Krebs-Ringer Bicarbonate Buffer Research Articles

Peptides originally derived from Chilobrachys jingzhao tarantula venom possess beneficial effects on pancreatic beta cell health and function

Clinical approval of the glucagon-like peptide-1 (GLP-1) mimetic exenatide for the treatment of type 2 diabetes highlights the therapeutic effectiveness of venom-derived peptides. In the present study, we examined and characterised the glucose-lowering potential of synthetic Jingzhaotoxin IX and Jingzhaotoxin XI peptides, which were originally isolated from the venom of the Chinese earth tarantula Chilobrachys jingzhao. Following confirmation of lack of beta-cell toxicity of synthetic peptides, assessment of enzymatic stability and effects on in vitro beta-cell function were studied, alongside putative mechanisms. Glucose homeostatic and appetite suppressive actions of Jingzhaotoxin IX and Jingzhaotoxin XI alone, or in combination with exenatide, were then assessed in normal overnight fasted C57BL/6 mice. Synthetic Jingzhaotoxin peptides were non-toxic and exhibited a decrease in mass of 6 Da in Krebs-Ringer bicarbonate buffer suggesting inhibitor cysteine knot (ICK)-like formation, but interestingly were liable to plasma enzyme degradation. The Jingzhaotoxin peptides evoked prominent insulin secretion from BRIN BD11 beta-cells, with activity somewhat characteristic of Kv2.1 channel binding. In addition, Jingzhaotoxin peptides enhanced beta-cell proliferation and provided significant protection against cytokine-induced apoptosis. When injected co-jointly with glucose, the Jingzhaotoxin peptides slightly decreased blood-glucose levels but had no effect on appetite in overnight fasted mice. Whilst the Jingzhaotoxin peptides did not enhance exenatide-induced benefits on glucose homeostasis, they augmented exenatide-mediated suppression of appetite. Taken together, these data highlight the therapeutic potential of tarantula venom-derived peptides, such as Jingzhaotoxin IX and Jingzhaotoxin XI either alone or in combination with exenatide, for diabetes and related obesity.

In vitro addition of epinephrine, norepinephrine, and carnitine alters palmitate oxidation and esterification in isolated ovine hepatocytes

Hepatocytes from 4 wethers were used to study the effects of carnitine and increasing concentrations of epinephrine and norepinephrine on palmitate oxidation and esterification. Liver cells were isolated from the wethers and incubated in Krebs-Ringer bicarbonate buffer with 1 mM [14C]-palmitate. Radiolabel incorporation was measured in CO2, acid-soluble products, and esterified products, including triglyceride, diglyceride, and cholesterol esters. Carnitine increased production of CO2 and acid-soluble products from palmitate by 41% and 216%, respectively, but had no effect on conversion of palmitate to esterified products. Epinephrine had a quadratic-increasing effect on palmitate oxidation to CO2, but norepinephrine did not increase palmitate oxidation to CO2. Neither epinephrine nor norepinephrine affected the production of acid-soluble products from palmitate. Increasing concentrations of norepinephrine and epinephrine linearly increased rates of triglyceride formation from palmitate. Increasing norepinephrine concentrations linearly increased diglyceride and cholesterol ester formation from palmitate in the presence of carnitine; epinephrine did not affect diglyceride or cholesterol ester formation. In general, catecholamine treatment had the greatest effect on the formation of esterified products from palmitate, and effects of norepinephrine were more pronounced than epinephrine. Conditions that result in catecholamine release might lead to fat accumulation in liver.

Early introduction of exercise prevents insulin resistance in postnatal overfed rats.

