Non-vascular Smooth Muscle Cells Research Articles

Melanopsin in the human and chicken choroid

The choroid embedded in between retina and sclera is essential for retinal photoreceptor nourishment, but is also a source of growth factors in the process of emmetropization that converts retinal visual signals into scleral growth signals. Still, the exact control mechanisms behind those functions are enigmatic while circadian rhythms are involved. These rhythms are attributed to daylight influences that are melanopsin (OPN4) driven. Recently, OPN4-mRNA has been detected in the choroid, and while its origin is unknown we here seek to identify the underlying structures using morphological methods.Human and chicken choroids were prepared for single- and double-immunohistochemistry of OPN4, vasoactive intestinal peptide (VIP), substance P (SP), CD68, and α-smooth muscle actin (ASMA). For documentation, light-, fluorescence-, and confocal laser scanning microscopy was applied. Retinal controls proved the reliability of the OPN4 antibody in both species.In humans, OPN4 immunoreactivity (OPN4-IR) was detected in nerve fibers of the choroid and adjacent ciliary nerve fibers. OPN4+ choroidal nerve fibers lacked VIP, but were co-localized with SP. OPN4-immunoreactivity was further detected in VIP+/SP + intrinsic choroidal neurons, in a hitherto unclassified CD68-negative choroidal cell population thus not representing macrophages, as well as in a subset of choroidal melanocytes. In chicken, choroidal nerve fibers were OPN4+, and further OPN4-IR was detected in clustered suprachoroidal structures that were not co-localized with ASMA and therefore do not represent non-vascular smooth-muscle cells. In the choroidal stroma, numerous cells displayed OPN4-IR, the majority of which was VIP-, while a few of those co-localized with VIP and were therefore classified as avian intrinsic choroidal neurons. OPN4-immunoreactivity was absent in choroidal blood vessels of both species.In summary, OPN4-IR was detected in both species in nerve fibers and cells, some of which could be identified (ICN, melanocytes in human), while others could not be classified yet. Nevertheless, the OPN4+ structures described here might be involved in developmental, light-, thermally-driven or nociceptive mechanisms, as known from other systems, but with respect to choroidal control this needs to be proven in upcoming studies.

Genetic Deletion of Emp2 Does Not Cause Proteinuric Kidney Disease in Mice.

Nephrotic syndrome is one of the most common glomerular diseases in children and can be classified on the basis of steroid responsiveness. While multiple genetic causes have been discovered for steroid resistant nephrotic syndrome, the genetics of steroid sensitive nephrotic syndrome remains elusive. Mutations in Epithelial Membrane Protein 2 (EMP2), a member of the GAS3/PMP22 tetraspan family of proteins, were recently implicated as putative monogenic cause of steroid sensitive nephrotic syndrome. We investigated this hypothesis by developing Emp2 reporter and knockout mouse models. In lacZ reporter mice (engineered to drive expression of the enzyme β-galactosidase under the control of the endogenous murine Emp2 promoter), Emp2 promoter activity was not observed in podocytes but was particularly prominent in medium- and large-caliber arterial vessels in the kidney and other tissues where it localizes specifically in vascular smooth muscle cells (vSMCs) but not in the endothelium. Strong Emp2 expression was also found in non-vascular smooth muscle cells found in other organs like the stomach, bladder, and uterus. Global and podocyte-specific Emp2 knockout mice were viable and did not develop nephrotic syndrome showing no evidence of abnormal glomerular histology or ultrastructure. Altogether, our results do not support that loss of function of EMP2 represent a monogenic cause of proteinuric kidney disease. However, the expression pattern of Emp2 indicates that it may be relevant in smooth muscle function in various organs and tissues including the vasculature.

Intrinsic Neurons and Nonvascular Smooth Muscle Cells in the Choroid of a Chimpanzee (Pan troglodytes)

Intrinsic Neurons and Nonvascular Smooth Muscle Cells in the Choroid of a Chimpanzee (Pan troglodytes)

SUBFOVEAL CHOROIDAL THICKNESS AND VASCULAR DIAMETER IN ACTIVE AND RESOLVED CENTRAL SEROUS CHORIORETINOPATHY.

