, 2003; Gafan et al., 2005). In fact, the application of PCR-DGGE analysis to the biliary sludge occluding our stents allowed the identification of a large additional number of bacterial and fungal species that were not revealed by culture.

The only partial overlapping selleck compound between the species identified by PCR-DGGE and those isolated by culturing is presumably due to the different stent portions analyzed by both techniques as well as the PCR-DGGE analysis performed on only 50% of stents. In fact, the number of isolated species, as well as the ratio between aerobic and anaerobic species, may vary considerably depending on the portion analyzed. However, our findings of such a large number of anaerobic species, both isolated by culturing or identified by PCR-DGGE, Talazoparib can be considered of particular interest. Apart from the paper of Leung et al., (2000), which reported the isolation from unblocked biliary stents of strains belonging to only three anaerobic species (C. perfringens, C. bifermentans and B. fragilis), this is the first report on the isolation from blocked biliary stents of anaerobic strains belonging to 14 different species as well as on the identification of five additional species by PCR-DGGE. Our SEM observations of sessile microorganisms remaining tightly attached to the surface of stent lumen after detachment of the covering

amorphous material occurring during the dehydration process seem

to significantly support the hypothesis that biliary stent clogging starts with the bacterial colonization of the stent lumen. This hypothesis finds a significant confirmation in the light micrograph of a cross-section of an occluded biliary stent recently published by Costerton (2007), in which concentric layers of a bacteria-rich biofilm are visible close to the inner surface of the stent lumen while large amounts of bile salts, Rebamipide mixed with dispersed small bacterial clusters, occupy the central part of the lumen, the remaining space allowing a slow bile flow. The isolation of anaerobic bacteria in 57% of the analyzed stents and the demonstrated ability of the majority of them to form a biofilm in vitro strongly suggest that anaerobic species presumably play a significant role in biliary stent clogging. On the basis of these evidences and the well-known antibiotic tolerance of biofilm-growing bacteria, further studies should be focused on strategies to prevent biofilm development on the inner surfaces of biliary stents in order to prolong their patency with important medical and economical outcomes. The authors gratefully acknowledge the collaboration of Professors Antonio Basoli and Fausto Fiocca for providing the clogged stents to be analyzed for their microbiological content.

Conclusion:  Higher intakes of fluid appear to protect against CKD. CKD may be preventable at a population level with low-cost increased fluid intake. “
“Haemodialysis, by design, uses a semipermeable membrane to separate blood from dialysate. The qualities of this membrane determine the nature of the ‘traffic’ between the blood and dialysate. In this sense, the qualities of the membrane determine what size molecules move from one compartment to the other, the amount and rate at which they might move and the amount and rate of water movement across the membrane. In addition, the nature of the membrane influences the biological response of the patient both in terms of what

is or is not removed Staurosporine concentration by the dialysis process and by way of the reaction to the biocompatibility of the membrane. This brief review will explore aspects of dialysis membrane Selleck ACP-196 characteristics. To digress before

paying attention to the membrane itself, it must be remembered that dialysers are comprised of more than just membranes – the geometry of the dialyser, the blood path, the potting compound, the sterilant used and spacers between the hollow fibres are all important and influence dialysis clearances and potentially induce reactions in the patient. As an example, ethylene oxide was used as a dialyser sterilant for many years, but itself induced an inflammatory reaction in the patient.1 Although gamma sterilization is still used, most modern dialysers now use steam as the prime sterilizing agent, which is inert. The geometry of the dialyser may influence the blood path and the matching of blood flow to dialysate flow – such aspects as the design of the header of the dialyser and spacing not yarns between the hollow fibres – thus influencing the ‘efficiency’ of the dialyser and the achieved clearance for a given dialyser surface area. The presence of spacer yarns between dialyser fibres, to optimize dialysate flow and dialysate: membrane contact results in approximately 10% improved small molecule clearance.2 Similarly, moire structure of the

fibres (a purposeful wrinkling of the membrane) also improves clearances. The internal diameter of the fibres can be reduced to increase surface shear pressures, thus reducing the resistance of the more static blood layers close to the walls of the fibre – blood exhibits laminar flow in hollow fibres with the peripheral layers exhibiting slower flow and these may create relative resistance to solute transfer. In one study, decreasing the internal fibre diameter by 7.5% and the wall thickness by 12.5% resulted in improved middle molecule clearance by almost 50%, with very little change in small molecule clearance.3 To return to the membranes – early dialysis membranes (see Table 1) were based on cellulose, with cuprophane (a copper-substituted cellulose) being one of the most commonly used early membranes.4 These were cheap to produce and had advantages of being thin-walled.

