Incubation Of Tissue Slices Research Articles

A novel immunofluorescence detection method for renal cell-type specific in situ cytokine production by confocal microscopy

The detection of cytokines production in tissues is subjected to significant limitations: (1) Cytokine protein production frequently does not correlate with mRNA levels. (2) Cytokines are secreted rapidly and dissipate from the cellular source, thus making detection difficult. (3) The synthetic rate of many cytokines are low. (4) Tissue fixation ablates antigenic sites and diminishes detection signals. The identification of the cellular sources of cytokines poses an additional challenge because of the lack of suitable and readily available cellular markers. In our renal cytokine production studies in lupus nephritis, we have established methods to resolve problems associated with the identification of cellular sources of pertinent cytokines in the glomerulus and interstitium. Four-color confocal microscopy was used to colocalize cell-type specific markers with cytokines. The cytokine signal was amplified by the incubation of tissue slices in medium containing pan-specific stimulants plus secretion blockers. Tissue fixation was optimized to provide sharp crisp signals. Commercially available Ab suitable for fluorochrome labeling were used to establish cell-specific markers in the tubules and glomeruli. This combination of optimizations allowed us to define the cellular sources of important glomerular cytokines including TNF-α, IL-6, and IL-1β which appear to form a cytokine circuit in glomerulonephritis pathogenesis.● Tissue stimulation and secretion blocking for cytokine detection● Fixation optimization and Ab source identification for direct staining● Colocalization of cytokines and renal cell-type specific markers

Evaluation of the tetrodotoxin uptake ability of pufferfish Takifugu rubripes tissues according to age using an in vitro tissue slice incubation method

The tetrodotoxin (TTX) uptake ability of pufferfish Takifugu rubripes tissues and its growth-associated changes were investigated using an in vitro tissue slice incubation method. Tissue slices prepared from the liver, skin, and intestine of a non-toxic cultured adult T. rubripes (20 months old) and incubated with incubation buffer containing 25 μg/mL TTX for 1–48 h showed a time-dependent increase in the TTX content in all tissues. The TTX contents of the skin and intestine slices were comparable to or slightly higher than that of the liver slices, with a similar transition pattern, suggesting similar TTX uptake ability among the skin, intestine, and liver. The TTX uptake ability of the liver and intestine did not differ significantly between young (8 months old) and adult (20 months old) fish, but the skin slices of young fish took up approximately twice as much TTX as that of adult fish, suggesting that the TTX uptake ability of the skin is involved in the growth-dependent changes in the toxin distribution inside the body in T. rubripes. To estimate the TTX uptake pathway in each tissue, an immunohistochemical technique was used to observe temporal changes in the intra-tissue microdistribution of TTX during incubation. The findings suggested that TTX is transferred and accumulates from pancreatic exocrine cells to hepatic parenchymal cells in the liver, from connective tissues to basal cells in the skin, and from villi epithelial cells via the lamina propria to the muscle layer in the intestine.

Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues.

Chelatable, or mobile, forms of zinc play critical signaling roles in numerous biological processes. Elucidating the action of mobile Zn(II) in complex biological environments requires sensitive tools for visualizing, tracking, and manipulating Zn(II) ions. A large toolbox of synthetic photoinduced electron transfer (PET)-based fluorescent Zn(II) sensors are available, but the applicability of many of these probes is limited by poor zinc sensitivity and low dynamic ranges owing to proton interference. We present here a general approach for acetylating PET-based probes containing a variety of fluorophores and zinc-binding units. The new sensors provide substantially improved zinc sensitivity and allow for incubation of live cells and tissue slices with nM probe concentrations, a significant improvement compared to the μM concentrations that are typically required for a measurable fluorescence signal. Acetylation effectively reduces or completely quenches background fluorescence in the metal-free sensor. Binding of Zn(II) selectively and quickly mediates hydrolytic cleavage of the acetyl groups, providing a large fluorescence response. An acetylated blue coumarin-based sensor was used to carry out detailed analyses of metal binding and metal-promoted acetyl hydrolysis. Acetylated benzoresorufin-based red-emitting probes with different zinc-binding sites are effective for sensing Zn(II) ions in live cells when applied at low concentrations (∼50–100 nM). We used green diacetylated Zinpyr1 (DA-ZP1) to image endogenous mobile Zn(II) in the molecular layer of mouse dorsal cochlear nucleus (DCN), confirming that acetylation is a suitable approach for preparing sensors that are highly specific and sensitive to mobile zinc in biological systems.

