholinergic impairment is discussed.

Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures.

Astrocyte activity may be modulated by steroid hormones and GFs. This study investigates the interaction between glucocorticoids or estrogens and GFs on the expression of heme oxygenase-1 (HO-1) and cyclin D1 in astrocyte cultures at 14 days treated for 48 or 60 hr with dexamethasone (DEX) or 48 hr with 17ß-estradiol (E2) alone or with GFs added only in the last 12 or 24 hr. Twelve- or twenty-four-hour epidermal growth factor (EGF) treatment significantly enhanced HO-1 expression in astrocyte cultures pretreated for 48 hr with DEX. A highly significant increase in HO-1 expression was obtained after the last-12-hr EGF treatment in 48-hr E2-pretreated astrocyte cultures; this enhancement was particularly significant in 48-hr E2-pretreated cultures as well as in the last-12-hr insulin-treated ones pretreated for 48 hr with E2. Sixty-hour DEX-alone pretreatment as well as the last-12-hr EGF treatment in 60-hr DEX-pretreated astrocyte cultures showed a significant increase of cyclin D1 expression. A significant decrease of cyclin D1 expression in the last-12-hr insulin-like growth factor-I (IGF-1)-treated cultures pretreated for 60 hr with DEX was observed. A highly significant enhancement in cyclin D1 expression in 14 days in vitro astrocyte cultures pretreated with E2 alone for 48 hr and treated in the last 12 hr with IGF-1 in 48-hr E2-pretreated cultures was found. Finally, the data highlight an interactive dialogue between the growth factors and glucocorticoids or estrogens during the maturation of astroglial cells in culture that may control the HO-1 and cyclin D1 expression as well as proliferating astroglial cells during the cell cycle.

Effect of lipoic acid and α-glyceryl-phosphoryl-choline on astroglial cell proliferation and differentiation in primary culture.

Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in glucose metabolism of different cell types. Choline alphoscerate (α-glyceryl-phosphoryl-choline [αGPC]) is a semisynthetic derivative of phosphatidylcholines representing, among acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte cultures treated with 50 µM (+)lipoic acid or (+/-)lipoic acid and/or 10 mM αGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of DNA status detected by alkaline comet assay. The addition of single drugs [(+)lipoic acid, (+/-)lipoic acid, or αGPC] induced an "upward modulation" of the expression of biomarkers used in our study. On the contrary, the cotreatment with either (+)lipoic acid + αGPC or (+/-)lipoic + αGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers. This latter finding demonstrated no additional effect after the cotreatment with both drugs with respect to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the processing of these neuroprotective agents in our in vitro models. Finally, these preliminary findings may represent a good tool with which to clarify the antioxidant and metabolic roles played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after brain lesions or damage correlated with oxidative stress that may occur in some degenerative diseases.

Cholinergic precursors modulate the expression of heme oxigenase-1, p21 during astroglial cell proliferation and differentiation in culture.

Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell differentiation. Further, some proteins related to cell cycle including cyclins and p21 are important markers of astrocyte cultures. Aim of investigation was to study the effects of cholinergic precursors (choline, CDP-choline, Acetylcholine and α-Glyceril-Phosphorylcholine) on HO-1 and p21 expression during astroglial cell proliferation and differentiation in primary cultures at 14 and 35 days in vitro (DIV) treated for 24 h with choline metabolites. Our results showed a slight reduction of HO-1 expression (data not statistical significant) in astroglial cell cultures treated with CDP-choline at 14 DIV and 35 DIV. On the contrary, ACh and choline induced a significant increase of HO-1 expression in 14 DIV astrocyte cultures. Surprisingly, choline and ACh dramatically reduced HO-1 expression at 35 DIV. A slight decrease not statistical significant was detectable for α-GPC at 14 DIV and particularly significant at 35 DIV. Data concerning p21 expression, a well known protein inhibiting cell cycle, evidenced a significant increase at 14 and 35 DIV after α-GPC treatment. CDP-choline treatment caused a high increase of p21 expression in 14 DIV astrocyte cultures, but no modification at 35 DIV. Instead, ACh treatment induced a marked increment of p21 expression at 35 DIV. Our data suggest that cholinergic precursors modulate HO-1 and p21 expression during astroglial cell proliferation and differentiation in culture and could be considered a tool to study the induced effects of ischemia and hypoxia diseases in some in vitro models to prevent and reduce its effects after treatment with cholinergic drugs.

Awareness of health risks at the workplace and of risks of contracting communicable diseases including those related to food hygiene, among seafarers.

