Krebs-Ringer Bicarbonate Buffer Research Articles

Signal transduction pathways used by EGF to stimulate protein secretion in rat lacrimal gland.

To determine the effects of epidermal growth factor (EGF) on lacrimal gland secretion of proteins and characterize its signal-transducing components. Both exorbital lacrimal glands were removed from male Sprague-Dawley rats. Dispersed acini were isolated by collagenase digestion in Krebs-Ringer bicarbonate (KRB) buffer at 37 degrees C. Acini were incubated with EGF (10(-7) M), the cholinergic agonist carbachol (10(-4) M), or the alpha(1)-adrenergic agonist phenylephrine (10(-4) M), and peroxidase secretion was measured by a fluorescence assay. To measure intracellular calcium ([Ca(2+)](i)), acini were incubated in fura-2 tetra-acetoxymethyl ester for 60 minutes at 22 degrees C, and fluorescence was measured at 340 and 380 nm with an emission wavelength of 505 nm. Extracellular Ca(2+) was chelated with KRB-BSA without CaCl(2) and with 2 mM EGTA before measurement of peroxidase secretion. Protein kinase C (PKC) was downregulated by incubating acini overnight, with or without the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-6) M), and peroxidase secretion was measured. EGF-stimulated peroxidase secretion in a concentration-dependent manner with a significant increase at 10(-7) M. EGF-stimulated secretion was inhibited by the EGF receptor (EGFR) inhibitor AG1478, but not by the phosphoinositide-3 kinase inhibitor LY292004 or the mitogen-activated kinase kinase (MEK) inhibitor U0126. EGF increased [Ca(2+)](i), whereas chelation of extracellular Ca(2+) inhibited EGF-induced peroxidase secretion by 90%. Downregulation of PKC also inhibited EGF-stimulated peroxidase secretion. EGF stimulates lacrimal gland secretion of protein by activating the EGFR to increase [Ca(2+)](i) and activate PKC.

Visualizing Superoxide Production in Normal and Diabetic Rat Islets of Langerhans

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.

Sildenafil citrate does not affect cardiac contractility in human or dog heart

SUMMARYObjective: This study evaluated whether sildenafil citrate, an oral treatment for erectile dysfunction and a selective inhibitor of phosphodiesterase type 5 (PDE5) with modest vasodilating properties, affects cardiac contractility in vitro.Research design and methods: Slices of freshly obtained human (n = 2) or dog (n = 3) atrial appendage were suspended in organ baths containing Krebs—Ringer bicarbonate buffer (pH 7.4, 37°C) bubbled continuously with 95% O2 and 5% CO2, and isometric tension was recorded using a Gould physiograph. Contractions were elicited by 1-Hz electric pacing. After 15min of equilibration, 1\\M sildenafil was added to the bath, followed 15min later (human and dog) by 5|iM epinephrine, an inotropic agent, and 10min later (dog) by 88|iM 3-isobutyl-1 -methylxanthine(IBMX), a nonselective PDE inhibitor. In a separate experiment, cyclic guanosine monophosphate levels and PDE, protein kinase G, and protein kinase A activities were determined.Results: Addition of 1 |iM sildenafil to isolated dog or human atrial tissue had no significant effect on force of cardiac contraction, whereas epinephrine produced a robust increase in contractile force in the same muscle strip. The addition of IBMX produced a marked stimulation of contractile force in dog atrial tissue. Very low amounts of PDE5 were found in extracts of human heart, consistent with its known primary location in the smooth muscle of systemic vasculature.Conclusions: Sildenafil is unlikely to directly produce inotropic effects on cardiac muscle in patients being treated for erectile dysfunction.