Rab3a Is Critical for Trapping Alpha-MSH Granules in the High Ca2+-Affinity Pool by Preventing Constitutive Exocytosis

Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of α-MSH.

Effects of Eicosapentaenoic Acid (EPA) Treatment on Insulin Sensitivity in an Animal Model of Diabetes: Improvement of the Inflammatory Status

In addition to decreased insulin sensitivity, diabetes is a pathological condition associated with increased inflammation. The ω-3 fatty acids have been proposed as anti-inflammatory agents. Thus, the major goal of this study was to analyze the effects of fatty acid supplementation on both insulin sensitivity and inflammatory status in an animal model of type 2 diabetes. Diabetic rats (Goto-Kakizaki model) were treated with eicosapentaenoic acid (EPA) or linoleic acid at 0.5 g/kg body weigh (bw) dose. In vivo incorporation of (14)C-triolein into adipose tissue was improved by the ω-3 administration. In vitro incubations of adipose tissue slices from EPA-treated rats showed an increase in (14)C-palmitate incorporation into the lipid fraction. These observations were linked with a decreased rate of fatty acid oxidation. EPA treatment resulted in a decreased fatty acid oxidation in incubated strips from extensor digitorum longus (EDL) muscles. The changes in lipid utilization were associated with a decrease in insulin plasma concentration, suggesting an improvement in insulin sensitivity. These changes in lipid metabolism were associated with an activation of AMP-activated protein kinase (AMPK) in white adipose tissue. In addition, EPA treatment resulted in a decreased content of peroxisome proliferator-activated receptor-α (PPARα) and PPARδ and in increased GLUT4 expression in skeletal muscle. Moreover, EPA increased 2-deoxy-D-[(14)C]glucose (2-DOG) uptake in C2C12 myotubes, suggesting an improvement in glucose metabolism. Concerning the inflammatory status, EPA treatment resulted in a decreased gene expression for both tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) both in skeletal muscle and adipose tissue. The data suggest that EPA treatment to diabetic rats clearly improves lipid metabolism although the evidences on insulin sensitization are less clear.

Metabolic Adaptations During Lactogenesis

1. Mammary-tissue biopsies were obtained from multiparous cows at 30 and 7 days pre partum and 7 and 40 days postpartum. Investigations of the effect of lactogenesis on fatty acid and lactose synthesis involved measurements of biosynthetic capacity (tissue-slice incubations in vitro) and activities ofrelevant enzymes. 2. Fatty acid synthesis from acetate increased over 20-fold from 30 days pre partum to 40 days post partum. Changes in the lipogenic capacity of mammary-tissue slices more closely paralleled increases in the activities of acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) than of other enzymes involved in acetate incorporation into fatty acids or in NADPH generation. 3. Lactose biosynthesis by mammary-tissue slices, lactose synthetase activity (EC 2.4.1.22) and α-lactalbumin concentration were all negligible at 30 days pre partum but increased 2.5-4-fold between 7 days pre partum and 40 days post partum. Phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDPglucose 4-epimerase (EC 5.1.3.2) had substantial activities at 30 days pre partum and increased less dramatically during lactogenesis. 4. Results are consistent with acetyl-CoA carboxylase and perhaps acetyl-CoA synthetase representing the regulatory enzyme(s) in fatty acid synthesis, with lactose synthetase (α-lactalbumin) serving a similar function in lactose biosynthesis.

Rapid and fine adjustment of corticosteroidogenesis operating in the adrenal gland of the Mongolian gerbil (Meriones unguiculatus) superfused in-vitro.