To determine the subfoveal choroidal thickness and analyze Haller's layer, Sattler's layer, and large choroidal vessel diameter in eyes with active central serous chorioretinopathy (CSC) and after resolution of CSC. Ocular and clinical features of 32 eyes with CSC were analyzed retrospectively from October 2014 to September 2015. Subfoveal choroidal thickness and thicknesses of Haller's layer and Sattler's layer were measured in the active and resolved states. The diameter of the subfoveal choroidal hyporeflective lumen (i.e., the large choroidal vessel in Haller's layer) was also measured. The mean subfoveal choroidal thickness, mean thickness of Haller's layer, and mean choroidal vessel diameter were significantly less after the resolution of CSC (P < 0.001). However, the thickness of Sattler's layer did not change after the resolution of CSC (P = 0.731). There were no significant differences among the different treatment modalities. After the resolution of CSC, the subfoveal choroidal thickness and thickness of Haller's layer declined, but the reduced diameter of subfoveal choroidal vessels accounted for only about half of the total thickness changes in the choroid. These results suggest that nonvascular smooth muscle cells might play a role in the thickening of the choroid during CSC and possibly in the pathogenesis and progression of CSC.

Large-scale identification of human cerebrovascular proteins: Inter-tissue and intracerebral vascular protein diversity.

The human cerebrovascular system is responsible for regulating demand-dependent perfusion and maintaining the blood-brain barrier (BBB). In addition, defects in the human cerebrovasculature lead to stroke, intracerebral hemorrhage, vascular malformations, and vascular cognitive impairment. The objective of this study was to discover new proteins of the human cerebrovascular system using expression data from the Human Protein Atlas, a large-scale project which allows public access to immunohistochemical analysis of human tissues. We screened 20,158 proteins in the HPA and identified 346 expression patterns correlating to blood vessels in human brain. Independent experiments showed that 51/52 of these distributions could be experimentally replicated across different brain samples. Some proteins (40%) demonstrated endothelial cell (EC)-enriched expression, while others were expressed primarily in vascular smooth muscle cells (VSMC; 18%); 39% of these proteins were expressed in both cell types. Most brain EC markers were tissue oligospecific; that is, they were expressed in endothelia in an average of 4.8 out of 9 organs examined. Although most markers expressed in endothelial cells of the brain were present in all cerebral capillaries, a significant number (21%) were expressed only in a fraction of brain capillaries within each brain sample. Among proteins found in cerebral VSMC, virtually all were also expressed in peripheral VSMC and in non-vascular smooth muscle cells (SMC). Only one was potentially brain specific: VHL (Von Hippel-Lindau tumor suppressor). HRC (histidine rich calcium binding protein) and VHL were restricted to VSMC and not found in non-vascular tissues such as uterus or gut. In conclusion, we define a set of brain vascular proteins that could be relevant to understanding the unique physiology and pathophysiology of the human cerebrovasculature. This set of proteins defines inter-organ molecular differences in the vasculature and confirms the broad heterogeneity of vascular cells within the brain.

CHOROIDAL THICKNESS IN PATIENTS WITH CENTRAL SEROUS CHORIORETINOPATHY: Assessment of Haller and Sattler Layers.

To investigate subfoveal choroidal thickness and subanalyze Haller and Sattler layers in eyes with central serous chorioretinopathy (CSC), uninvolved fellow eyes, and eyes of healthy controls using enhanced depth imaging optical coherence tomography. Ocular findings and clinical features of 31 eyes with CSC, 24 fellow eyes and eyes of 30 healthy controls were analyzed retrospectively from October, 2014 to March, 2015. Subfoveal choroidal thickness was measured using enhanced depth imaging optical coherence tomography, and the thicknesses of Haller and Sattler layers were analyzed. Mean subfoveal choroidal thickness and mean thickness of Haller layer were significantly greater in CSC than in fellow eyes (P = 0.043 and P = 0.036, respectively) and in normal control eyes (P < 0.001 each), and those of fellow eyes in CSC patients were significantly thicker than those in normal control eyes (P = 0.018 and P = 0.017, respectively). The thickness of Sattler layer did not differ significantly among these groups (P = 0.519). Subfoveal choroidal thickness and the thickness of Haller layer were increased not only in affected but also in uninvolved fellow eyes of CSC patients. Nonvascular smooth muscle cells of the choroid may play a role in the pathophysiology of CSC, in response to increased sympathetic tone.