BACKGROUND: The awareness of health risks on board ships in terms of knowledge of dangers and discomfort at the workplace, and of risks of contracting communicable diseases including those related to food hygiene was assessed in a sample of workers of an Italian shipping company. Analysis was performed on crew members and on ashore personnel of the same firm to assess possible differences in risk perception. MATERIALS AND METHODS: The study was conducted by proposing an anonymous questionnaire to the crew members of 9 tankers and to the office staff of the shipping company Finaval S.p.A., which has its headquarters in Rome. RESULTS: People living ashore have a better knowledge of infectious risks than seafarers. Both ashore workers and seafarers have a reasonable awareness of blood-borne and sexually-transmitted diseases. Seafarers are more concerned about the risks of psychological problems due to isolation than are office personnel. The risk of not being adequately cared for in case of disease or injury on board is also perceived as a major problem by seafarers. Ashore personnel, eating raw fish more than their mates on board, are at a greater risk of communicable gastrointestinal diseases. CONCLUSIONS: Seafarers should be the target of specific informative campaigns about health risks, possible consequences, and how to minimize exposure to them during travel/life at sea.

Aldosterone does not modify gene expression in human endothelial cells.

The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

Survey on HIV risk perception and sexual behaviours among seafarers.

Because the nature of their work seafarers spend long periods of time away from their families and therefore represent a group at risk for sexually transmitted diseases, including HIV infection. This paper reports the results of a survey to evaluate awareness and knowledge of the risk of HIV infection among seafarers. Risky behaviours for HIV transmission were also assessed. The survey was conducted using anonymous questionnaires on 197 workers of 9 vessels and 26 office employees of an Italian shipping company (FINAVAL S.p.A., Rome). The respondents considered HIV/AIDS as one of the diseases with a high risk of transmission. Most respondents had a good general knowledge of HIV/AIDS and on the ways of its transmission. However, there is still lack of knowledge on the basics of this disease. On the other hand, in spite of knowledge and awareness about the risks of the disease, only 56.35% of the interviewed crewmembers used protection in sexual intercourse with occasional partners. Compared to data available in literature, the percentage of self-protecting people is increasing, but the number of seafarers exposing themselves to risky behaviours is still high. As expected, condoms are used with regular partners with lower frequency compared to occasional intercourse. The results of this survey indicate that adequate prevention campaigns and major attention paid to seafarers health is useful for stimulating responsible conduct for the prevention of infectious diseases, including HIV infection. Nevertheless, it is still necessary to increase information about the risk of sexually transmitted diseases and how to reduce it.

Alpha-lipoic acid modulates GFAP, vimentin, nestin, cyclin D1 and MAP-kinase expression in astroglial cell cultures.

In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 DIV astrocyte cultures pretreated or not with 0.5 mM glutamate for 24 h and than maintained under chronic or acute treatment with 50 µM R(+)enantiomer or raceme alpha-lipoic acid (ALA). GFAP expression significantly increased after (R+)enantiomer acute-treatment and also in glutamate-pretreated ones. Vimentin expression increased after R(+)enantiomer acute-treatment, but it decreased after raceme acute-treatment. Nestin expression drastically increased after acute raceme-treatment in glutamate-pretreated or not cultures, but significantly decreased after (R+)enantiomer acute and chronic-treatments. Cyclin D1 expression increased in raceme acute-treated cultures pretreated with glutamate. MAP-kinase expression slightly increased after (R+)enantiomer acute treatment in glutamate-pretreated or unpretreated ones. These preliminary findings may better clarify antioxidant and metabolic role played by ALA in proliferating and differentiating astrocyte cultures suggesting an interactive cross-talk between glial and neuronal cells, after brain lesions or damages.

Oral health in Alzheimer's disease: a review.

The main data on oral health and dental pathologies affecting Alzheimer's disease (AD) patients were reviewed. Oral health declines and dental pathologies increase with progression of AD. Poor oral hygiene, difficulty in wearing dentures, and the inability to self-care, including carrying out oral hygiene procedures are the most probable cause of impaired oral health in AD. Collection of information on oral/dental conditions from AD patients or their caregivers/relatives is often difficult and scientific literature on the topic is sparse . The majority of data on the subject consist in retrospective studies affected to some extent by subjective views of dental professionals involved. Appropriate dental interventions in adult-onset dementia disorders will decrease pain and oral pathology and consequently could contribute to maintain enough oral and nutritional health in these patients. Dental treatment in early stages of the disease are important and should be finalized at producing a stable oral condition. This could improve the quality of life and contribute to decrease worsening of oral situations in the later stages of the disease when dental treatment may be difficult. The problem of awareness of good oral health for keeping quality of life more acceptable in adult-onset dementia disorders is discussed.