Electroencephalogram is activated by addition of pentobarbital in the isolated perfused rat head

To evaluate the direct effects of a barbiturate on cerebral functions without its influence on brain perfusion pressure, circulatory hormones and metabolites, the electroencephalogram (EEG) was studied in the isolated rat head. Male Wistar rats were anesthetized, and EEG electrodes were inserted into the cranium. A Krebs-Ringer bicarbonate buffer solution containing heparinized rat whole blood, 20 mmol/l glucose, 200 mmol/l mannitol and 0.1 mg/ml dexamethasone was used for the perfusate. The bilateral common carotid arteries were cannulated, pumped at a rate of 6 ml/min and the head was isolated. The venous effluent was reoxygenated and recirculated into the brain. Twenty-five min after isolation of the heads pentobarbital was added to the perfusate at concentrations of 0.1, 0.5 and 2.5 mg/ml. EEG was recorded before and during perfusion. EEG activity could be recorded for more than 25 min after the beginning of perfusion. EEG activity gradually declined from 42±5 μV before perfusion (in vivo) to 4±1 μV at 25 min after the beginning of perfusion. Then, 3 min after the addition of pentobarbital, the EEG activity became significantly higher in the high dose groups; 12±3 μV in the 0.5 mg/ml group (p<0.05) and 12±1 μV in 2.5 mg/ml group (p<0.05) compared with the group without pentobarbital (2±2 μV). The present study suggests that a barbiturate has mitigating effects on the brain damage induced by the in vitro brain perfusion.

Glutaminolysis and insulin secretion: from bedside to bench and back.

Identification of regulatory mutations of glutamate dehydrogenase (GDH) in a form of congenital hyperinsulinism (GDH-HI) is providing a model for basal insulin secretion (IS) and amino acid (AA)-stimulated insulin secretion (AASIS) in which glutaminolysis plays a key role. Leucine and ADP are activators and GTP is an inhibitor of GDH. GDH-HI mutations impair GDH sensitivity to GTP inhibition, leading to fasting hypoglycemia, leucine hypersensitivity, and protein-induced hypoglycemia, indicating the importance of GDH in basal secretion and AASIS. The proposed model for glutaminolysis in IS is based on GDH providing NADH and alpha-ketoglutarate (alpha-KG) to the Krebs cycle, hence increasing the beta-cell ATP-to-ADP ratio to effect insulin release. The process operates with 1) sufficient lowering of beta-cell phosphate potential (i.e., fasting) and when 2) AAs provide leucine for allosteric activation and glutamate from transaminations. To test this hypothesis, IS studies were performed in rat and GDH-HI mouse models. In the rat study, rat islets were isolated, cultured, and then perifused in Krebs-Ringer bicarbonate buffer with 2 mmol/l glutamine using 10 mmol/l 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) or a BCH ramp after 50 or 120 min of glucose deprivation. In the GDH-HI mouse study, the H454Y GDH-HI mutation driven by the rat insulin promoter was created for H454Y beta-cell-specific expression. Cultured, isolated islets were perifused in leucine 0-10 mmol/l with 2 mmol/l glutamine 0-25 mmol/l, AA 0-10 mmol/l, or glucose 0-25 mmol/l. Rat islets displayed enhanced BCH-stimulated IS after 120 min of glucose deprivation, but not when energized by fuel. H454Y and control islets had similar glucose-stimulated IS, but H454Y mice had lower random blood glucose. Leucine-stimulated IS and AASIS occurred at lower thresholds and were greater in H454Y versus control islets. Glutamine stimulated IS in H454Y but not control islets. The clinical manifestations of GDH-HI and related animal studies suggest that GDH regulates basal IS and AASIS. Energy deprivation enhanced GDH-mediated IS, and H454Y mice were hypoglycemic, substantiating roles for GDH and its regulation by the phosphate potential in basal IS. Excessive IS from H454Y islets upon exposure to GDH substrates or stimuli indicate that regulation of GDH by the beta-cell phosphate potential plays a critical role in AASIS. These findings provide a foundation for defining pathways of basal secretion and AASIS, augmenting our understanding of beta-cell function.

Excitation- and beta(2)-agonist-induced activation of the Na(+)-K(+) pump in rat soleus muscle.