Lymphatic Markers in the Adult Human Choroid.

Reports of lymphatics in the posterior human uvea are contradictory. We systematically analyzed the choroid by combining various lymphatic markers, following recently established guidelines for the immunohistochemical detection of ocular lymphatics. Human choroids were prepared for flat mount serial cryosectioning. Sections were processed for immunohistochemistry of the lymphatic markers LYVE-1, PDPN, PROX1, FOXC2, VEGFR3, CCL21, and combined with α-smooth muscle-actin and 4',6-diamidino-2-phenylendole (DAPI). Single, double, and triple marker combinations were documented using confocal microscopy. Messenger RNA analysis for CCL21, FOXC2, LYVE-1, PDPN, PROX, and VEGFR3 was performed in choroid and skin. In the choroid, CCL21 immunoreactivity was detected in choroidal blood vessels, intrinsic choroidal neurons, and numerous small cells of the choroidal stroma. These small cells were not colocalized with PROX1 and PDPN, while a subpopulation of cells showed immunoreactivity for CCL21 and LYVE-1, and very occasionally PDPN-only+ cells were detected. Nuclei positive for PROX1 were never detected in the choroid, and vessel-like structures immunoreactive for LYVE-1, PDPN, or CCL21 (other than blood vessels) were never observed. Immunoreactivity of VEGFR3 was absent in the majority of choroidal blood vessels, but present in choriocapillaris, while other structures positive for VEGFR3 were not detected. Nonvascular smooth muscle cells were lacking VEGFR3-immunoreactivity. Messenger RNA analysis detected all lymphatic markers investigated and confirmed immunohistochemical results. By combining several lymphatic markers, single cells expressed these markers, but classical lymphatic vessels were not detected in the human choroid. Therefore, the healthy adult human choroid must be considered alymphatic, at least with the markers applied here.

Inhibitory Role of PIP2 on Calcium-Activated Chloride Channel Activity

Ca2+-activated Cl- channels (CaCCs) in vascular and many non-vascular smooth muscle cells are thought to be encoded by the gene TMEM16A. Our group recently showed that the amplitude and pharmacology of CaCCs currents (IClca) are influenced by cholesterol depletion (Sones et al. Cardiovasc Res 2010;87:476-484). The aim of this study was to investigate the mechanism by which native CaCCs are regulated by cholesterol depletion and the possible role of phosphatidylinositol 4, 5-bisphosphate (PIP2). Freshly isolated pulmonary artery myocytes were obtained from male Wistar rats (200-225g), sacrificed in accordance with Schedule 1 of the United Kingdom Animals Act (1986). In whole cell patch clamp recording mode, macroscopic IClca were elicited by dialysing the cells with a pipette solution set to 500nM ‘free’ [Ca2+]. Single channel inside-out recordings were also induced by 500nM [Ca2+] and displayed similar characteristics to earlier characterisation. These currents displayed the distinct voltage- and time-dependent properties described in previous studies. Cholesterol depletion from the cell membrane through the application of 3mg.ml−1 methyl-β-cyclodextrin (mβcd) augmented IClca, which was prevented by application of the TMEM16A specific inhibitor T16Ainh-A01. Augmentation by mβcd was also sensitive to the phospholipase C inhibitor U73312, but not the inactive analogue U73343. Inhibiting PIP2 synthesis by incubation with 20µM wortmannin, a PI-4 kinase inhibitor augmented IClca and prevented the stimulatory effects of mβcd. Enrichment of the pipette solution with 1μM diC-8 PIP2 attenuated IClcaand suppressed the effect of mβcd. Wortmannin and PIP2 effects on IClca were replicated in single channel inside-out recordings. Furthermore, the application of the poly-cation Poly-L-lysine (50µg/ml), which scavenges PIP2, augmented IClca. This study suggests that cholesterol depletion of the cell membrane increases TMEM16A/CaCC activity, partly through the removal of the inhibitory effect of PIP2.