[109-111] No effective treatment is currently available except for acetylcholinesterase inhibitors which augment cholinergic function but this is not curative and only a temporary measure. As for the pathogenesis of AD, the amyloid cascade hypothesis postulates that memory deficits are caused by increased levels of both soluble and insoluble amyloid β (Aβ) peptides, which are derived from the larger amyloid precursor protein (APP) sequential proteolytic processing.[109-111] A recent study has reported that treatment of PDAPP mice, a transgenic mouse model of AD, with anti-Aβ antibody completely restored hippocampal acetylcholine release

and high-affinity choline uptake and improved habituation learning.[112] Based on the study, a clinical trial in AD patients is underway in the USA. Chronically decreasing Aβ levels Rapamycin clinical trial in the brain has been suggested as a possible therapeutic approach for AD, and experimental evidence indicates that proteinases such as neprilysin,[113] insulin degrading enzyme,[114, 115] plasmin[116] and cathepsin B[117] could be used as therapeutic

agents to reduce Aβ levels in AD brain. Recent studies have shown that intracerebral injection of a lentivirus vector expressing human neprilysin in transgenic mouse models of amyloidosis reduced Aβ deposits in the brain and blocked neurodegeneration in the fronal cortex Panobinostat solubility dmso and hippocampus,[118] and that intracerebrally injected fibroblasts over-expressing the human neprilysin gene were found to significantly reduce amyloid plaque burden in the brain of Aβ transgenic mice.[119] These studies support

the use of Aβ-degrading proteases as a tool to therapeutically lower Aβ levels and encourage further investigation of ex vivo delivery of protease genes using human NSCs for the treatment of AD. We have recently generated a human NSC line encoding the human neprylysin gene, transplanted these cells into the lateral ventricle of AD transgenic mouse brain, and results are expected PAK5 shortly. Ealier studies have indicated that nerve growth factor (NGF) prevents neuronal death and improves memeory in animal models of aging, excitotoxicity and amyloid toxicity,[120-124] and could be used for treating neuronal degeneration and cell death in the AD brain. However, delivery of NGF into the brain is not possible via peripheral administration. Because of its size and polarity, NGF does not cross the blood–brain barrier. In order to overcome this difficulty, a gene therapy approach could be adopted. Using an ex vivo gene therapy approach (genetically modify cells), NGF can be directly inserted into the brain and diffuse for a distance of 2–5 mm.[125] Previously, a phase 1 clinical trial of ex vivo NGF gene delivery was performed in eight mild AD patients, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After a mean follow-up of 22 months in six subjects, long-term adverse effects were not found.

This work was funded by IOC and IPEC-FIOCRUZ, PAPES 6, FUNASA/MS, CNPq and FAPERJ, Brazil. M.R.P. is a fellow from Fiocruz-CNPq. We thank to Rodrigo Mexas for the final artwork. this website A.O.S is recipient of fellowships from CNPq and FAPERJ. Table S1. Percentage of positive cells and CD4/CD8 ratio in healthy control donors and patients with mucosal leishmaniasis. Table S2. Number of positive cells/mm2 tissue in healthy donors and patients with

mucosal leishmaniasis. “
“Recent studies show that proteinase-activated receptor-2 (PAR2) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR2 activation in the anti-microbial

defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR2 to the anti-microbial response of isolated human innate immune cells. We found that PAR2 agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that selleck antibody of interferon-γ (IFN-γ). However, co-application of PAR2-cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR2 agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 Cediranib (AZD2171) kinase and janus kinase molecules are involved in the synergistic effect of PAR2 agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role

of PAR2 agonists in the anti-microbial defence established by human monocytes and neutrophils. Proteinase-activated receptor-2 (PAR2) plays a role in the development of allergic diseases of the skin1 and in certain inflammatory disorders.2 The impact of PAR2 activation on inflammation can be pro- or anti-inflammatory, depending on the stage of disease and the primary cell type involved in disease progression.2 During receptor activation, serine protease cleavage of PAR2 unmasks the N-terminal sequence of the ‘tethered ligand’. This unmasked sequence further serves as a receptor activator.3 The PAR2 is activated by trypsin and tryptase, and also by proteases derived from immune cells and pathogens.4 However, serine proteases cause PAR-dependent as well as PAR-independent effects.5,6 As a result, specific synthetic activating peptides are important probes for investigating the role of PAR activation in different processes.