Corticosteroid release from adrenals of male adult Mongolian gerbils (Meriones unguiculatus) has been studied during continuous and discontinuous in-vitro superfusion. Corticosteroid output from glands of untreated animals with plasma corticosteroid levels below 300 ng/ml was 3.3 +/- 0.3 ng/adrenal pair/min and decreased only slightly with the length of superfusion (60 min: 3.1 +2- 0.3 ng/adrenal pair/min). In-vitro secretion was significantly higher from adrenals of animals which had corticosteroid plasma levels of over 300 ng/ml, underwent clonic-tonic seizures, or were injected with 6 IU/animal (1-24) ACTH. On the other hand, injection of 2 X 50 micrograms dexamethasone markedly decreased corticosteroid plasma levels but had no significant effect on in-vitro secretion of corticosteroids. In contrast to the slow and small, but long-lasting stimulation of corticosteroid secretion elicited by (1-24) ACTH, secretion could be changed within much shorter time periods, either by the addition of plasma proteins to superfusion medium or by stops of superfusion flow. While a significant stimulation of corticosteroid output occurred after the addition of 1% or 10% BSA or rat plasma, stops of superfusion flow for 1.5 or 10 min resulted in a strong inhibition of steroidogenesis, as is evident from corticosteroid amounts found in the first 1-min samples after re-start of superfusion. Within 4-5 min after re-start of superfusion, secretion returned to basal values. Corticosteroid amounts secreted from adrenals superfused in-vitro were significantly higher than those secreted from adrenals incubated in-vitro. In addition, prolonged incubation suppressed corticosteroidogenesis (30 min: 100%, 60 min: 64%, 90 min: 56%, 120 min: 59%). The results demonstrate that superfusion of sliced adrenal tissue gives insights into aspects of adrenal function, including the rapid changes in synthesis and secretion after flow stops which cannot be investigated by incubation of either tissue slices or isolated cells. The possibility that the observed decline in corticosteroidogenesis during flow stops may be due to a feedback inhibition resulting from corticosteroids accumulating within slices is discussed.

Distribution and functional characterization of pituitary adenylate cyclase–activating polypeptide receptors in the brain of non-human primates

The distribution and density of pituitary adenylate cyclase–activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [ 125I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase–activating polypeptide–specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase–activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase–activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.

9cis,11trans conjugated linoleic acid (CLA) is synthesised and desaturated into conjugated 18:3 in bovine adipose tissues

Although endogenous synthesis of conjugated linoleic acid (CLA) in the mammary gland of lactating cows has been already well documented, no study has determined so far as to which tissue and/or organ is involved in CLA synthesis in the growing ruminant except one study showing that CLA synthesis does not occur in ruminant liver. In this context, adipose tissue appears to be a good candidate for endogenous synthesis of CLA in the growing ruminant. The aim of this study was to compare the respective metabolisms of 11trans 18:1 (vaccenic acid, VA) and 9cis,11trans 18:2 (rumenic acid) to that of stearic acid (the preferential substrate of Δ9 desaturase) in adipose tissues (subcutaneous, SC and intermuscular, IM) of six Charolais steers by using the in vitromethod of incubated tissue slices. Samples of SC and IM adipose tissues were incubated at 37°C for 16 h under an atmosphere of 95% O2/5% CO2 in a medium supplemented with 0.75 mM of fatty acid (FA) mixture (representative of circulating non-esterified FA) and 186 μM [1-14C]-18:0 or 58.6 μM [1-14C]-VA or 56 μM [1-14C]-9cis,11trans CLA. Viability of explants was verified by measuring metabolic functions (glucose uptake and glucose-6-phosphate dehydrogenase activity). After 16 h of incubation, FA uptake was similar for all FA (18:0, VA and 9cis,11trans 18:2) in both SC and IM adipose tissues (around 40%). Once in adipose tissue, all FA were preferentially esterified (>80% of cell FA) favouring neutral lipid synthesis (around 90% of esterified FA). Stearic acid was highly (27%) desaturated into oleic acid in SC adipose tissue whereas this desaturation was much lower (6.8%) in IM adipose tissue (P < 0.0001). VA was desaturated into 9cis,11trans CLA at a low extent of about 2.5% to 4.4% in both adipose tissues probably because of a limited affinity of Δ9 desaturase for VA. 9cis,11trans CLA was itself converted by desaturation into 6cis, 9cis,11trans 18:3 at the intensity of 10.8% and 14.5% of cell 9cis,11trans CLA in SC and IM adipose tissues, respectively. In conclusion, bovine adipose tissues of the growing ruminant were especially involved in the endogenous synthesis of CLA from VA and in its desaturation into conjugated derivative, mainly 6cis, 9cis,11trans 18:3, of which biological properties need to be elucidated.