In rat skeletal muscle, Na(+)-K(+) pump activity increases dramatically in response to excitation (up to 20-fold) or beta(2)-agonists (2-fold), leading to a reduction in intracellular Na(+). This study examines the time course of these effects and whether they are due to an increased affinity of the Na(+)-K(+) pump for intracellular Na(+). Isolated rat soleus muscles were incubated at 30 (o)C in Krebs-Ringer bicarbonate buffer. The effects of direct electrical stimulation on (86)Rb(+) uptake rate and intracellular Na(+) concentration ([Na(+)](i)) were characterized in the subsequent recovery phase. [Na(+)](i) was varied using monensin or buffers with low Na(+). In the [Na(+)](i) range 21-69 mM, both the beta(2)-agonist salbutamol and electrical stimulation produced a left shift of the curves relating (86)Rb(+) uptake rate to [Na(+)](i). In the first 10 s after 1 or 10 s pulse trains of 60 Hz, [Na(+)](i) showed no increase, but (86)Rb(+) uptake rate increased by 22 and 86 %, respectively. Muscles excited in Na(+)-free Li(+)-substituted buffer and subsequently allowed to rest in standard buffer also showed a significant increase in (86)Rb(+) uptake rate and decrease in [Na(+)](i). Na(+) loading induced by monensin or electroporation also stimulated (86)Rb(+) uptake rate but, contrary to excitation, increased [Na(+)](i). The increase in the rate of (86)Rb(+) uptake elicited by electrical stimulation was abolished by ouabain, but not by bumetanide. The results indicate that excitation (like salbutamol) induces a rapid increase in the affinity of the Na(+)-K(+) pump for intracellular Na(+). This leads to a Na(+)-K(+) pump activation that does not require Na(+) influx, but possibly the generation of action potentials. This improves restoration of the Na(+)-K(+) homeostasis during work and optimizes excitability and contractile performance of the working muscle.

Rapid effect of testosterone on rat Sertoli cell membrane potential. Relationship with K+ATP channels.

For this study, we investigated the changes in the electrophysiological parameters of Sertoli cells in seminiferous tubules from 17 - 19 day-old rats induced by testosterone. Using conventional intracellular microelectrode techniques, we analysed the membrane potential and its input resistance. The entire tubules were fixed in a superfusion chamber continuously perfused with Krebs-Ringer bicarbonate buffer (pH 7.4, 32 degrees C). Visual control of cell impalement was achieved using an inverted microscope. The parameters analysed were passed through an amplifier and recorded using a proprietary software system. The topical application of testosterone (0.1 to 10 microM) led to an immediate (within 30 seconds) and significant dose-dependent depolarization of the membrane potential of the cell at all concentrations used. Concomitantly, the input resistance of the cell membrane underwent a significant increment at 30 seconds. These changes returned to resting values after washout. Topical administration of 17beta-estradiol or progesterone (10 microM) did not change the membrane potential. The addition of the K +ATP channel agonist diazoxide to the perfusion buffer nullified the depolarization effect of testosterone at 30 seconds. This result suggests that the immediate action of testosterone is associated with the closing of K +ATP channels, thereby depolarizing the membrane.

The Mechanism for Transcriptional Activation of the Human ATA2 Transporter Gene by Amino Acid Deprivation is Different than That for Asparagine Synthetase

After amino acid deprivation, the mRNA content for both asparagine synthetase (AS) and the system A transporter ATA2 is increased. The purpose of the reported experiments was to characterize the molecular mechanism for the ATA2 gene and to contrast the ATA2 regulatory characteristics with those of AS. Amino acid limitation was initiated by incubation of HepG2 human hepatoma cells in either amino acid-free Krebs-Ringer bicarbonate buffer or culture medium lacking the single amino acid histidine. For ATA2, like AS, the elevated mRNA content was due to increased transcription. However, there were fundamental differences between the mechanisms for nutrient regulation of the AS and ATA2 genes. When cells were deprived of amino acids, there was a lag period of approximately 4 h before an increase in AS mRNA occurred, whereas the elevation of ATA2 mRNA was readily detectable at 2-4 h. Consistent with these observations, de novo protein synthesis was absolutely required for the activation of the AS gene, but the increase in ATA2 mRNA was largely independent of protein synthesis. Furthermore, in contrast to AS, transcription from the ATA2 gene was not increased by glucose deprivation. Given this lack of ATA2 transcriptional activation by glucose starvation and that the induction of the AS gene by glucose or amino acid starvation is mediated by common genomic elements, it is likely that the ATA2 gene does not contain the same genomic amino acid-responsive cis-elements as the AS gene.