The effects of structurally disparate Kv7 activators in the rat vasculature

Kv7 voltage‐gated potassium channels, encoded by KCNQ1–5 genes, are present functionally in vascular and non‐vascular smooth muscle cells. The aim of the present study was to compare the effect of structurally different activators of Kv7.2–7.5 channels: S‐1, BMS‐204352, ICA27243 and retigabine in the rat mesenteric artery, thoracic aorta and coronary circulation. Isometric tension recording was performed on segments of mesenteric artery and thoracic aorta. In addition the coronary circulation was studied using the Langendorff heart preparation. In mesenteric arteries the Kv7 activators demonstrated EC50 values of: 1.9 μM (BMS204352), 2.5 μM (S‐1), 5.4 μM (ICA27243), and 14.3 μM (retigabine). Application of all Kv7 activators at 10 μM relaxed the thoracic aorta albeit with varying efficacies. In the isolated heart increasing concentrations of Kv7 activators produced a concentration‐dependent increase in coronary flow. This study is the first to fully characterise the effects of various Kv7 channel activators in the rat mesenteric artery, thoracic aorta and coronary artery.Work was funded by BBSRC‐CASE studentship with NeuroSearch A/S.

Expression pattern of endothelin system components and localization of smooth muscle cells in the human pre-ovulatory follicle

Whether ovarian follicular rupture involves contractile activity or not has been debated for decades. Recently, study in the rodents has indicated that an endogenously produced potent vasoconstrictive peptide, endothelin-2 (EDN2), may induce follicular constriction immediately prior to ovulation. This study was aimed to systematically characterize the human ovarian endothelin system and localize smooth muscle cells to assess the possible involvement of contractile activity in human ovulation. This is a prospective experimental study. Study subjects were 20 women aged 20-38 years who underwent IVF owing to tubal or male factors. Expression patterns of messenger RNAs (mRNAs) for EDN1, EDN2, EDN3, endothelin-converting enzyme-1 (ECE1 and ECE2), endothelin receptor A (ET(A)) and ET(B) in the granulosa cells (GCs) and cumulus cells and endothelin peptide concentration in the pre-ovulatory follicles were measured at 36 h after hCG injection. In addition, localization of ovarian smooth muscle cells and endothelin receptor expression were determined in normal (non-IVF patient) ovaries. Pre-ovulatory follicles express mRNA for EDN1 and EDN2, ECE1, ECE2, ET(A) and ET(B), but not EDN3, contain highly concentrated endothelin peptides (105.9 pg/ml) and are surrounded by theca externa that are made mostly of multicell layer non-vascular smooth muscle cells. ET(A) expression is localized in the smooth muscle cells of theca externa, theca interna and GC, whereas ET(B) expression is confined to theca interna. Pre-ovulatory follicles contain highly concentrated endothelins and are surrounded by non-vascular smooth muscle cells that express endothelin receptor, suggesting involvement of endothelin-induced contractile action in ovulation in the human ovary.

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-beta-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and alpha-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.

The multifunctional choroid.

The choroid of the eye is primarily a vascular structure supplying the outer retina. It has several unusual features: It contains large membrane-lined lacunae, which, at least in birds, function as part of the lymphatic drainage of the eye and which can change their volume dramatically, thereby changing the thickness of the choroid as much as four-fold over a few days (much less in primates). It contains non-vascular smooth muscle cells, especially behind the fovea, the contraction of which may thin the choroid, thereby opposing the thickening caused by expansion of the lacunae. It has intrinsic choroidal neurons, also mostly behind the central retina, which may control these muscles and may modulate choroidal blood flow as well. These neurons receive sympathetic, parasympathetic and nitrergic innervation. The choroid has several functions: Its vasculature is the major supply for the outer retina; impairment of the flow of oxygen from choroid to retina may cause Age-Related Macular Degeneration. The choroidal blood flow, which is as great as in any other organ, may also cool and warm the retina. In addition to its vascular functions, the choroid contains secretory cells, probably involved in modulation of vascularization and in growth of the sclera. Finally, the dramatic changes in choroidal thickness move the retina forward and back, bringing the photoreceptors into the plane of focus, a function demonstrated by the thinning of the choroid that occurs when the focal plane is moved back by the wearing of negative lenses, and, conversely, by the thickening that occurs when positive lenses are worn. In addition to focusing the eye, more slowly than accommodation and more quickly than emmetropization, we argue that the choroidal thickness changes also are correlated with changes in the growth of the sclera, and hence of the eye. Because transient increases in choroidal thickness are followed by a prolonged decrease in synthesis of extracellular matrix molecules and a slowing of ocular elongation, and attempts to decouple the choroidal and scleral changes have largely failed, it seems that the thickening of the choroid may be mechanistically linked to the scleral synthesis of macromolecules, and thus may play an important role in the homeostatic control of eye growth, and, consequently, in the etiology of myopia and hyperopia.