Early childhood obesity increases the risk of developing metabolic diseases. We examined the early introduction of exercise in small-litter obese-induced rats (SL) on glucose metabolism in the epididymal adipose tissue (AT) and soleus muscle (SM). On day 3 post-birth, pups were divided into groups of ten or three (SL). On day 22, rats were split into sedentary (S and SLS) and exercise (E and SLE) groups. The rats swam three times/week carrying a load for 30 min. In the first week, they swam without a load; in the 2nd week, they carried a load equivalent to 2% of their body weight; from the 3rd week to the final week, they carried a 5% body load. At 85 days of age, an insulin tolerance test was performed in some rats. At 90 days of age, rats were killed, and blood was harvested for plasma glucose, cholesterol, and triacylglycerol measurements. Mesenteric, epididymal, retroperitoneal, and brown adipose tissues were removed and weighed. SM and AT were incubated in the Krebs-Ringer bicarbonate buffer, 5.5 mM glucose for 1 h with or without 10 mU/mL insulin. Comparison between the groups was performed by 3-way ANOVA followed by the Tukey post-hoc test. Sedentary, overfed rats had greater body mass, more visceral fat, lower lactate production, and insulin resistance. Early introduction of exercise reduced plasma cholesterol and contained the deposition of white adipose tissue and insulin resistance. In conclusion, the early introduction of exercise prevents the effects of obesity on glucose metabolism in adulthood in this rat model.

Effects of Transport Medium Composition on In vitro Drug Permeation Across Excised Pig Intestinal Tissue

Background:: Knowledge of the permeation characteristics of new chemical entities across biological membranes is essential to drug research and development. Transport medium composition may affect the absorption of compounds during in vitro drug transport testing. To preserve the predictive values of screening tests, the possible influence of transport media on the solubility of model drugs and on the activities of tight junctions and efflux transporter proteins (e.g., Pglycoprotein) must be known. Objective:: The aim of this study was to compare the impact of different transport media on the bidirectional transport of standard compounds, selected from the four classes of the Biopharmaceutical Classification System (BCS), across excised pig intestinal tissue. Methods:: The Sweetana-Grass diffusion apparatus was used for transport studies. Krebs-Ringer bicarbonate (KRB) buffer and simulated intestinal fluids in the fed (FeSSIF) and fasted (FaSSIF) states were used as the three transport media, while the chosen compounds were abacavir (BCS class 1), dapsone (BCS class 2), lamivudine (BCS class 3) and furosemide (BCS class 4). Results:: Abacavir exhibited lower permeability in both the simulated intestinal fluids than in the KRB buffer. Dapsone showed similar permeability in all media. Lamivudine exhibited lower permeability in FaSSIF than in the other two media. Furosemide exhibited improved transport with pronounced efflux in FaSSIF. Conclusion:: Different permeation behaviors were observed for the selected drugs in the respective media, which may have resulted from their different physico-chemical properties, as well as from the effects that dissimilar transport media components had on excised pig intestinal tissue.

Mechanism of endocytic regulation of intestinal tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells.

Intestinal epithelial cells are tightly bound by tight junction proteins (TJP) which are dynamic and sensitive to environmental stress. However, the role of the endocytic pathway in the regulation of TJP abundance and tight junction integrity during nutrient stress is poorly understood. Therefore, this study was conducted to investigate the regulation of TJP abundance during nutrient starvation and the role of the endocytic mechanism in this process. IPEC-J2 cells were subjected to nutrient starvation in Krebs-Ringer bicarbonate buffer (KRB) and abundance of TJP, an indication of tight junction remodeling, was characterized with RT-PCR, western blotting and immunofluorescence. Abundance of TJP was dynamically regulated by nutrient starvation. The protein levels of claudin-1, 3, and 4 were initially downregulated within the first 6 hours of starvation, and then, increased thereafter (P<.01). However, there was no change in occludin and ZO-1. Lysosome and proteasome inhibitors were used to determine the contribution of these protein degradation pathways to the TJP remodeling. Short-term starvation-induced degradation of claudin-1, 3, and 4 was found to be lysosome dependent. Specifically, the downregulation of claudin-3 and 4 was via a dynamin-dependent, but clathrin and caveolae independent, endocytic pathway and this downregulation was partly reversed by amino acids supplementation. Interestingly, the re-synthesis of TJP with prolonged starvation partly depended on proteasome function. Collectively, this study, for the first time, elucidated a major role for dynamin-dependent endocytosis of claudin-3 and 4 during nutrient stress in intestinal epithelial cells. Therefore, transient endocytosis inhibition may be a potential mechanism for preserving tight junction integrity and function in metabolic or pathological states such as inflammatory bowel disease that involves destruction of intestinal epithelial TJP.