Delta(9)-tetrahydrocannabinol increases endogenous extracellular glutamate levels in primary cultures of rat cerebral cortex neurons: involvement of CB(1) receptors.

The effects of the principal psychoactive component of marijuana, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), on endogenous extracellular glutamate levels in primary cultures of rat cerebral cortex neurons were investigated. Locally applied Delta(9)-THC (0.03, 3, 300, and 1,000 nM) concentration-dependently increased basal extracellular glutamate levels (+18% +/- 11%, +54% +/- 10%, +90% +/- 14%, +149% +/- 33% vs. basal). The facilitatory effects of Delta(9)-THC (3 and 300 nM) on cortical glutamate were fully counteracted in the presence of the selective CB(1) receptor antagonist SR141716A (10 nM) and by replacement of the normal Krebs-Ringer bicarbonate buffer with a low-Ca(2+) (0.2 mM) medium. Delta(9)-THC application also induced an enhancement in K(+)-evoked glutamate levels. These findings suggest that an increase in cortical glutamatergic transmission mediated by local CB(1) receptor activation may underlie some of the psychoactive and behavioral effects of acute marijuana consumption.

Phyllodulcin, a constituent of "Amacha", inhibits phosphodiesterase in bovine adrenocortical cells.

Phyllodulcin, a constituent of "Amacha", inhibits phosphodiesterase in bovine adrenocortical cells.

The effect of nucleotides (ADP and ADP + V i) on the thermal stability of rat uterus

Thermal unfolding of stripes prepared from rat uterus has been studied in the presence of nucleotides by differential scanning calorimetry (DSC). Using ADP, ATP and inorganic phosphate analogue orthovanadate, three intermediate states of the ATP hydrolysis cycle were simulated in the uterus stripes. In the main transition of the DSC pattern at least four overlapping endotherms were detected in rigor (AM), in strongly binding (AM·ADP) and weakly binding state (AM·ADP·V i) of myosin to actin. It was found that nucleotide binding induced a shift of the main melting temperatures (from 60.7 to 61.1°C) and produced changes in the total calorimetric enthalpies (0.45 J/g for rigor, 0.4 J/g for strong binding, and 0.6 J/g for weak binding state). In the Krebs–Ringer bicarbonate buffer containing 100 nM estrogen (Oe) the main transition temperature shifted to 62.4°C and the total enthalpy change was 0.56 J/g. It seems to be an intermediate phase between the strong and weak binding state. The changes of the parameters of the peak functions suggest global rearrangements of the internal structure in myosin heads in the intermediate states.

The cannabinoid receptor agonist WIN 55,212-2 regulates glutamate transmission in rat cerebral cortex: an in vivo and in vitro study.

The effects of the cannabinoid receptor agonist WIN 55,212-2 on endogenous extracellular glutamate levels in the prefrontal cortex of the awake rat and in primary cultures of rat cerebral cortex neurons were investigated. In the prefrontal cortex WIN 55,212-2 (0.1 and 1 mg/kg i.p.) increased dialysate glutamate levels from of the awake rat, while the lower (0.01 mg/kg) and the higher (2 mg/kg) doses were ineffective. Furthermore, the WIN 55,212-2 (0.1 mg/kg)- induced increase of dialysate glutamate levels was counteracted by pretreatment with the selective CB(1) receptor antagonist SR141716A (0.1 mg/kg i.p.) and by the local perfusion with a low-calcium Ringer solution (Ca(2+) 0.2 mM). In primary cultures of rat cerebral cortex neurons, WIN 55,212-2 (0.01--100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. The facilitatory effect of WIN 55,212-2 (1 nM) was fully counteracted by SR141716A (10 nM), by the replacement of the normal Krebs Ringer-bicarbonate buffer with a low Ca(2+) medium (0.2 mM) and by the IP(3) receptor antagonist xestospongin C (1 microM). These in vivo and in vitro findings suggest an increase in cortical glutamatergic transmission by CB(1) receptors, an effect that may underlie some of the psychoactive and behavioural actions of acute exposure to marijuana.