Differential expression of immunohistochemical markers in bladder smooth muscle and myofibroblasts, and the potential utility of desmin, smoothelin, and vimentin in staging of bladder carcinoma

Distinguishing bladder muscularis propria from muscularis mucosae can be problematic especially in transurethral resection specimens performed for bladder carcinoma. Moreover, bladder carcinoma can be associated with a proliferative/desmoplastic myofibroblastic response that can resemble smooth muscle and potentially lead to overdiagnosis of muscularis propria invasion. The aim of this study was to investigate the potential role of immunohistochemistry in staging bladder carcinoma by evaluating the expression of different markers in myofibroblasts and nonvascular smooth muscle cells in 15 cases of invasive bladder carcinoma. Reactive myofibroblasts were consistently positive for vimentin and smooth muscle actin, consistently negative for caldesmon, desmin, and smoothelin, and had variable expression of actin and CD10. Nonvascular smooth muscle cells of the bladder were consistently positive for smooth muscle actin, actin, desmin, and caldesmon, and consistently negative for CD10. In contrast to smooth muscle cells of the muscularis propria, which displayed strong smoothelin expression in all 15 cases, the smooth muscle cells of the muscularis mucosae displayed moderate smoothelin expression in only 1 (9%) of 11 cases (P=10−7). Surprisingly, although strongly highlighting endothelial and endomysial cells, the smooth muscle cells of the muscularis propria weakly expressed vimentin in only 1 (7%) of 15 cases, whereas smooth muscle cells of the muscularis mucosae had moderate or strong expression in 9 (82%) of 11 cases (P=0.00016). The sensitivity and specificity of desmin or caldesmon expression for smooth muscle cells were 100%. The sensitivity and specificity of strong smoothelin expression for muscularis propria were 100%, whereas those of absent vimentin expression were 93 and 82%, respectively. Although morphology remains the gold standard, the findings suggest that immunohistochemistry, using a panel composed of desmin, smoothelin, and vimentin, may be potentially useful for staging of bladder carcinoma. Confirmatory larger-scale studies, especially on transurethral resection specimens, are warranted.

Elastic Fibers in Myocardial Scars in Rats: Development Teraction with Other Components

This work was designed to determine the course of development of elastic fibers in myocardial scars in rats and their relationship to other components of such structures. Light and electron microscopic observations were made on tissues from 24 rats, killed at sequent stages from 4 to 24 days postinjury. By both techniques, elastic fibers, shown to be forming by 4 days, had increased in size and number with maturation of the scar. At later stages they became interdigitated with the stumps of viable myocytes. We also saw that these fibers often had formed close contacts with the cell surfaces of myofibroblasts and nonvascular smooth muscle cells; a process found in some other situations but not previously in myocardial scars. This information is relevant, in particular, to the dynamics of myocardial scars and thus to the maintenance of function in the injured heart, but also to elastic fiber behavior in general. The integral role of elastic fibers in cell-matrix interactions as well as their biomechanical function is emphasized.

Antibody-independent localization of the electroneutral Na+-HCO3− cotransporter NBCn1 (slc4a7) in mice