WATER EXTRACT OF CAESALPINIA PULCHERRIMA FLOWERS INCREASES GLUCOSE UPTAKE IN INSULIN RESISTANT ADIPOCYTES IN A DOSEDEPENDENT MANNER

Caesalpinia pulcherrima (CP) flower extract has been shown to have antidiabetic effects on pancreatic beta cell regeneration and improved skeletal muscle glucose utilisation. However, little is known about its effect on adipocytes, particularly insulin resistant adipocytes. This study investigated the possibility of improving glucose uptake in adipocytes obtained from chickens which are naturally insulin resistant via direct effects of water extract of Caesalpinia pulcherrima flowers. The extract was prepared by boiling air dried CP flowers in a water based modified Krebs Ringer Bicarbonate buffer for 5mins and sterilizing the filtrate via vacuum filtration. A glucose load of 18mM and 10% ITS was added to the extract. In the absence of adipocytes, the glucose concentration of the CP extract remained unchanged (p=0.5087). Insulin resistance in the chicken adipocytes was confirmed by delayed and limited glucose uptake (maximum 2.9% + 1.62% in 60mins). This was reversed via the addition of the water extract of the CP flowers as concentrations as low as 2.8mg/ml doubled glucose uptake and concentrations as low as 5.6mg/ml increased the rate of glucose uptake to that of insulin sensitive cells. These results suggest that water extract of CP flowers directly modulate glucose uptake into insulin resistant cells at concentrations as low as 2.8mg/ml following a 5mins decoction preparation.

HYPOGLYCEMIC ACTIVITY OF THE AQUEOUS EXTRACT OF CAESALPINIA PULCHERRIMA FLOWERS IS INDEPENDENT OF INSULIN STIMULATION AND SUPPRESSED IN THE PRESENCE OF METFORMIN

Considering the high prevalence of insulin resistance, antidiabetic strategies that enhance insulin action or act independent of insulin are desirable. Caesalpinia pulcherrima (CP) flowers are known to have antidiabetic properties, but more work is required with respect to this action in insulin resistant adipocytes, particularly, its dependence on insulin and its therapeutic equivalence and/or interactions with other antidiabetic drugs. The purpose of this study was therefore to investigate the insulindependency of the water extract of CP flowers (CP extract) hypoglycemic effects, compare its antidiabetic action in diabetic and non-diabetic glucose loads, and explore its therapeutic equivalence and interactions with metformin. CP extract was prepared by boiling the air-dried flowers in cell culture media prepared in Krebs Ringer Bicarbonate buffer for 5mins. Metformin solution was prepared from a Metformin hydrochloride extended-release tablet to obtain low and therapeutic levels of metformin (0.8-2.4mg/L and). Insulin resistant (IR) adipocytes were exposed to CP extract in cell culture containing either 8mM or 18mM glucose and one of three insulin concentrations. CPextract allowed an efficient glucose disposal in the IR adipocytes in an insulin independent manner (p&lt;0.0001). The percentage of glucose uptake did not significantly differ by models of diabetic and non-diabetic conditions (p=0.4727) although the significantly higher glucose concentration taken up by the IR adipocytes in the presence of IR adipocytes suggest an enhancement of antidiabetic action in hyperglycemic conditions. Expectedly metformin had a higher potency than the CP extract with its therapeutic dose of 1.8-2.4mg/L corresponding to 280mg/l of CP extract (p=0.9996). Additionally, metformin and CP extract appear to compete for similar sites which suppressed the hypoglycemic activity of CPextract.