The histological score shows a significantly increased lymphocyte infiltration in the intestinal mucosa in Bim–/– animals compared to wild-type animals upon chronic DSS-induced colitis. First, we isolated Peyer’s patches by excising whole lymph nodes together with adherent mucosal tissue. We could show increased gene expression levels for Bim in wild-type mice when they had developed chronic colitis (control: 1·1 ± 0·3, n = 5; DSS: 1·5 ± 0·6, n = 5; Fig. 3d). As TCR Vβ8+ T cells from Bim–/– selleck kinase inhibitor mice were found to be resistant to enterotoxin-induced deletion [11], and apoptosis of TCR Vβ8+ T cells but not TCR Vβ6+ T cells is impaired in Bim–/– mice [12], we focused on the presence

of TCR Vβ8+ T cells in Peyer’s patches by flow cytometric analysis. The number of TCR Vβ8+ lymphocytes

was increased significantly in Peyer’s patches from Bim–/– mice compared to wild-type controls (10·5 ± 1·9% versus 7·3 ± 1·2%, respectively, P < 0·05; Fig. 4a). An increase of TCR Vβ8+ lymphocytes was confirmed by IF for Bim–/– mice compared to wild-type controls (Fig. 4b). Whole Peyer's patches were excised and snap-frozen. We assessed the DAPT manufacturer cytokine profile in whole Peyer’s patches without further pre-stimulation of lymphocytes on the level of mRNA. iNos gene expression was detectable in wild-type but almost absent in Bim–/– animals without chronic colitis (1·10 ± 1·00, n = 9 versus 0·34 ± 0·24, n = 12, respectively, Fig. 5a). There was a significant difference for wild-type mice upon chronic DSS-induced colitis compared to Bim–/– animals (1·00 ± 0·97, n = 15 versus 0·23 ± 0·14, n = 17, respectively, P < 0·05; Fig. 5a). Data could be confirmed by Western blot. Wild-type mice exhibited significantly higher

iNOS protein contents than Bim–/– mice for animals both with and without chronic DSS-induced intestinal inflammation (0·18 ± 0·04, n = 3, versus 0·02 ± 0·03, n = 5, respectively, for mice without DSS-induced chronic colitis and 0·12 ± 0·08, n = 7, versus 0·02 ± 0·05, n = 6, P < 0·05, respectively, for mice with DSS-induced chronic colitis; Fig. 5b). For IL-6, TNF and IL-1β mRNA expression, no significant changes were recorded between wild-type and Bim–/– mice with and without chronic mafosfamide DSS-induced colitis (not shown). Bim interacts with the pro-survival family member BCL-2. Bim is involved critically in negative selection of thymocytes during maturation processes and Bim plus Puma co-regulate lymphocyte homeostasis in the periphery [9]. Deletion of activated cells after antigenic challenge is impaired in Bim-deficient animals, thereby facilitating the development of systemic lupus erythematosus-like pathology [8]. As dysregulated apoptosis of lymphocytes contributes to the pathogenesis of IBD [14-17, 23], we analysed the role of Bim in lymphocytes in our mouse model of colitis.

