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Abstract Background Triptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp). Methods We investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice. Results We demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice. Conclusions We have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions. Graphical Abstract

Background: Triptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp). Methods: We investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice. Results: We demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice. Conclusions: We have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions. [ABSTRACT FROM AUTHOR]

Background: The atrial natriuretic peptide (ANP) released from the heart regulates intravascular volume and is suspected to increase capillary permeability. Contradictory results regarding ANP and glycocalyx degradation have been reported. The aim of this study was to investigate if an infusion of ANP causes degradation of the endothelial glycocalyx. Methods: Twenty pigs, pretreated with 250 mg methylprednisolone, were randomized to receive an infusion of either ANP (50 ng/kg/min) (n = 10) or 0.9% NaCl (n = 10) during 60 min. Endothelial glycocalyx components (heparan sulphate proteoglycan and hyaluronic acid), Hct, calculated plasma volume and colloid osmotic pressure were measured from baseline to 60 min. Results: There was no difference between the control and intervention groups for heparan sulphate proteoglycan and hyaluronic acid corrected for the change in plasma volume (P =.333 and 0.197). Hct increased with 1.8 ± 2.2% in the intervention group (P =.029) with no change −0.5 ± 2.3% in the control group (P =.504). The plasma volume decreased in the intervention group with −8.4 ± 10% (P =.034) with no change in the control group 3.1 ± 12% (P =.427). Median changes in colloid osmotic pressures in the control and intervention group were −0.39 [95% CI, −1.88‐0.13] and 0.9 [95% CI, 0.00‐1.58], respectively (P =.012). Conclusions: In this randomized porcine study, an ANP infusion did not cause endothelial glycocalyx degradation but decreased the plasma volume most probably due to precapillary vasodilation and increased filtration. [ABSTRACT FROM AUTHOR]

The heart is a metabolic omnivore that combusts a considerable amount of energy substrates, mainly long-chain fatty acids (FAs) and others such as glucose, lactate, ketone bodies, and amino acids. There is emerging evidence that muscle-type continuous capillaries comprise the rate-limiting barrier that regulates FA uptake into cardiomyocytes. The transport of FAs across the capillary endothelium is composed of three major steps—the lipolysis of triglyceride on the luminal side of the endothelium, FA uptake by the plasma membrane, and intracellular FA transport by cytosolic proteins. In the heart, impaired trans-endothelial FA (TEFA) transport causes reduced FA uptake, with a compensatory increase in glucose use. In most cases, mice with reduced FA uptake exhibit preserved cardiac function under unstressed conditions. When the workload is increased, however, the total energy supply relative to its demand (estimated with pool size in the tricarboxylic acid (TCA) cycle) is significantly diminished, resulting in contractile dysfunction. The supplementation of alternative fuels, such as medium-chain FAs and ketone bodies, at least partially restores contractile dysfunction, indicating that energy insufficiency due to reduced FA supply is the predominant cause of cardiac dysfunction. Based on recent in vivo findings, this review provides the following information related to TEFA transport: (1) the mechanisms of FA uptake by the heart, including TEFA transport; (2) the molecular mechanisms underlying the induction of genes associated with TEFA transport; (3) in vivo cardiac metabolism and contractile function in mice with reduced TEFA transport under unstressed conditions; and (4) in vivo contractile dysfunction in mice with reduced TEFA transport under diseased conditions, including an increased afterload and streptozotocin-induced diabetes.

The study of the structural basis of gas exchange function in the lung depends on the availability of quantitative information that concerns the structures establishing contact between the air in the alveoli and the blood in the alveolar capillaries, which can be entered into physiological equations for predicting oxygen uptake. This information is provided by morphometric studies involving stereological methods and allows estimates of the pulmonary diffusing capacity of the human lung that agree, in experimental studies, with the maximal oxygen consumption. The basis for this 'machine lung' structure lies in the complex design of the cells building an extensive air-blood barrier with minimal cell mass. [ABSTRACT FROM AUTHOR]