β-Adrenergic regulation of uncoupling protein expression in swine

This study examined the beta-adrenergic regulation of uncoupling protein (UCP) 2 and UCP3 gene expression in porcine tissues. In vitro experiments examined changes in UCP2 and UCP3 gene expression in middle (MSQ) and outer (OSQ) subcutaneous adipose tissues from crossbred neutered male pigs. Incubation of tissue slices (24 h) with 0 to 1000 nM isoproterenol increased UCP2 and UCP3 mRNA abundance in MSQ and OSQ, relative to 18S rRNA (P<0.05). For the in vivo experiment, nine randomly selected pigs (80 kg) were presented with a diet supplemented with 10.0 ppm ractopamine for 2 weeks. Another eight pigs were maintained on a control diet. Dietary ractopamine did not affect adipose UCP2 or UCP3 gene expression (P>0.05). However, UCP2 mRNA abundance was depressed in semitendinosus white (STW, P<0.05) and semitendinosus red (STR, P<0.001) by ractopamine feeding. Also, ractopamine decreased UCP3 mRNA abundance by 28% in STW (P<0.05). The in vitro data suggest that beta-adrenergic agonists directly affect adipose tissue UCP expression, although these adipose effects can be masked by the in vivo physiology. The in vivo data indicate that beta-adrenergic agonists may function in regulating UCP2 and UCP3 expression in selected muscles.

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M(3) muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M(3) mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca(2+) signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.

Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease.

Protein deposition diseases involve the aggregation of normally soluble proteins, leading to both fibrillar and amorphous deposits. The aggregation of alpha-synuclein is associated with Parkinson's disease, and the aggregation of the Abeta peptide is associated with Alzheimer's disease. Here we show that L-dopa, dopamine, and other catecholamines dissolve fibrils of alpha-synuclein and Abeta peptide generated in vitro. The catecholamines also inhibited the fibrillation of these proteins. In addition, intraneuronal alpha-synuclein deposits formed in a mouse model were dissolved by incubation of tissue slices with L-dopa. These catecholamines are susceptible to oxidative breakdown, and we show that oxidation products are more effective than the parent compounds in inhibition. The ability to dissolve fibrils provides a new approach for studying mechanisms and consequences (e.g., the relationship between fibril formation and neurodegeneration) of protein aggregation. It is also likely to help in the development of strategies for the prevention and treatment of protein deposition diseases.

Effects of steroid hormone treatment on mammary development in prepubertal heifers

The objective of this study was to determine the effects of steroid hormone implantation in heifer calves on the ability of mammary tissue to develop subsequently in organ culture. Twenty-four calves were paired by date of birth and assigned to groups (eight calves/group). At 4, 7, or 10 mo of age, calves were implanted subcutaneously (s.c.) with pellets containing cholesterol or cholesterol, 17β-estradiol, and progesterone for 9 or 18 d. The calves were euthanized and uteri and mammary glands were removed and weighed. Slices of mammary parenchymal tissue were incubated for 5 d at 37°C in a 50% O 2, 5% CO 2 humidified atmosphere in Waymouth’s 752/liter medium supplemented with insulin (5.0 μg/ml) or lactogenic hormone complex insulin (5.0 μg/ml), aldosterone (0.1 μg/ml), hydrocortisone (0.1 μg/ml), and prolactin (1.0 μg/ml) in the presence or absence of epidermal growth factor (EGF) (0.06 μg/ml) to promote lobulo-alveolar development. Tissue sections were stained and mounted on slides for morphologic and histologic analysis or prepared to evaluate expression of β-casein mRNA. There were no morphologic differences in slices from calf mammary tissues despite age, steroid hormone priming, or hormones used in tissue culture. The 4-mo-old calves required steroid priming followed by incubation of the tissue slices with the lactogenic complex with or without epidermal growth factor to induce cytological changes associated with lactogenesis but did not express β-casein mRNA. At 7 mo of age, steroid hormone priming was not necessary for induction of alveolar formation and secretion. Incubation of the tissue slices from 7-mo-old calves with the lactogenic complex was sufficient to induce alveolar formation and secretion. However, β-casein mRNA was not expressed. At 10 mo of age, exposure of tissue from calves to the lactogenic hormones caused histologic changes reminiscent of the ability to secrete milk regardless of hormone priming. However, estrogen and progesterone priming was necessary before incubation of the tissue slices with the lactogenic hormones to induce β-casein mRNA expression. When epidermal growth factor was added to the lactogenic hormone complex, β-casein mRNA expression decreased. These data support the concept that there is a sequential development of responsiveness of the mammary gland to various hormones. By 10 mo of age, prepubertal heifers reach a stage of maturity where steroid hormone priming followed by incubation of tissue slices with the lactogenic hormones is sufficient to induce both structural and functional differentiation.