PSIII-36 The endocytic pathway plays a major role in the regulation of tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells

Abstract Postweaning pigs are subjected to nutrient deprivation during which intestinal epithelial cells undergo increased turnover. To preserve intestinal function, intestinal epithelial cells must activate adaptive mechanisms that allow them to cope with starvation-induced stress; most importantly, the preservation of intestinal barrier function. The objective of this study was to investigate the underlying mechanisms involved in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were subjected to total nutrient starvation in Krebs-Ringer bicarbonate (KRB) buffer for 0, 3, 6, 12 and 24 h. Abundance of tight junction proteins was determined by RT-PCR, western blotting and immunofluorescence. Compared with control group (0 h), the protein expression of claudin 1, claudin 3 and claudin 4 protein was downregulated up to 6 h of starvation and then increased thereafter (P &amp;lt; 0.01). However, there was no change in the protein level of occludin and ZO-1. To determine the contribution of the lysosome and the ubiquitin proteasome pathways to regulation of tight junction protein abundance, the lysosome (Bafilomycin A1) and the proteasome (MG132) inhibitors were used in nutrient starved cells. Results showed the degradation of claudin 1, 3 and 4 up to 6 h of starvation was through the lysosomal pathway. Surprisingly, re-synthesis of claudins 4 and claudin 3 after prolonged starvation (12 and 24 h) was prevented when cells were treated with bafilomycin A1 and MG132, respectively. The autophagy-lysosome pathway inhibitors (Wortmannin and MHY1485) and endosome-lysosome pathway inhibitors (Dynasore and Pitstop 2) were further used to determine the specific roles of these pathways. In summary, the degradation of claudin 3 and claudin 4 during short-term starvation (up to 6 h) was through the dynamin-dependent endocytic pathway. However, re-synthesis of these proteins after prolonged starvation relies on both the lysosome and proteasome pathways.

PSVIII-1 Nutrient starvation induces autophagy and alters intestinal barrier function in IPEC-J2 cells

Abstract Autophagy is a cellular process of controlled degradation of damaged organelles and cytoplasmic macromolecules during stress for maintaining homeostasis. Intestinal epithelial cells are barriers against microorganisms, toxins and food antigens. Whether autophagy plays a role in regulating intestinal barrier function is unclear. The objective of this study was to characterize the role of autophagy in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were nutrient starved in Krebs-Ringer bicarbonate (KRB) buffer for 0, 0.5, 1, 2, 3, 6, 9 and 12 h. Expression of genes implicated in autophagy regulation (AMPK, MDM2, p53 and DRAM) was determined by RT-PCR. The ratio of protein abundance of microtubule-associated protein light chain 3 (LC3-II/LC3-I) and p62, positive and negative markers of autophagy induction, respectively, was determined by western blotting. Compared with control group (0 h), the relative mRNA expression level of AMPK, MDM2, p53 and DRAM significantly decreased by an average of 52.5% (P &amp;lt; 0.01). Relative to 0 h, the mRNA expression of claudin 1 and claudin 4 significantly increased (108.0%) up to 6 h of starvation and then decreased (31.4%) thereafter (P &amp;lt; 0.01). On the contrary, abundance of claudin 1 and claudin 4 protein was downregulated (49.5%) up to 3 h of starvation and then increased (82.6%) thereafter (P &amp;lt; 0.01). Protein expression of claudin 3 was reduced (P &amp;lt; 0.01) with duration of starvation. There was no change in the protein level of occludin and ZO-1. The ratio of LC3-II/LC3-I significantly increased with duration starvation (P &amp;lt; 0.01), whereas p62 and phospho-AMPK levels decreased up to 6 h of starvation and then increased thereafter (P &amp;lt; 0.01). In summary, autophagy may be implicated in the regulation of tight junction integrity during nutrient starvation in IPEC-J2 cells.

Leucine Regulates the Exocrine Function in Pancreatic Tissue of Dairy Goats In Vitro.