Abstract: Aims: The vanilloid type 4 transient receptor potential channel (TRPV4) is a potential environmental sensor to multiple stimuli in many types of cells. In this study, we show that TRPV4 activated by 4α-phorbol 12,13-didecanoate (4αPDD) and hypo-osmotic stimulation (HOS) is a regulator of intracellular calcium ([Ca2+]i) in human brain capillary endothelial cells (HBCEs), and its activation can partially regulate cell proliferation of HBCEs. Main methods: The expression of TRPV4 in HBCEs was analyzed at the mRNA and protein levels. The function of TRPV4 in HBCEs was evaluated using a TRPV4 agonist, 4αPDD, and HOS while measuring [Ca2+]i and membrane currents. Key findings: Analysis of the mRNA transcripts of the TRPV subfamily revealed that TRPV2 and TRPV4 were expressed in HBCEs. Immunoreactivity to the TRPV4 protein was also detected in HBCEs, which were positively stained by von Willebrand factor and CD31. When 4αPDD was applied, [Ca2+]i in HBCEs was elevated in a concentration-dependent manner. In addition, exposure of HBCEs to HOS at 228mOsm induced an elevation of [Ca2+]i. Application of 4αPDD also activated non-selective cation currents (NSCCs). Pretreatment of HBCEs with short interference RNA targeting TRPV4 (siRNA) significantly reduced the 4αPDD-induced elevation of [Ca2+]i. When HBCEs were treated for 24h with concentrations of 4αPDD between 0.3 and 3μM, the cell proliferation was potentiated in a concentration-dependent manner. The potentiation was partially inhibited in HBCEs treated with siRNA. Significance: These data suggest that endogenous TRPV4, which functions as a regulator of [Ca2+]i in HBCEs, partially controls cell proliferation. [Copyright &y& Elsevier]

Please cite this paper as: Wagner R, Modla S, Hossler F, Czymmek K. Three-dimensional analysis and computer modeling of the capillary endothelial vesicular system with electron tomography. Microcirculation 19: 477-484, 2012. Abstract Objective: We examined the three-dimensional organization of the endothelial vesicular system with TEM tomography of semi-thick sections. Materials and methods: Mouse abdominal muscle capillaries were perfused with terbium to label vesicular compartments open to the luminal surface. The tissue was prepared for TEM and semi-thick (250 nm) sections were cut. Dual axis tilt series, collected from +60° to −60° at 1° increments, were acquired in regions of labeled abluminal caveolae. These tomograms were reconstructed and analyzed to reveal three-dimensional vesicular associations not evident in thin sections. Results: Reconstructed tomograms revealed free vesicles, both labeled and unlabeled, in the endothelial cytoplasm as well as transendothelial channels that spanned the luminal and abluminal membranes. A large membranous compartment connecting the luminal and abluminal surfaces was also present. Computer modeling of tomographic data and video animations provided three-dimensional perspectives to these structures. Conclusions: Uncertainties associated with other three-dimensional methods to study the capillary wall are remedied by tomographic analysis of semi-thick sections. Transendothelial channels of fused vesicles and free cytoplasmic vesicles give credence to their role as large pores in the transport of solutes across the walls of continuous capillaries. [ABSTRACT FROM AUTHOR]