Lipoprotein and hormone-sensitive lipases in porcine adipose tissue.

Lipoprotein lipase (LPL) is an adipocyte enzyme that cleaves fatty acids from circulating lipoproteins. Fatty acids enter the cell to be oxidized or esterified. Hormone-sensitive lipase (HSL) is an adipocyte enzyme that cleaves fatty acids from intracellular triacylglycerol. The HSL is activated by phosphorylation. Assays for the two lipases are complex because the hydrophobic substrate, triacylglycerol, must be presented as a gum-based suspension or as a detergent-based emulsion to a relatively hydrophilic enzyme. A convenient, stable glycerol/phospholipid suspension of the substrate was used for measurement of porcine adipose tissue LPL and HSL in vitro. This substrate was excellent for LPL. It produced rates five times those using a more complex and less convenient gum-based substrate suspension. The LPL activity was released by heparin, had a pH optimum of approximately 8.5, was activated by serum, and was inhibited by NaCl and protamine. This LPL assay measures enzyme capacity. The same substrate was used to measure an adipose tissue lipase activity that had a pH optimum below 7, was not activated by serum, and was not inhibited by NaCl or protamine. These are all characteristics of HSL. Despite the convenience, this substrate was not appropriate for porcine adipose tissue HSL because the rates were only 30 to 50% of those produced with a more complex, less convenient gum-based substrate suspension. Furthermore, incubation of enzyme or tissue slices with insulin, or agents that elevate cAMP concentration, did not modulate this lipase activity, as expected. These incubations poorly modulated LPL activity.

Use of low temperature and high K+ incubation media for in vitro tissue preparation for X-ray microanalysis.

Incubation of tissue slices in physiological buffers gives rise to significant changes in the intracellular ion concentrations, which may disturb subsequent X-ray microanalysis. In the present study it was attempted to design incubation conditions that retain the in vivo conditions better. The following variables were investigated: (1) exchange of Na+ in the incubation medium for K+, and exchange of Cl- for the less permeable gluconate anion; (2) incubation at 4 degrees C rather than at 37 degrees C; and (3) addition of dextran to the incubation medium. Brief exposure (a few seconds) of liver slices to a buffer causes changes in the intracellular Na, Cl and K concentrations, depending on the ionic composition of the buffer. Incubation in a normal physiological (high NaCl) buffer at 37 degrees C results in a further increase of Na and Cl and a further decrease in K in liver cells. The changes reach a maximum at 30 min and the concentrations then remain stable throughout a 2-h incubation. Incubation in sodium gluconate medium or addition of dextran to the physiological buffer somewhat reduces the changes in the intracellular ion composition (compared to the standard physiological incubation medium). Incubation in potassium gluconate medium results in a decrease in cellular Na and an increase in K. Quantitative morphological studies show that tissue oedema is observed to the same extent in hepatocytes incubated in sodium gluconate, potassium gluconate and physiological buffer containing 10% dextran. However, these buffers cause significantly less cell oedema than the physiological (high NaCl) buffer. Incubation of liver, cerebral cortex or submandibular gland slices in physiological (high NaCl) solutions at 4 degrees C for 4 h caused a more extensive increase in Na+ and decrease in K+ than incubation at 37 degrees C for 2 h. This suggests inhibition of the Na+, K(+)-ATPase under these conditions. As compared to incubation at 37 degrees C for 2 h, tissues incubated in potassium gluconate buffer at 4 degrees C for 4 h have a cellular K concentration closer to the in situ value. Cholinergic stimulation of tissue slices from cerebral cortex and submandibular gland at room temperature for 1 min shows the best physiological response in tissue slices preincubated at 4 degrees for 4 h in high KCl, potassium gluconate and high NaCl, in this order. The response can, however, only be seen, when cholinergic stimulation is carried out in a standard physiological buffer with a high NaCl concentration. It is concluded that in vitro storage of tissue for X-ray microanalysis is best carried out at 4 degrees C in a solution with a high K+ concentration.