Vesicular acetylcholine transporter (VAChT) in the brain of spontaneously hypertensive rats (SHR): effect of treatment with an acetylcholinesterase inhibitor.

The cholinergic marker vesicular acetylcholine transporter (VAChT) was investigated in different cerebral areas of spontaneously hypertensive rats (SHR) by immunochemistry (Western blot analysis) and by immunohistochemistry. SHR were used as an animal model of hypertensive brain damage. The sensitivity of manipulation of cholinergic system on VAChT was assessed in rats treated for four weeks with the acetylcholinesterase (AChE) inhibitor galantamine (3 mg/Kg/day). VAChT concentrations were increased in the brain of control SHR compared to age-matched normotensive Wistar-Kyoto rats. This increase probably represents an up-regulation of VAChT to oppose cholinergic deficits reported in SHR and is countered by galantamine administration. The possibility that cholinergic neurotransmission enhancement may represent a therapeutic strategy in cerebrovascular disease is discussed.

Effect of acetylcholine precursors on proliferation and differentiation of astroglial cells in primary cultures.

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5'-diphosphocholine (CDP-choline) and alpha-glyceryl-phosphorylcholine (alpha-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 microM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 microM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 microM alpha-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 microM choline or alpha-GPC, whereas in 24 h 1 microM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 microM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 microM alpha-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture.

Pathways of acetylcholine synthesis, transport and release as targets for treatment of adult-onset cognitive dysfunction.

Acetylcholine (ACh) is a neurotransmitter widely diffused in central, peripheral, autonomic and enteric nervous system. This paper has reviewed the main mechanisms of ACh synthesis, storage, and release. Presynaptic choline transport supports ACh production and release, and cholinergic terminals express a unique transporter critical for neurotransmitter release. Neurons cannot synthesize choline, which is ultimately derived from the diet and is delivered through the blood stream. ACh released from cholinergic synapses is hydrolyzed by acetylcholinesterase into choline and acetyl coenzyme A and almost 50% of choline derived from ACh hydrolysis is recovered by a high-affinity choline transporter. Parallel with the development of cholinergic hypothesis of geriatric memory dysfunction, cholinergic precursor loading strategy was tried for treating cognitive impairment occurring in Alzheimer's disease. Controlled clinical studies denied clinical usefulness of choline and lecithin (phosphatidylcholine), whereas for other phospholipids involved in choline biosynthetic pathways such as cytidine 5'-diphosphocholine (CDP-choline) or alpha-glyceryl-phosphorylcholine (choline alphoscerate) a modest improvement of cognitive dysfunction in adult-onset dementia disorders is documented. These inconsistencies have probably a metabolic explanation. Free choline administration increases brain choline availability but it does not increase ACh synthesis/or release. Cholinergic precursors to serve for ACh biosynthesis should be incorporate and stored into phospholipids in brain. It is probable that appropriate ACh precursors and other correlated molecules (natural or synthesized) could represent a tool for developing therapeutic strategies by revisiting and updating treatments/supplementations coming out from this therapeutic stalemate.

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures.

Type-2 transglutaminase (TG-2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin-like growth factor-I (IGF-I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron-glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17beta-estradiol (E(2)) on TG-2 activity and their expression in cultured astrocytes. We observed a significant increase in TG-2 activity and expression in astroglial cells treated for 24 hr with IGF-I, EGF, or INS. Priming of the cells with DEX or E(2), for 48 hr also led to an increase in TG-2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG-2 expression and activity and a different subcellular TG-2 distribution were found. Our data indicate that steroid hormone-GF interaction may play an important role in astroglial function. The effect on TG-2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.

Observational study of hypertension in Matelica, Italy (Matelica hypertension study).

This study summarizes the results of an epidemiological investigation carried out on the occasion of the Second World Hypertension Day (May 13, 2006) in the city of Matelica in the Region of the Marches, Central Italy. In all, 518 subjects (298 males, average age 52.3 years; 220 females, average age 55 years) with either diagnosed hypertension or who were thought to be normotensive had arterial blood pressure measured. Other cardiovascular risk factors and the costs of pharmacological treatment for hypertension were assessed as well. In 72.46% of examined subjects, arterial blood pressure levels averaged > or =140-90 mmHg if non-diabetic and > or =130-80 mmHg if diabetics. A total of 48.14% of individuals assumed in anamnesis to be normotensive had arterial blood pressure levels higher than the above values and were therefore found to have hypertensive values. The cost of anti-hypertensive treatment in the area of Matelica averages Euro 543.7/patient/year. The present data, which are in line with those of other epidemiological studies performed in Italy, confirm the view that arterial hypertension control in Italy is still largely unsatisfactory. This observation should stimulate both health and specific medical measures to counter the risk of complications of arterial hypertension in aged populations, such as those present in the territory examined.