The time course of blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) responses to hyperosmolar mannitol infusion (HMI; 1.6 M) during chronic hypertension was investigated using 14C-sucrose as a marker of barrier integrity. 14C-sucrose entry into CSF of both spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats 2 min after HMI increased ∼7-fold compared to their respective control. The volume of distribution (Vd) of 14C-sucrose into brain cortex of SHR increased 13-fold 2 min after HMI while that in WKY rats increased only 4-fold. After HMI Vd of 14C-sucrose into the cortex of WKY, and CSF of both SHR and WKY remained steadily greater than their corresponding control for up to 30 min (p < 0.01), whereas in the cortex of SHR the Vd of 14C-sucrose reached control values 20 min after HMI (p > 0.05), indicating that after HMI the increase in paracellular diffusion of 14C-sucrose into SHR cortex was not persistent, in contrast to WKY rats and CSF of both SHR and WKY rats. Electron microscopy of the brain cortex after HMI showed capillary endothelial cell shrinkage and perivascular swellings in the brain cortex, and in the choroid plexus opening of tight junctions were observed. Our results indicate disruption of both the BBB and the BCSFB after HMI in both SHR and WKY rats. The disruption remained persistent up to 25 min after HMI at the BBB of WKY rats and BCSFB in both animal groups, while in SHR the protective function of the BBB returned to control values 20 min after HMI. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: We report the characteristics of CD300LG, a member of the CD300 antigen like family. Its genomic structure is similar in both mouse and human, and at least four isoforms exist in both species. The amino acid sequence of the immunoglobulin (Ig) V like domain of CD300LG showed approximately 35% identity to those of the polymeric Ig receptor (pIgR) and Fcα/μR. Interestingly, mouse CD300LG proteins were uniquely expressed on capillary endothelium. Immunoelectron microscopy revealed that mouse CD300LG is localized on both apical and basolateral plasma membranes, as well as on intracellular vesicular structures, in the capillary endothelium. Transcytosis assays using polarized MDCK epithelial cells showed that CD300LG could be transcytosed bidirectionally. Furthermore, CD300LG exogenously expressed on HeLa cells could take up IgA2 and IgM, but not IgG. These results suggest that CD300LG might play an important role in molecular traffic across the capillary endothelium. [Copyright &y& Elsevier]

The water channel aquaporin-1 (AQP1) is considered as the molecular counterpart of the ultrasmall pore predicted by the three-pore model of fluid transport across the peritoneal membrane. However, the definitive proof of the implication of AQP1 in solute-free water transport, sodium sieving, and ultrafiltration (UF) during peritoneal dialysis (PD) is lacking, and the effects of its deletion on the structure of the membrane are unknown. Using real-time reverse transcriptase-polymerase chain reaction and immunogold electron microscopy, we showed that AQP1 is the most abundant member of the AQP gene family expressed in the mouse peritoneum, and the only one located in the capillary endothelium. Transport studies during a 2-h dwell demonstrated that, in comparison with Aqp1+/+ littermates, Aqp1−/− mice had no sodium sieving; an ∼70% decrease in the initial, solute-free UF; and an ∼50% decrease in cumulative UF. These modifications occurred despite unchanged osmotic gradient and transport of small solutes in the Aqp1−/− mice. Heterozygous Aqp1+/− mice showed intermediate values in sodium sieving and initial UF, whereas cumulative UF was similar to Aqp1+/+ mice. The deletion of AQP1 had no effect on the expression of other AQPs and on the density, structure, or diameter of peritoneal capillaries. These data provide direct evidence for the role of AQP1 during PD. They validate essential predictions of the three-pore model: (i) the ultrasmall pores account for the sodium sieving, and (ii) they mediate 50% of UF during a hypertonic dwell.Kidney International (2006) 69, 1518–1525. doi:10.1038/sj.ki.5000285; published online 1 March 2006 [ABSTRACT FROM AUTHOR]

Shen G, Hägg U, Rabie AB, Kaluarachchi K Based on the phenomenon that expression of type X collagen and capillary endothelium correlates with endochondral ossification, the prime aim of this study was to establish the temporal pattern of condylar growth in Sprague–Dawley rats by biochemically identifying the expression of these two factors. Sprague–Dawley rats were divided into five groups representing five different stages during somatic pubertal growth. In situ hybridization and immunoperoxidase were performed to examine expression of type X collagen in hypertrophic zone and capillary endothelium in erosive zone of condylar cartilage. Computer-assisted imaging analyses were conducted to allow for a quantitative assessment of the expression of these two factors, from which the temporal pattern of condylar growth was inferred. (1) Synthesis of type X collagen and emergence of capillary endothelium were critical factors during the transition of condylar cartilage from chondrogenesis into osteogenesis, a biological pathway that leads to endochondral bone formation, the mode through which the condyle grows. (2) Quantitative analyses revealed the temporal pattern of the expression of these two factors, indicating that the thrust of natural growth of the condyle in the rats occurred in concomitance with somatic pubertal growth, featured by an acceleration starting from day 38, a maximum growth rate on day 56, followed by a decrease afterwards. It is suggested that the biochemical examination of growth markers, such as type X collagen, might be a new approach to accurately depict temporal pattern of condylar growth which is too delicate to be reflected by gross measurement not only in Sprague–Dawley rats but potentially also in other species. [ABSTRACT FROM AUTHOR]