The expression pattern of the electroneutral Na(+)-HCO(3)(-)cotransporter NBCn1 (slc4a7) was investigated by beta-galactosidase staining of mice with a LacZ insertion into the NBCn1 gene. This method is of particular value because it is independent of immunoreactivity. We find that the NBCn1 promoter is active in a number of tissues where NBCn1 has previously been functionally or immunohistochemically identified, including a broad range of blood vessels (vascular smooth muscle cells and endothelial cells), kidney thick ascending limb and medullary collecting duct epithelial cells, the epithelial lining of the kidney pelvis, duodenal enterocytes, choroid plexus epithelial cells, hippocampus, and retina. Kidney corpuscles, colonic mucosa, and nonvascular smooth muscle cells (from the urinary bladder, trachea, gastrointestinal wall, and uterus) were novel areas of promoter activity. Atrial but not ventricular cardiomyocytes were stained. In the brain, distinct layers of the cerebral cortex and cerebellar Purkinje cells were stained as was the dentate nucleus. No staining of skeletal muscle or cortical collecting ducts was observed. RT-PCR analyses confirmed the expression of NBCn1 and beta-galactosidase in selected tissues. Disruption of the NBCn1 gene resulted in reduced NBCn1 expression, and in bladder smooth muscle cells, reduced amiloride-insensitive Na(+)-dependent HCO(3)(-) influx was observed. Furthermore, disruption of the NBCn1 gene resulted in a lower intracellular steady-state pH of bladder smooth muscle cells in the presence of CO(2)/HCO(3)(-) but not in its nominal absence. We conclude that NBCn1 has a broad expression profile, supporting previous findings based on immunoreactivity, and suggest several new tissues where NBCn1 may be important.

Molecular characterization and expression analysis of the gene coding for the porcine β 3 integrin subunit (CD61)

Integrins are heterodimeric cell adhesion molecules with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the β 3 integrin subfamily are composed of a β 3 subunit (CD61) non-covalently associated with two α subunits, α IIb (CD41) and α v (CD51), to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. This investigation has focused on the molecular characterization of the cDNA encoding the porcine β 3 integrin subunit. The deduced 762-amino acid sequence was 93, 92, 91, 89, 79 and 73% homologous to human, dog, rabbit, mouse, chicken and Xenopus laevis CD61 protein, respectively. Porcine CD61 molecule shares many structural features with human CD61, including a region containing a metal ion-dependent adhesion site (MIDAS) folding into an I domain-like structure. Through PCR-SSCP analysis and sequencing, six polymorphic positions were detected in the cDNA sequence of porcine CD61, and their frequencies were observed from a collection of 47 pigs. Expression analysis was done at two different levels: expression of the CD61 mRNA by RT-PCR and localization of the protein by immunohistochemistry. Our results show that CD61 transcripts were detected mainly in platelets and hematopoietic tissues. The immunohistochemical tissue localization of CD61 protein by a specific monoclonal antibody against CD61 recombinant protein showed that CD61 was expressed on vascular and non-vascular smooth muscle, epithelium and myeloid cells, being undetectable in cells of the lymphoid lineage. Furthermore, pulmonary intravascular macrophages (PIM), a subpopulation of macrophages which seem to play an important role in blood clearance, expressed much more CD61 when compared to pulmonary alveolar macrophages (PAM). The knowledge of the structure and distribution of the CD61 provides insight into the physiological function of the porcine β 3 integrins and should be of importance in understanding the role of this integrin family in biological processes.

Localization of NBCn1 (slc4a7) by a non‐immunological method

The expression pattern of NBCn1 (slc4a7) was analyzed by β-galactosidase staining of mice with a LacZ gene insertion after the NBCn1 promoter. The applied method is of particular value as it is independent of immunoreactivity. No color was seen in wild-type mice. The NBCn1 promoter was active in several tissues where functional importance of NBCn1 has been shown; including numerous arterial beds, kidney medulla (inner stripe of outer medulla and inner medulla), duodenal enterocytes, hippocampus (CA1 through CA4 and the dentate gyrus) and retina. Kidney glomeruli and the epithelial lining of the kidney pelvis were novel areas of promoter activity. Colonic enterocytes and non-vascular smooth muscle (from esophagus, stomach, intestine, bladder, trachea and uterus) were stained in addition to heart atria but not ventricles. In the brain, distinct layers of the cerebral and cerebellar cortex were stained as was the dentate nucleus. In conclusion, NBCn1 has a broad expression profile being abundant in a broad range of arteries, in non-vascular smooth muscle, in duodenal, colonic and kidney epithelial cells, in retina, as well as in distinct brain structures. This study supports previous findings based on immunoreactivity and suggests several new tissues where NBCn1 may be important. This work was supported by the Danish National Research Foundation.