This study aimed to investigate the effects of leucine (Leu) on the synthesis and secretion of digestive enzymes in cultured pancreatic tissue of dairy goats and on the signaling molecules. Fresh pancreatic tissue from dairy goats was cut into approximately 2 mm × 2 mm pieces and incubated in oxygenated Krebs-Ringer bicarbonate buffer containing 0 (the control), 0.40, 0.80, or 1.60 mM Leu at 39°C in a CO2 incubator for 180 min. The results showed that Leu increased the release of α-amylase, trypsin, and chymotrypsin in the buffer and tissue, as well as the total activity (P < 0.05), especially at 0.40 and 0.80 mM. Compared with the control, 1.60 mM Leu increased the release of α-amylase and the total activity of trypsin and chymotrypsin (P < 0.05) but had no effect on the tissue concentration of α-amylase, trypsin, and chymotrypsin or the total activity of α-amylase (P > 0.05). Leu improved the mRNA expression of α-amylase, trypsin, and chymotrypsin (P < 0.05), especially at 0.80 and 1.60 mM. The activity and mRNA expression of lipase were not affected (P > 0.05). Compared with the control, 0.40 and 0.80 mM Leu increased the expression of the γ isoform of 4EBP1 (P < 0.05), implying increased phosphorylation of 4EBP1. Leu increased the phosphorylation of S6K1 (P < 0.05). Compared with the control, 0.40 and 0.80 mM Leu decreased the eEF2 phosphorylation level (P < 0.05). Conclusively, these results suggested that Leu could regulate the synthesis of pancreatic enzymes by increasing the mRNA expression and phosphorylation level of protein factors in the mammalian target of rapamycin pathway and the optimal Leu level in this experiment was 0.80 mM.

68: Rapid Viability and Potency Assessment of Encapsulated Islets and Insulin-producing Cells

Introduction: Diabetes and its insidious complications sicken and kill millions of people worldwide. Our group has developed macroencapsulation devices capable of providing supplemental oxygen to macroencapsulated transplanted tissue. These devices house and protect islets and insulin-producing cells (IPCs) from the immune system, eliminating the need for immunosuppression of the transplant recipient. We predict these devices will facilitate transplant of small footprint devices containing high cell densities of islets or IPCs that can reverse diabetes in humans by fostering increased viability and functionality of transplanted islets and IPCs. However, in order to a) determine the number of cells encapsulation devices can support, b) assess the effects of encapsulation on islets and IPCs, c) meet FDA standards, and d) ensure the quality of a cellular transplant product, the viability and potency of islets and IPCs within encapsulation devices must be determined before clinical application. Materials and Methods: We have developed 2 bioreactor systems integrated with BioRep perifusion machines capable of simultaneous measurement of cellular oxygen consumption rate (OCR) and hormone secretion. One bioreactor can be used to assay free cells and the other, macroencapsulated cells. Each bioreactor contains two integrated FOSPOR oxygen sensors (Ocean Optics) at the inlet and outlet of the bioreactor. Oxygen concentration is derived from the fluorescence lifetime of the sensor measured by fiber optic cables linked to a phase fluorometer (Ocean Optics). Media flow, temperature and collection are controlled by the BioRep perifusion machine. Figure 1 depicts the general design of the integrated bioreactor systems. Results and Discussion: We have determined that cellular density and encapsulation can affect the viability and function of islets. Islet density greater than 500 islet equivalents/cm2 (without supplemental oxygen) was detrimental to islet viability during a 7-day transplant period. In vitro, densely encapsulated islets displayed delayed and diminished glucose-stimulated insulin secretion (GSIS) relative to free islets (Figure 2A), an important consideration for physiological control of glycemia with engrafted tissue. Furthermore, as expected in Krebs Ringer Bicarbonate Buffer, glucose-stimulated insulin secretion (GSIS) was correlated with an increased OCR (Figure 2B) highlighting the link between OCR and GSIS in this medium and the utility of OCR, an inexpensive real time assay as an islet potency indicator. Conclusions: In concert, oxygen consumption and hormone secretion are excellent indicators of islet and IPC health and function. Our bioreactor will allow us to optimize encapsulation densities, cellular function, and supplemental oxygen delivery strategies ensuring a clinical high-grade cellular product and, together with our encapsulation devices, improve the treatment of diabetes. BioRep, Inc. Juvenile Diabetes Research Foundation Award # #2-SRA-2018-685-S-B. NIH Diabetes Impact Award #1DP3DK106933-01.

Electrochemical Aptamer-Based Sensor for Real-Time Monitoring of Insulin.