The lung endothelium layer is exposed to continuous CO2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na+/H+ exchanger and HCO3--dependent H+-transporting mechanisms regulate intracellular pH (pHcyt) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H+-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na+/H+ exchanger and HCO3--based H+-transporting mechanisms, to maintain pHcyt homeostasis. Immunocytochemical studies revealed V-H+-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na+ and HCO3- that were similar to those observed in the presence of either Na+, or Na+ and HCO3-. The Na+- and HCO3--independent pHcyt recovery was inhibited by bafilomycin A1, a V-H+-ATPase inhibitor. These studies show a Na+- and HCO3--independent pHcyt regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases. [Copyright &y& Elsevier]

We report the identification and characterization of a novel cytokine-like gene family using structure-based methods to search for novel four-helix-bundle cytokines in genomics databases. There are four genes in this family, FAM3A, FAM3B, FAM3C, and FAM3D, each encoding a protein (224–235 amino acids) with a hydrophobic leader sequence. Northern analysis indicates that FAM3B is highly expressed in pancreas, FAM3D in placenta, and FAM3A and FAM3C in almost all tissues. Immunohistochemistry showed that FAM3A is expressed prominently in the vascular endothelium, particularly capillaries. We found that FAM3A and FAM3B protein were both localized to the islets of Langerhans of the endocrine pancreas. Recombinant FAM3B protein has delayed effects on β-cell function, inhibiting basal insulin secretion from a β-cell line in a dose-dependent manner. [Copyright &y& Elsevier]

Hyperhomocyst(e)inemia has been associated with the development of hypertension, stroke, and cardiovascular, cerebral/neuronal, renal, and liver diseases. To test the hypothesis that homocyst(e)ine plays an integrated role in multiorgan injury in hypertension, we employed: (1) spontaneously hypertensive rats (SHR) in which endogenous homocyst(e)ine levels are moderately high (18.1 ± 0.5 μM); (2) control age- and sex-matched Wistar Kyoto (WKY) rats in which homocyst(e)ine levels are normal (3.7 ± 0.3 μM). To create the pathophysiological condition of hyperhomocyst(e)inemia, 20 mg/day homocyst(e)ine was administered for 12 weeks in (3) SHR (SHR-H) and in (4) WKY (WKY-H) rats. (5) Endogenous homocyst(e)ine levels were reduced slightly but not significantly from 18.1 ± 0.5 μM to 12.5 ± 0.7 μM in SHR by folic acid administration (SHR-F). Plasma and tissue levels of homocyst(e)ine were determined by HPLC and spectrophotometric methods. Plasma and sympathetic ganglion (neuronal) matrix metalloproteinase (MMP) activity was measured by zymography. Activity of neuronal MMP was increased in hyperhomocyst(e)inemic rats as compared with controls. Mean arterial pressure (mmHg) was 95 ± 5, 126 ± 8, 157 ± 10, 188 ± 5, and 165 ± 12 in WKY, WKY-H, SHR, SHR-H, and SHR-F, respectively. Urinary protein (mg/day) was 0.11 ± 0.03, 0.88 ± 0.22, 0.47 ± 0.10, 0.89 ± 0.21, and 0.81 ± 0.21 in WKY, WKY-H, SHR, SHR-H, and SHR-F, respectively, as measured by the Bio-Rad dye binding assay. The relationships between increased arterial pressure, plasma homocyst(e)ine, and urinary protein were delineated. Plasma and neuronal creatinine phosphokinase (CK) isoenzymes were measured by agarose gel electrophoresis. All three CK isoenzymes, i.e., MM, MB, and BB, specific for skeletal, cardiac, and nerve tissue, respectively, were induced following 12 weeks' hyperhomocyst(e)inemia, suggesting multiorgan injury by homocyst(e)ine. Homocyst(e)ine induces endocardial endothelial cell (capillary) apoptosis and may reduce capillary cell density. Structural damage to aorta, myocardium, kidney, and renal-ureter was analyzed by histology. Results suggested an integrated physiological role of homocyst(e)ine in injury to the endothelial/epithelial cell lining in the respective organs. [ABSTRACT FROM AUTHOR]