Liver metabolism during cold ischemic incubation in UW solution in the rat model

Simple cold storage of livers for transplantation activates glycolysis due to lack of oxygen. Energy derived from glycolysis may be critical for cell survival and liver cell death may occur once glycolysis is inhibited in the liver due to accumulation of end products or lack of substrates (glycogen). The relationship between cell death (lactate dehydrogenase, LDH release), anaerobic glycolysis (lactate production), and glycogen content of liver tissue was studied during cold incubation of liver slices in UW solution. Rat livers slices from male Sprague Dawley rats were incubated at 4 degrees C in UW solution, with continuous gentle shaking, under conditions of chemical hypoxia (KCN, 5 mM). The rate of lactate production, LDH release-ATP and glycogen content were measured spectrophotometrically and by HPLC. Lactate increased nearly linearly for the first 48 h of incubation; total lactate which had accumulated after 48 h was 33.9 +/- 0.81 mumol/g and at 96 h nearly the same, 31.3 +/- 1.2 mumol/g. Glycolysis stopped, apparently, because of the depletion of liver slice glycogen which was initially 228.8 +/- 1.7 mumol/g wet wt. It decreased to 34.7 +/- 2.7 mumol/g at 48 h and to 18.7 +/- 1.1 mumol/g at 72 h and remained at this level for the next 24 h. An increased leakage of LDH occurred once glycogen metabolism (and accumulation) ceased. LDH release could be stimulated after only a few hours of cold incubation of liver tissue slices by adding glycolysis inhibitor (iodoacetic acid) to the medium. After 24 h. LDH release was 24.4 +/- 1.8% and increased to 52.8 +/- 5.2% (P < 0.05, Student's t-text) with iodoacetic acid. Adding a glycolytic substrate (fructose, 10 mM) to the medium maintained lactate production for 96 h. The stimulation of glycolysis by fructose also reduced cell death: LDH release was significantly lower at 72- and 96-h incubation (P < 0.001, two-way ANOVA). The ATP content was significantly higher with fructose (P < 0.001). Adding glucose (20 mM) and fructose (10 mM) in combination resulted in prolonged cell survival, significantly delayed glycogen depletion and significantly higher ATP content at 48 and 72 h (two-way ANOVA). Livers from rats who had fasted for 24 h demonstrated the same LDH release at 48 h when incubated with glucose (20 mM) and fructose (10 mM). In conclusion, LDH leakage from hypoxic cold-stored liver slices is related to anaerobic glycolysis. Anaerobic glycolysis appears to continue slowly under hypothermia and provides sufficient energy for maintenance of cell viability. A stimulation of glycolysis in the cold is possible by fructose and results in prolonged cell survival under hypothermic conditions. Glycogen depletion can be slowed down by combining glucose and fructose.

Effect of somatostatin and octreotide acetate on OP-CCK-stimulated exocrine secretion in the denervated canine pancreas.