In this paper, we developed an electrochemical, aptamer-based (E-AB) for the real-time monitoring of insulin. The sensor utilizes a redox label-modified guanine-rich aptamer which folds into a G-quadruplex for specific recognition of insulin. To develop a reproducible E-AB sensor employing insulin aptamer probes for the detection of insulin, 10% sodium dodecyl sulfate (SDS) pretreatment is crucial as it disrupts interstrand G-quartets. After sensor pretreatment with 10% SDS, a more uniform sensor response is obtained. Upon introduction of the insulin target, binding-induced steric hindrance quantitatively reduces the efficiency of electron transfer of a distal-end redox label leading to the rapid signal change within ∼60 s. Testing demonstrates that the E-AB insulin exhibits a limit of detection of 20 nM and can be used to discriminate against both glucagon and somatostatin in Krebs-Ringer bicarbonate buffer, typically used in perfusion experiments. These results demonstrate that this assay has potential for rapid, specific, and quantitative analysis of insulin.

Phenylalanine regulates initiation of digestive enzyme mRNA translation in pancreatic acinar cells and tissue segments in dairy calves.

As new nutritional strategies for ruminant are designed to change production efficiency by improving the supply of rumen protect protein, lipid, and even starch, the digestive system must fit to utilize these increased nutrient supplies, especially the pancreas. The objective of this study was to investigate the effects of phenylalanine (Phe) on digestive enzymes synthesis or secretion and cellular signaling in pancreatic acinar (PA) cells of dairy calves. The PA cells isolated from fresh pancreas of dairy calves, and cultured in completed RIPA 1640 medium with no fetal serum but 0, 0.15 and 0.45 mM Phe at 37°C in CO2 incubator for 120 min. The pancreatic tissue segments (PTS) was cut approximately 2 × 2 mm from the fresh pancreas, and incubated in oxygenated Krebs-Ringer bicarbonate (KRB) buffer containing 0 or 0.35 mM Phe at 39°C for 180 min, and the samples were collected every 60 min after incubation. In PA cells, Phe increased (P < 0.05) the α-amylase secretion and mRNA expression, the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). In PTS, the Phe increased (P < 0.05) α-amylase and trypsin synthesis, secretion and mRNA expression, as well as the phosphorylation of S6K1 and 4EBP1. Conclusively, these results suggested that Phe regulates the synthesis or secretion of α-amylase, trypsin and lipase through mRNA translation initiation factors – S6K1 and 4EBP1.

Real-Time Monitoring of Insulin Using a Graphene Field-Effect Transistor Aptameric Nanosensor.

This paper presents an approach to the real-time, label-free, specific, and sensitive monitoring of insulin using a graphene aptameric nanosensor. The nanosensor is configured as a field-effect transistor, whose graphene-based conducting channel is functionalized with a guanine-rich IGA3 aptamer. The negatively charged aptamer folds into a compact and stable antiparallel or parallel G-quadruplex conformation upon binding with insulin, resulting in a change in the carrier density, and hence the electrical conductance, of the graphene. The change in the electrical conductance is then measured to enable the real-time monitoring of insulin levels. Testing has shown that the nanosensor offers an estimated limit of detection down to 35 pM and is functional in Krebs-Ringer bicarbonate buffer, a standard pancreatic islet perfusion medium. These results demonstrate the potential utility of this approach in label-free monitoring of insulin and in timely prediction of accurate insulin dosage in clinical diagnostics.

Antibacterial activity and biofilm inhibition by surface modified titanium alloy medical implants following application of silver, titanium dioxide and hydroxyapatite nanocoatings

One of the most common causes of implant failure is peri-implantitis, which is caused by bacterial biofilm formation on the surfaces of dental implants. Modification of the surface nanotopography has been suggested to affect bacterial adherence to implants. Silver nanoparticles are also known for their antibacterial properties. In this study, titanium alloy implants were surface modified following silver plating, anodisation and sintering techniques to create a combination of silver, titanium dioxide and hydroxyapatite (HA) nanocoatings. Their antibacterial performance was quantitatively assessed by measuring the growth of Streptococcus sanguinis, proportion of live/dead cells and lactate production by the microbes over 24 h. Application of a dual layered silver–HA nanocoating to the surface of implants successfully inhibited bacterial growth in the surrounding media (100% mortality), whereas the formation of bacterial biofilm on the implant surfaces was reduced by 97.5%. Uncoated controls and titanium dioxide nanocoatings showed no antibacterial effect. Both silver and HA nanocoatings were found to be very stable in biological fluids with material loss, as a result of dissolution, to be less than 0.07% for the silver nanocoatings after 24 h in a modified Krebs-Ringer bicarbonate buffer. No dissolution was detected for the HA nanocoatings. Thus, application of a dual layered silver–HA nanocoating to titanium alloy implants creates a surface with antibiofilm properties without compromising the HA biocompatibility required for successful osseointegration and accelerated bone healing.