[38] With regard to blood pressure management new evidence reviewed in this updated guideline has led to an upward revision

of the recommended BP targets. These new targets are in line with those recommended by the NHMRC.[39] There are a number of epidemiological studies[40, 41] which have established that asymptomatic hyperuricaemia is associated with both CKD and ESKD. However, hyperuricaemia is a ubiquitous finding Selleckchem Trichostatin A in CKD[42] and could be a consequence of reduced excretion, diuretic therapy, or oxidative stress. Although it is not clear whether urate plays a causative role or is an indirect marker of kidney function, uric acid lowering therapy has emerged as a potentially novel therapeutic treatment for slowing the progression of CKD.[41] In the CKD population, both vitamin D deficiency and insufficiency are common. As GFR falls, hydroxylation/activation of vitamin D is impaired leading DNA Damage inhibitor to hyperparathyroidism and

CKD mineral and bone disorder (CKD-MBD). Retention of phosphate may begin to occur when renal function falls below 80% of normal. Changes in any of these laboratory values may begin in stage CKD 3, although the presence, rate of change and severity of these abnormal parameters are highly variable among individuals. In a study of 168 consecutive new referrals of patients with stages 2–5 CKD to a CKD clinic, Ravani et al.[43] observed that both 25-hydroxyvitamin D and 1,25-dihydroxyvitamin-D levels were significantly, inversely associated with eGFR. Consequently, the prevalence rates of vitamin D insufficiency and deficiency increased from 62% and 25% in stage 2 CKD to 88% and 56% in stage 5 CKD. Similarly, a cross-sectional study of 15 068 adults participating in the Third National Health and Nutrition Examination Survey (NHANES) reported a strong, inverse association between albuminuria

and serum 25-hydroxyvitamin D concentrations.[44] The objective of this guideline is to review currently available evidence with regards to medical therapies for the management of: hypertension, hypercholesterolaemia, diabetes mellitus, CVD, hyperuricaemia and vitamin D insufficiency Venetoclax in vivo and deficiency in patients with stage 1–3 CKD. Evidence for lifestyle modification and nutrition is also reviewed. a. We suggest that patients with progressive CKD have individualized diet intervention involving an appropriately qualified dietitian (2C). e. We recommend that early CKD patients restrict their dietary sodium intake to 100 mmol/day (or 2.3 g sodium or 6 g salt per day) or less, as it reduces blood pressure and albuminuria in patients with CKD (1C). g. We suggest that early CKD patients (stages 1–3) should not restrict dietary phosphate intake as restriction of dietary phosphate does not influence renal or cardiovascular outcomes in these patients (2C). h.

It may be plausible that β-defensins and cathelicidins could contribute to reduce parasite burden from the bite of an infected

tsetse because of Metformin research buy the expression in neutrophils or keratinocytes at the locality of the bite. However, no data exist on the killing of metacyclic form trypanosomes by AMPs. Motivated by the desire to identify novel agents to treat HAT, several groups have identified synthetic trypanolytic AMPs and AMPs from diverse sources such as insects, fish and soil microorganisms (20–22,36). With the exception of the fungal-derived AMPs and the cell-penetrating peptide TP10, these peptides are directly derived from known trypanolytic defensins or cathelicidins. The peptide antibiotics leucinostatin A and B, alamethicin and tsushimycin are natural products isolated from fungi. These peptides differ from the canonical AMPs by the virtue of the presence of unusual amino acids,

acylation or both. The Selleckchem MDV3100 leucinostatins, named for their high leucine content, kill trypanosomes in vitro at low nanomolar concentrations (20). The potency of these peptides might be attributable to pleiotropic effects. Studies with model liposomes indicate that leucinostatins increase the permeability of lipid bilayers (37). The leucinostatins have also been shown to inhibit mitochondrial ATP synthesis and uncouple oxidative phosphorylation (38). The relevance of these activities to killing BSF trypanosomes is not clear, because of the lack of functional electron transport chain in this developmental form; however, disrupting the mitochondrial membrane potential may contribute to toxicity. A comparative analysis with the trypanocidal drug D-malate dehydrogenase suramin indicates greater potency of the leucinostatins in mice. However, these mycological metabolites exhibit high oral toxicity (20). Alamethicin exhibits strong trypanolytic activity in vitro, killing BSF trypanosomes at nanomolar concentrations (20).

The membrane permeabilizing activity of alamethicin has been well established. Alamethicin monomers orient perpendicular to the lipid membrane and oligomerize in the bilayer forming cylindrical pores that facilitate the passage of ions and water (39). Studies in mice indicate that alamethicin does not provide greater in vivo activity than suramin (20). The in vitro trypanolytic activity of tsushimycin may be attributed to its structural similarity to amphomycin, which exhibits activity against T. b. gambiense and T. b. rhodesiense in mice (40). Amphomycin has been shown to inhibit the formation of dolichol–phosphate–sugar complexes, molecules that donate sugar moieties for protein glycosylation and GPI anchors. This potential mechanism is particularly relevant to African trypanosomes. A relatively large portion of proteins are GPI-anchored including the VSG coat, and it has been shown that inhibition of GPI modification is toxic (41).