Somatostatin and its analogue, octreotide acetate (Sandostatin), have been demonstrated to suppress exocrine secretion in a denervated canine pancreatic autograft model. To help define this inhibitory mechanism, the effect of these agents on cholecystokinin (CCK)-stimulated acinar cell secretion was evaluated. In vitro assessment evaluated the effect of somatostatin on octapeptide (OP)-CCK-stimulated amylase release of pancreatic tissue slices. In vivo assessment employed animals with pancreatic autografts and pancreaticocystostomies, evaluating the effect of a bolus intravenous injection of 100 micrograms of octreotide acetate on the basal and OP-CCK-stimulated (125 ng/kg/h) secretion of urinary (autograft) amylase and bicarbonate. Incubation of tissue slices with 0.16, 0.24, or 0.32 microgram/ml somatostatin had no significant effect on in vitro OP-CCK-simulated amylase release. Intravenous octreotide acetate resulted in a significant decrease in the basal rate of amylase secretion but had no significant effect on OP-CCK-stimulated autograft amylase or bicarbonate release. These studies demonstrate that octreotide acetate has an in vivo inhibitory effect on basal amylase release of pancreatic autografts but cannot counteract maximal stimulation with exogenous OP-CCK. Also, somatostatin does not inhibit OP-CCK-stimulated acinar cell secretion of pancreatic tissue slices. These results indicate that the exocrine inhibition produced by somatostatin analogues in the grafted pancreas occurs via an indirect mechanism.

In VitroBiosynthesis of Androgens in the Australian Lungfish,Neoceratodus forsteri

The serum concentration of testosterone was estimated from a population of wild lungfish over 6–7 years of sampling. Male lungfish were found to have high circulating levels of testosterone (∼50 ng/ml) which varied seasonally and could be correlated with spermatogenesis as judged by testis histology. Incubation of testis tissue slices with [3H]progesterone, [3H]17-hydroxyprogesterone, or [3H]testosterone confirmed that testosterone is the major androgen inNeoceratodus.Not even trace amounts of 11-keto- or 11β-hydroxytestosterone or 5α-dihydrotestosterone could be identified by TLC separations. There was little or no conjugation of steroids by the testes, except during the spawning season, when glucuronides of androstenedione and testosterone were produced.

Use of post mortem and in vitro tissue specimens for X‐ray microanalysis

Post mortem changes in the distribution of elements, as well as changes induced by dissection and incubation of tissue slices, were investigated by X-ray microanalysis of brain tissue, liver, pancreas and submandibular gland. Dissection itself causes minor changes in the intracellular ionic concentrations, but even a brief exposure of dissected tissue slices to a physiological buffer causes an increase in intracellular Na and Cl and a decrease in intracellular K concentration. The effect is most marked in brain tissue and least marked in submandibular gland slices. Incubation in fluid resembling the extracellular compartment in its ion composition results in a further increase of Na and Cl in brain tissue (cortex and hippocampus) and liver; in pancreas and submandibular gland, on the other hand, a stable situation throughout 2 h of incubation can be obtained. Incubation at lower temperature, and exchanging NaCl in the incubation medium for Na gluconate, has only relatively minor effects on the intracellular K/Na ratio. Exchanging the NaCl for K gluconate results in a high intracellular K/Na ratio throughout the incubation, but morphological evidence of tissue oedema was nevertheless observed. Physiological changes in intracellular ion content induced by cholinergic stimulation are similar in in vitro preparations as compared with stimulation in situ. The effect of dissection and brief incubation on the ionic composition of brain tissue is less pronounced 6 h after death than in a living, anaesthetized animal, but this is largely due to the post mortem changes that already have taken place.

The Effect of Sublethal Doses of Insecticides on Triatoma infestans Lipid Synthesis

The effect of sublethal amounts of d-phenothrin (pyrethroid), phenitrothion (organophosphate), and propoxur (carbamate) on the lipid synthesis in the nymphal integument and fat body of the blood-sucking bug Triatoma infestans was examined in vivo, in tissue slices, and in cytosolic fractions. De novo synthesis of lipids was compared for insecticide-pretreated and untreated insects. Lipid biosynthesis, measured as [1- 14C]acetate incorporation, is significantly affected. d-Phenothrin inhibits in vivo incorporation of [1- 14C]propionate, the precursor of methyl-branched hydrocarbons in insects. The incorporation of [1- 14C]propionate after incubation of integumental tissue slices is inhibited by both d-phenothrin and phenitrothion. d-Phenothrin has no influence on the cytosolic fatty acid synthetase (FAS) activity of the fat body or integument. Phenitrothion alters both fat body and integumental FAS activities. A total inhibition of the cytosolic integumental FAS is produced in the presence of propoxur, but no alteration of the fat body FAS was detected. These data suggest a different mode of action of the organophosphorate-, pyrethroid-, or carbamate-derived insecticides on the insect lipogenic enzymes.