Central Stimulatory Influence of Oxytocin on Preovulatory Gonadotropin-Releasing Hormone Requires More than the Median Eminence

The study was designed to determine whether the ability of central oxytocin (OT) to stimulate gonadotropin-releasing hormone (GnRH) on the afternoon of proestrus (PE) in the cycling female rat is mediated at the level of GnRH terminals within the median eminence (ME), or at higher hypothalamic levels where GnRH cell bodies and axons are located. Determining the location of this OT effect in vivo has proven difficult. Therefore, an in vitro system utilizing ME or basal hypothalamic (BH) explants containing GnRH terminals, or GnRH neurons including the cell bodies, axons and terminals, respectively, were harvested from regular cycling female rats at 15:00 h on PE or diestrus (DI). The explants were allowed to preincubate in Krebs Ringer Bicarbonate Buffer containing glucose, ascorbic acid, calcium, and a metalloprotease inhibitor (KRBG) and enriched with 95% O₂/5% CO₂ at 37°C until a stable baseline release of GnRH was achieved (30 min). The 0.05 level of probability was used as the minimum criterion of significance in all experiments. The ability of OT (10^–15–10^–9M) to stimulate the release of GnRH was determined in both ME and BH explants on PE and DI. The results demonstrated a sensitive, dose-dependent ability of OT to stimulate GnRH release from PE BH explants which was observed only in PE. Furthermore, OT failed to significantly stimulate GnRH release from ME explants on either PE or DI. The data indicate that the PE BH explant paradigm can be used to examine the manner and mechanisms by which OT influences GnRH release on the afternoon of PE. Furthermore, the results indicate for the first time that the stimulatory action of OT by itself on preovulatory GnRH release in cycling female rats is not mediated at the level of the GnRH terminals within the ME, but requires neuronal interactions and mechanisms within the BH explants.

Effect of 3,5,3′-triiodo-L-thyronine on amino acid accumulation and membrane potential in Sertoli cells of the rat testis

The purpose of this study was to investigate the effect of T 3 on amino acid accumulation and on the membrane potential of Sertoli cells of immature rat testes. Testes of pre-pubertal and pubertal rats were pre-incubated (30 min) in Krebs-Ringer bicarbonate buffer and incubated in the presence of [ 14C]methylaminoisobutyric acid with and without T 3 for 15, 45 and 60 min. The hormone (10 −6 M and 10 −7M) significantly stimulated amino acid accumulation in 6 and 13-day old rat testes but did not have any effect in neonatal and pubertal animals. T 3 produced a dose-dependent hyperpolarizing effect at concentrations of 10 −6 M, 10 −5 M, 2 × 10 −5 M and 10 −4 M. We conclude that T 3 induces a membrane hyperpolarization in Sertoli cells and stimulates amino acid accumulation in immature rat testes, demonstrating that the hormone has a rapid plasma membrane action.

Melanin-concentrating hormone stimulates the release of luteinizing hormone-releasing hormone and gonadotropins in the female rat acting at both median eminence and pituitary levels.

The purpose of this study was to investigate whether melanin-concentrating hormone (MCH) acts directly on the median eminence and on the anterior pituitary of female rats regulating LHRH and gonadotropin release. In addition, immunohistochemistry was used to examine the density and distribution of MCH-immunoreactive fibers in the median eminence of proestrous rats. MCH-immunoreactive fibers were found in both the internal and external layers of the median eminence and in close association with hypophysial portal vessels. In the first series of in vitro experiments, median eminences and anterior pituitaries were incubated in Krebs-Ringer bicarbonate buffer containing two MCH concentrations (10(-10) and 10(-8) M). The lowest MCH concentration (10(-10) M) increased (P < 0.01) LHRH release only from proestrous median eminences. Anterior pituitaries incubated with both MCH concentrations also showed that 10(-10) M MCH increased gonadotropin release only from proestrous pituitaries. In the second series of experiments, median eminences and pituitaries from proestrous rats were incubated with graded concentrations of MCH. MCH (10(-10) and 10(-9) M) increased (P < 0.01) LHRH release from the median eminence, and only 10(-10) M MCH increased (P < 0.01) LH and FSH release from the anterior pituitary. The effect of MCH on the stimulation of both gonadotropins from proestrous pituitaries was similar to the effect produced by LHRH. Simultaneous incubation of pituitaries with MCH and LHRH did not modify LH but increased the FSH release induced by LHRH. The present results suggest that MCH could be involved in the regulation of preovulatory gonadotropin secretion.