Dopamine receptor subtypes in the human pulmonary arterial tree.

Dopamine induces vasorelaxation of pulmonary artery primarily through an endothelium-dependent mechanism, but dopamine receptor subtypes involved in these mechanisms have not been identified yet. The expression and localization of dopamine D1-like (D1 and D5) and D2-like (D2, D3 and D4) receptors were investigated in hilar, lobar and intrapulmonary branches of human pulmonary artery by immunoblotting and immunohistochemistry. Pulmonary artery expresses dopamine D1, D2, D4 and D5 receptor subtypes, but not the D3 receptor subtype. Dopamine D1 and to a lesser extent D5 receptors were accumulated primarily in the endothelium of extrapulmonary branches of pulmonary artery. A faint dopamine D1 and D5 receptor immunoreactivity was found in the inner media of extrapulmonary and of large sized intrapulmonary branches of pulmonary artery, but not in medium- or small-sized intrapulmonary artery branches. Dopamine D2 and to a lesser extent D4 receptor immunoreactivity co-localized with the tyrosine hydroxylase-immunoreactive sympathetic plexus supplying pulmonary artery was found in the adventitia and in the adventitia-media of both extra- and different-sized intrapulmonary branches of pulmonary artery. These findings suggest the possible role of dopamine receptors in the pulmonary endothelium-dependent vasorelaxing activity. The D1 receptor subtype seems to be the most involved in this mechanism. Dopamine D2-like receptors are prejunctional and are located at the level of sympathetic neuroeffector plexus. The heterogeneous distribution and density of dopamine receptor subtypes along the human pulmonary arterial tree may be related to the different functional roles of dopamine at various levels of the pulmonary circulation.

Dopamine plasma membrane transporter (DAT) in rat thymus and spleen: an immunochemical and immunohistochemical study.

The expression of the dopamine plasma membrane transporter (DAT) was investigated in rat thymus and spleen by immunochemical and immunohistochemical techniques. Antibodies raised against a peptide mapping near the amino terminus of DAT were bound to a single band of approximately 76 kDa in thymus and spleen membranes as well as in striatal and kidney membranes which were used as dopaminergic reference tissues. Reverse transcription-polymerase chain reaction analysis revealed that both thymus and spleen expressed DAT mRNA. Immunohistochemistry revealed in rat thymus a DAT immune reaction in the wall of arteries located in septa of connective tissue as well as in the medulla, with a reticular localization and an apparent negative reaction of thymocytes. In the spleen, DAT immunoreactivity was located primarily in the red-white pulp marginal zone, within small cells, likely corresponding to lymphocytes and in the wall of white pulp arteries. The presence of a dopamine transporter suggests that dopamine released in the lymphoid microenvironment may contribute to neuroimmune modulation. It cannot be excluded a different activity of dopamine in primary and secondary immune organs, such as maturation and selection of lymphocytes and activation of immune responses in the spleen.

Localization of the m5 muscarinic cholinergic receptor in rat circle of Willis and pial arteries.

The expression and microanatomical localization of the muscarinic cholinergic m5 receptor subtype was investigated in rat circle of Willis and pial arteries by in situ hybridization, immunoblotting and immunohistochemistry. In situ hybridization histochemistry revealed a strong signal in the endothelium of circle of Willis and pial arteries and a moderate signal in the tunica media of the same arteries, within smooth muscle. Exposure of membranes of arteries to anti-m5 receptor protein antibodies caused the development of a band of approximately 81 kDa. Immunohistochemistry revealed the accumulation of m5 receptor protein immunoreactivity primarily within endothelium of circle of Willis and cerebral arteries and to a lesser extent in the tunica media, within smooth muscle. Medium (external diameter 200-100 microm) and small-sized (external diameter smaller than 100 microm) pial arteries displayed a significantly higher immune staining than large-sized pial arteries or circle of Willis arteries. The above data that are consistent with recent functional studies reporting cholinergic dilation of cerebral blood vessels mediated via a m5 receptor, have shown that both endothelial and muscular components of cerebral arteries synthesize and express a muscarinic m5 receptor. In view of the peculiar localization in cerebral vessels, handling of the muscarinic m5 receptor may be considered as an approach in the treatment of cerebrovascular disease.