It is possible that pre-clustering is a general feature of antigen receptors, as it has been reported for BCR as well.36,37 However, not much is known about how proteins partition into the islands and how the localization of the islands themselves is regulated. How do Src kinases specifically recognize the antigen receptors engaged with antigens? It seems that the access EX 527 nmr of Src kinases to at least some of the ITAMs may be controlled by conformational changes in the receptors’

cytoplasmic domains. Evidence of conformational changes in the cytoplasmic domains of the TCR came from studies of CD3ε.38 CD3ε contains a proline-rich sequence in its cytoplasmic tail that is inaccessible in resting T cells, but is exposed upon peptide–MHC (pMHC) binding. A recent study suggests that the accessibility may be related to binding of the CD3ε ITAMs to the inner leaflet of the plasma membrane.39 In this study Xu et al.39 showed that synthetic CD3ε cytoplasmic tail bound to acidic liposomes in vitro. Similar binding had been Panobinostat nmr observed with the TCR-ζ chain.40 Both CD3ε and TCR-ζ contain a group of basic residues, which were required for binding to lipids. In cells,

FRET measured between the end of the cytoplasmic tails of CD3ε and fluorescent probes embedded in the plasma membrane showed that the CD3ε tail was close to the membrane and was therefore probably bound to the inner leaflet in vivo as well.39 Using nuclear magnetic resonance measurements, Xu et al.39 determined ID-8 the structure of the cytoplasmic domain of CD3ε bound to bicelles, flat nanoscopic pieces of bilayers. This structure showed that the ITAM was folded into a partially helical structure, with the canonical tyrosines inserted into the hydrophobic interior of the phospholipid bilayer. Presumably, unfolding of the cytoplasmic domain is necessary for the access of Src kinases. It will be interesting in the future to determine how the membrane binding of the cytoplasmic tails changes

after pMHC binding. Although the positively charged residues that were required for the membrane binding are unique to CD3ε and TCR-ζ, it is possible that other ITAMs may fold in the presence of plasma membrane as well. Notably, earlier FRET measurements in the BCR showed that cytoplasmic tails lose FRET between each other after initial clustering.41 This signifies an ‘opening’ of the BCR cytoplasmic domains and was dependent on the phosphorylation of the ITAM tyrosines. However, understanding of the specific structure of the BCR ITAMs will require more experimental work. Currently, there is little understanding of the mechanisms by which the changes in the cytoplasmic domains are triggered by antigen binding. In principle, the cytoplasmic domains can be released from the membrane by perturbations of the local composition of the bilayer, or of its physical properties. It is also possible that these changes originate at the receptors’ extracellular domains after antigen binding.

In histological sections, the occurrence of numerous alcian blue–positive mucous cells was observed among the intestinal epithelial cells of infected fish notably within the epithelia in close proximity to the nodule (Figure 2a). RCs in variable numbers (Figure 3a) were seen among the epithelia of both M. wageneri-infected Ensartinib mouse tench (i.e. in close proximity to the point of cestode attachment and at a distance) and in uninfected specimens. Interestingly, within the parasitized intestines, RCs were found to co-occur with granulocytes within the submucosa of the nodule (Figure 3b) and in close proximity to blood vessels and/or within the capillaries. The inflammatory swellings surrounding the M. wageneri

primarily consisted of fibroblasts but also included a large number of neutrophils and MCs. Neutrophils (Figure 3c) and MCs were seen within the connective tissue surrounding capillaries and within the blood vessels within the submucosa and muscularis layer.