Selectivity of repaglinide and glibenclamide for the pancreatic over the cardiovascular KATP channels

Sulfonylureas and glinides close beta cell ATP-sensitive K(+) (K(ATP)) channels to increase insulin release; the concomitant closure of cardiovascular K(ATP) channels, however, leads to complications in patients with cardiac ischaemia. The insulinotrope repaglinide is successful in therapy, but has been reported to inhibit the recombinant K(ATP) channels of beta cells, cardiocytes and non-vascular smooth muscle cells with similar potencies, suggesting that the (patho-)physiological role of the cardiovascular K(ATP) channels may be overstated. We therefore re-examined repaglinide's potency at and affinity for the recombinant pancreatic, myocardial and vascular K(ATP) channels in comparison with glibenclamide. K(ATP) channel subunits (i.e. inwardly rectifying K(+) channels [Kir6.x] and sulfonylurea receptors [SURx]) were expressed in intact human embryonic kidney cells and assayed in whole-cell patch-clamp and [(3)H]glibenclamide binding experiments at 37 degrees C. Repaglinide and glibenclamide, respectively, were >or=30 and >or=1,000 times more potent in closing the pancreatic than the cardiovascular channels and they did not lead to complete inhibition of the myocardial channel. Binding assays showed that the selectivity of glibenclamide was essentially based on high affinity for the pancreatic SUR, whereas binding of repaglinide to the SUR subtypes was rather non-selective. After coexpression with Kir6.x to form the assembled channels, however, the affinity of the pancreatic channel for repaglinide was increased 130-fold, an effect much larger than with the cardiovascular channels. This selective effect of coexpression depended on the piperidino substituent in repaglinide. Repaglinide and glibenclamide show higher potency and efficacy in inhibiting the pancreatic than the cardiovascular K(ATP) channels, thus supporting their clinical use.

Non‐vascular smooth muscle cells in the human choroid: distribution, development and further characterization

To characterize further non-vascular smooth muscle cells (NVSMC) in the choroid of the human eye, extensive morphological studies were performed including a three-dimensional distribution of NVSMC in the adult human eye and their appearance during development. Whole mounts and sections through the choroid and sclera of eyes of 42 human donors (between the 13th week of gestation and 89 years of age) were stained with antibodies against smooth muscle actin and other markers for smooth muscle cells. On the basis of their morphological localization, three groups of NVSMC could be distinguished in the adult eyes: (a) a semicircular arrangement of NVSMC in the suprachoroid and inner sclera, around the entry of posterior ciliary arteries and nerves; (b) NVSMC parallel to the vessels in the posterior eye segment between the point of entry of the posterior ciliary arteries and the point of exit of the vortex veins; and (c) a dense plaque-like arrangement of NVSMC in the suprachoroid, overlying the foveal region. The last of these groups showed most pronounced interindividual differences. During development, the first NVSMC to be observed at the 20th week of gestation belonged to group b. A complete NVSMC network was first observed in a 6-year-old donor eye. All three groups stained positive for smoothelin, caldesmon and calponin in all localizations. The NVSMC show a distinct distribution that might reflect different aspects of their function in the choroid and suprachoroid. All cells could be histochemically characterized as truly contractile.

Low-Energy Laser Irradiation Promotes Cellular Redox Activity

Low-energy visible light (LEVL) has been shown to stimulate cell functions. This is called "photobiostimulation" and has been used successfully over the last three decades for treating a range of conditions, including soft tissue injuries, severe wounds, chronic pain, and more. Nevertheless, the mechanism of photobiostimulative processes is still being debated. It is obvious that, in order to interact with the living cell, light has to be absorbed by intracellular chromophores. In a search for chromophores responsible for photobiostimulation, endogenous porphyrins, mitochondrial and membranal cytochromes, and flavoproteins were found to be suitable candidates. The above-mentioned chromophores are photosensitizers that generate reactive oxygen species (ROS) following irradiation. As the cellular redox state has a key role in maintaining the viability of the cell, changes in ROS may play a significant role in cell activation. In the present review, we summarize evidence demonstrating that various ROS and antioxidants are produced following LEVL illumination. We found that very little evidence for NO formation in illuminated non-vascular smooth muscle cells exists in the literature. We suggest that the change in the cellular redox state which plays a pivotal role in maintaining cellular activities leads to photobiostimulative processes.