The heart is a metabolic omnivore that combusts a considerable amount of energy substrates, mainly long-chain fatty acids (FAs) and others such as glucose, lactate, ketone bodies, and amino acids. There is emerging evidence that muscle-type continuous capillaries comprise the rate-limiting barrier that regulates FA uptake into cardiomyocytes. The transport of FAs across the capillary endothelium is composed of three major steps—the lipolysis of triglyceride on the luminal side of the endothelium, FA uptake by the plasma membrane, and intracellular FA transport by cytosolic proteins. In the heart, impaired trans-endothelial FA (TEFA) transport causes reduced FA uptake, with a compensatory increase in glucose use. In most cases, mice with reduced FA uptake exhibit preserved cardiac function under unstressed conditions. When the workload is increased, however, the total energy supply relative to its demand (estimated with pool size in the tricarboxylic acid (TCA) cycle) is significantly diminished, resulting in contractile dysfunction. The supplementation of alternative fuels, such as medium-chain FAs and ketone bodies, at least partially restores contractile dysfunction, indicating that energy insufficiency due to reduced FA supply is the predominant cause of cardiac dysfunction. Based on recent in vivo findings, this review provides the following information related to TEFA transport: (1) the mechanisms of FA uptake by the heart, including TEFA transport; (2) the molecular mechanisms underlying the induction of genes associated with TEFA transport; (3) in vivo cardiac metabolism and contractile function in mice with reduced TEFA transport under unstressed conditions; and (4) in vivo contractile dysfunction in mice with reduced TEFA transport under diseased conditions, including an increased afterload and streptozotocin-induced diabetes. [ABSTRACT FROM AUTHOR]

To dermine the effect of post-ischaemic reperfusion on the ultrastructure of the endothelial glycocalyx and the role of oxygen free radicals, isolated working rat hearts were subjected to 20 min ischaemia followed by 3 or 30 min of reperfusion. Ruthenium red and lanthanum chloride were used to delineate the endothelial glycocalyx, and histochemical manganese/diaminobenzidine (Mn/DAB) or iron/diaminobenzidine (Fe/DAB) techniques were applied to visualize superoxide and hydrogen peroxide in myocardial capillaries. We found that ischaemia alone led to only a slightly flocculent appearance of the glycocalyx and its disruption was not observed until the onset of reperfusion. Prolongation of reperfusion to 30 min had no further effect on the ultrastructure of the glycocalyx. The ultrastructure of endothelial cells was normal. The disruption of the glycocalyx correlated in time and place with the appearance of Mn/DAB and Fe/DAB reaction products on the luminal surface of endothelial cells. Treatment with 5 mM N-(2-mercaptopropionyl)-glycine (MPG), an ·OH radical scavenger, starting before ischaemia prevented the disruption of the glycocalyx, while 100 mM 3-morpholinosydnonimine (SIN-1), capable of generating both NO andO simultaneously when applied at the time of reperfusion, increased the mean density of capillaries positively stained with Mn/DAB and Fe/DAB, and caused substantial disruption of the glycocalyx and damage to endothelial cells, which was not prevented by MPG. Our results suggest that the onset of reperfusion is critical for injury to the endothelial glycocalyx. Most probably the hydroxyl radical derived from the Fenton reactionis responsible for this injury. Peroxynitrite and/or nitric dioxide, if present upon reperfusion, may also account for damage of the endothelial. glycocalyx. [ABSTRACT FROM AUTHOR]