In some intestinal sections taken from infected tench, neutrophils were also observed within the epithelia (not shown). Neutrophils appeared round to oval in shape although their outline was commonly irregular (Figure 3c). These cells also contained a round nucleus and a cytoplasm HDAC inhibitor that contained dark, elongated granules that were fibrous in appearance (Figure 3c). Very few mitochondria and fragments of rough endoplasmic reticulum were observed in the cytoplasm of the neutrophils. The MCs, which were frequently observed within the epithelia of

infected hosts (Figure 3a), were irregular in shape with an eccentric, polar nucleus, and a cytoplasm characterized by numerous large, electron-dense, membrane-bounded granules (Figure 3d). The cytoplasm typically contained two to three mitochondria and an inconspicuous Golgi apparatus. Accurate counts of MCs and neutrophils were obtained from two intestinal grids from each infected fish. Neutrophils were found to be numerous within the nodule, in close proximity to the tegument of the cestode, but their number was seen to decrease towards the periphery of the nodule. Neutrophils were significantly more abundant than MCs (Table 1; anova, P < 0·01) second in host tissue close to the point of cestode attachment. At a distance of 200 μm from the site of parasite attachment, however, the number of neutrophils was significantly lower than the MCs (Table 1; anova, P < 0·01). There were significant differences in the number of neutrophils in close proximity to and at a distance of 200 μm from the point of cestode attachment (Table 1; anova, P < 0·01). Likewise, there were significant differences in the number of MCs at the site of infection and 200 μm away (Table 1; anova, P < 0·01). Commonly, the neutrophils and MCs adjacent to the M. wageneri scolex tegument had a cytoplasm that appeared vacuolized (Figure 4a) and contained very few organelles.

The sequestration of BMCs in coronary capillaries occurred independent of WI, generalized atherosclerosis, or adhesion molecule function. This is the first study allowing direct assessment Protein Tyrosine Kinase inhibitor of BMC homing to the postischemic myocardium. Heterotopic heart transplantation and IVM are proper means to study the myocardial sequestration of BMCs after direct antegrade intracoronary injection in vivo. We show for the first time that intracoronarily injected BMCs sequester exclusively in nutritive myocardial capillaries. “
“Endothelium-dependent vasodilation of coronary arterioles is impaired in obese rats and may be improved by a LCD. The aim of this study is to elucidate the mechanism by which this improvement

occurs. We used four groups of male Zucker rats: lean and obese on either SD or LCD. Coronary arterioles were cannulated and pressurized for diameter measurements during administration of acetylcholine or sodium nitroprusside or during flow. Real-time PCR was performed to quantify mRNA expression of CuZnSOD and catalase. The LCD significantly selleck chemical increased endothelium-dependent dilation in the obese rats. l-NAME and indomethacin reduced responses to flow and acetylcholine in the lean rats without any effect on the obese

on either diet. In contrast, TEA and catalase blocked flow-dependent and acetylcholine-induced dilation in the obese on either diet, while no effect was observed on the lean. The LCD in the obese significantly up-regulated catalase mRNA expression and slightly increased CuZnSOD mRNA levels. A LCD improves endothelium-dependent CYTH4 vasodilation of coronary arterioles in obese rats through the production of H2O2 which acts as a hyperpolarizing factor, independent of nitric oxide and PGI2. “
“Please cite this paper as: Bagher, Davis and Segal (2011). Intravital Macrozoom Imaging and Automated Analysis of Endothelial Cell Calcium Signals Coincident with Arteriolar Dilation in Cx40BAC-GCaMP2 Transgenic Mice. Microcirculation 18(4), 331–338. Objective:  Calcium

signaling is integral to endothelium-dependent vasodilation. Our goal was to develop methods enabling automated analyses for accurately and objectively determining the dynamic relationship between EC Ca2+ responses and arteriolar diameter in vivo. Methods:  User-friendly software (DiaFluor) written in LabView was applied to images acquired at 15 fps with a custom macrozoom intravital microscope to evaluate changes in EC Ca2+ concomitant with arteriolar diameter. Transgenic Cx40BAC-GCaMP2 mice expressing a fluorescent Ca2+ indicator molecule in arteriolar ECs enabled resolution of EC Ca2+ signaling in response to ACh microiontophoresis (500 nA, 100–1000 msec pulse) from a micropipette (1 μm tip) positioned adjacent to an arteriole in the superfused cremaster muscle preparation. Results:  A 100-msec pulse of ACh (1 M) had little effect on EC Ca2+ or arteriolar diameter.