Cross Regulation Between cGMP-dependent Protein Kinase and Akt in Vasodilatation of Porcine Pulmonary Artery

cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

Metabolic oxygen consumption measurement with a single-cell biosensor after particle microbeam irradiation.

A noninvasive, self-referencing biosensor/probe system has been integrated into the Columbia University Radiological Research Accelerator Facility Microbeam II end station. A single-cell oxygen consumption measurement has been conducted with this type of oxygen probe in 37°C Krebs-Ringer Bicarbonate buffer immediately before and after a single-cell microbeam irradiation. It is the first such measurement made for a microbeam irradiation, and a six fold increment of oxygen flux induced during a 15-s period of time has been observed following radiation exposure. The experimental procedure and the results are discussed.

YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

Testicular Perfusion Apparatus

A reproductive biology laboratory requires to provide reliable instruments for regular use of research workers. To understand an organ and perform scientific study and reach on a conclusion good instruments and techniques are essential. Such a suitable one is not available in case of testicular study. The present study is aimed to clear this lacuna. A new testicular perfusion apparatus for rabbit and rat is designed and fabricated with glass. The glass blowing instrument available is used. The perfusion fluid preferred is Krebs Ringer bicarbonate buffer (pH 7.4). Quartz wool provided in stem tube filters emboli present in it. Perfusate temperature is maintained to that of testis. A 26-gauge needle is introduced into the spermatic artery to allow entry of perfusate into testis. Method for the collection of perfusate leaving testis is available. This permits to measure the level of an injected substance before its entry through perfusion fluid into the testis, which shall be compared in the fluid after completion of perfusion. Initially few animal testes were used and found the instrument to be functioning well. A new simple perfusion apparatus for testes of rabbit and rat is designed. The apparatus made out of glass could be prepared with the help of glass blowing facility and used regularly in any laboratory.

Propofol-induced relaxation of rat aorta is altered by aging.

Propofol causes vasodilation via endothelium-dependent and -independent mechanisms. Because endothelial function is impaired with aging, the effects of propofol on endothelium-dependent vasodilation might be altered by aging. The aim of this study was thus to determine the effects of aging on vascular responses to propofol. Young (4-6 weeks old) or adult (16-25 weeks old) rats were anesthetized with sevoflurane. The thoracic aorta was dissected and cut into pieces 3-4 mm in length. In some rings, the endothelium was deliberately removed. The ring segment of the aorta was mounted for isometric force recording at a resting tension of 0.5-1.0 g in a 2 ml organ bath, containing Krebs-Ringer bicarbonate buffer. Arteries were precontracted with phenylephrine, and the function of endothelium was confirmed with acetylcholine. Then, we studied the concentration-dependent effects of propofol in endothelium-intact (control group) and -denuded aortic rings (denuded group), as well as those treated with N(ω)-nitro-L-arginine methylester (L-NAME group). Relaxation due to propofol was observed in the control groups of both young and adult rats in a concentration-dependent manner, but the magnitude of relaxation was significantly greater in young rats. In addition, in young rats, relaxation due to propofol was significantly and equally reduced in both L-NAME and denuded groups at all propofol concentrations that we studied (10(-6)-10(-3) M). In adult rats, relaxation due to propofol was quite similar between control and L-NAME groups at all propofol concentrations, whereas it was significantly reduced in the denuded group. These results suggest that endothelium-derived nitric oxide plays an important role in propofol-induced vasodilation in young rats, but not in adult rats.