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Research has suggested a role for insulin delivery through capillaries in muscle in the regulation of insulin sensitivity. Therefore, the formation and turn-over of capillary endothelial cells in muscle were studied in relation to exposure to moderately elevated insulin concentrations with or without concomitant increase of corticosterone concentrations. Female rats were exposed to a moderate, physiological hyperinsulinaemia (~ 450 pmol/l) for 24 h 48 h, 3 days, 7 days and 7 weeks. Propranolol was used to inhibit elevated adrenergic activity. In one insulin-exposed group, corticosterone secretion was controlled by adrenalectomy with substitution of corticosterone to maintain normal concentrations, while another group was left with adrenal corticosterone secretion intact. Rats were exposed to insulin with controlled, non-elevated corticosterone concentrations after adrenalectomy and corticosterone substitution; compared to controls, the number of mitoses in capillary endothelial cells in the soleus and extensor digitorum longus muscle were approximately doubled after 24 h, reaching a maximum, about fivefold higher than controls, after 3 days. After 7 weeks of insulin exposure there were no longer any significant differences between control and insulin-exposed rats. The number of capillaries per unit muscle surface area was moderately (10-15%) but significantly increased at 7 days (only the extensor digitorum longus muscle) and 7 weeks (the extensor digitorum longus and the soleus muscles). In rats exposed to insulin, with intact adrenals, endogenous corticosterone production resulted in concentrations about threefold higher than in rats adrenalectomized with subsequent corticosterone substitution. In these rats the increase in mitoses in capillary endothelium was totally abolished. The results of this study suggest that exposure to insulin in this rat model is followed by a dramatic short-term increase in the formation of new capillary endothelial cells in muscle. It is also suggested that this growth factor-like effect of insulin is abolished by corticosterone. It is suggested that insulin and corticosterone exert opposite effects on the capillary network in muscles, which might be important for the insulin supply to this tissue, and hence for regulation of insulin sensitivity. [ABSTRACT FROM AUTHOR]

Objective: The aim was to determine the effect of hypoxia on the ultrastructure of the endothelial glycocalyx of cardiac capillaries. Methods: Isolated rat hearts were perfused with oxygenated or hypoxic Krebs solution for 30 min after equilibration with oxygenated medium. They were then perfused with a cationic marker (ruthenium red, lanthanum nitrate, or cationised ferritin) to delineate the cardiac endothelial glycocalyx in the electron microscope. With ruthenium red and lanthanum, perfusions were carried out both in the presence and absence of bovine serum albumin. Ferritin perfused hearts were used to quantify changes in the glycocalyx as a result of hypoxia and to measure the cross sectional area of the endothelial cells. Results: In all the hearts perfused with well oxygenated solution, all three markers showed an even, electron dense layer on the luminal surface of the capillaries. With ruthenium red and lanthanum (but not with ferritin), the marker was occasionally observed throughout the length of the interendothelial clefts and on the albuminal surface. After 30 min hypoxic perfusion, both ruthenium red and lanthanum showed disruption and irregular clumping of the glycocalyx, with or without albumin. Ferritin, however, showed a sparse and uneven layer. Measurements of endothelial cell area showed that some cells from hypoxic hearts were swollen when compared with controls. Measurements of the percentage of luminal membrane covered by ferritin molecules showed a significant loss of glycocalyx in hypoxic hearts. There was, however, no correlation between loss of glycocalyx and endothelial cell swelling. Conclusions: The endothelial cell glycocalyx of continuous capillaries is sensitive to changes in Po2. The disruption of this surface coat may explain the reported increase in capillary permeability in hypoxia.Cardiovascular Research 1993;27:384-389 [ABSTRACT FROM PUBLISHER]

Transport of palmitate from the albumin-palmitate complex in the plasma to inside mitochondria where it undergoes β-oxidation is a multistep process. Albumin's large size prevents permeation via interendothelial clefts. Palmitate dissociation from albumin in solution is too slow to provide an adequate supply of the unbound palmitate. The discovery that the dissociation occurs upon albumin binding to an endothelial surface receptor resolves the conundrum. Palmitate transport across the luminal surface membrane may be either carrier-mediated or passive. Fatty-acid binding protein inside endothelial and cardiac muscle cells facilitates diffusion through cytosol while maintaining the unbound palmitate concentration at a very low level. Within the interstitium, albumin is again the palmitate carrier. Still controversial is whether or not there is a saturable sarcolemmal transporter or simply passive exchange. Inside the myocyte palmitate is again bound to the fatty acid binding protein which buffers the free palmitate concentration, facilitates diffusion, and may facilitate further intracellular reactions. [ABSTRACT FROM AUTHOR]