Capacitation and acrosome reaction in buffalo bull spermatozoa assessed by chlortetracycline and [formula omitted] agglutinin fluorescence assay

Studies on buffalo sperm capacitation have been limited because of the non-availability of a direct assay system. We describe two methods for detecting the acrosomal status of buffalo spermatozoa, namely chlortetracycline (CTC) fluorescence assay and Pisum sativum agglutinin (FITC-PSA) stain. We also test them under various treatment regimens and simultaneously standardize and calibrate them with transmission electron microscopy. An initial comparison of three physiological media, such as Krebs-Ringer bicarbonate buffer, Tyrode solution and Brackett & Oliphant medium (having different calcium concentrations and osmolality) used for studying the capacitation of buffalo spermatozoa and assessed by CTC, FITC-PSA, Giemsa stain and TEM, revealed Brackett & Oliphant medium to be marginally better than the other two media. When stained with chlortetracycline, three distinct fluorescent patterns were visible in buffalo spermatozoa under capacitating conditions. These were ‘F’ with fluorescence in the post acrosomal region characteristic of uncapacitated acrosome-intact cells; ‘B’ with fluorescence on the anterior portion of the sperm head and a dark band in the post-acrosomal region, characteristic of capacitated, acrosome intact cells and ‘AR’ with a fluorescent band on the posterior portion of the head, characteristic of acrosome-reacted cells. The FITC-PSA intensely labels the acrosomal region of acrosome intact buffalo sperm. Acrosome reacted sperms had diminished acrosomal labelling by both the probes used. Buffalo spermatozoa was not capacitated when calcium was either omitted from the medium or chelated with EGTA. In the presence of Ca 2+ ionophore, A23187, 68% at 4 h and 85% at 8 h completed the acrosome reaction. Time course studies revealed a 4 h incubation period at 1.71 mM Ca 2+ concentration to be necessary before transformation of ‘F’ to ‘B’ cells could take place. Spontaneous acrosome reaction induced at 6 and 8 h incubation of buffalo spermatozoa in KRB medium resulted in conversion of ‘B’ cells to ‘AR’ while ‘F’ cells remained unchaged. A simultaneous evaluation of acrosome intact and acrosome-reacted cells using FITC-PSA, Giemsa and TEM gave results similar to examination by CTC stain. Both the assays are rapid, reproducible, reliable and they detect an increase in physiological acrosome reactions. They thus can be used to study effects of calcium and prove to be good monitoring systems to identify buffalo sperm capacitation and acrosome reaction in individual buffalo bulls for fertility studies.

Nitric oxide inhibits the release of acetylcholine in the isolated retina.

Recent studies have revealed that administration of nitric oxide (NO) donors increases the release of neurotransmitters in various brain regions. In the retina, NO synthetase (NOS) is found in retinal amacrine and ganglion cells, and it is evident that NO is involved in encoding visual information. In the present study, therefore, NO donors were used to study the effect of exogenous NO on the high K(+)-evoked release of endogenous acetylcholine (ACh) in the rat retina. Isolated rat retinal preparations were superfused with modified Krebs-Ringer bicarbonate buffer solution. In each experiment, stimulation for 10 min with 30 mM KCl was done twice. The amounts of ACh released by the first or second KCl stimulation were termed S1 and S2 respectively. Test agents were applied just before the second KCl stimulation. The effects of test agents were evaluated as S2 divided by S1. ACh was converted to hydrogen peroxide and electrochemically assayed by high-performance liquid chromatography. S-Nitro-N-acetyl-DL-penicillamine (SNAP), an NO donor, dose-dependently inhibited the high K(+)-evoked release of endogenous ACh. Such inhibition by NO was confirmed also by another NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxy imino]-5-nitro-3-hexenamide (NOR3). The inhibitory effect of SNAP was abolished by both carboxy-PTIO, an NO scavenger, and bicuculline, an antagonist of GABAA receptors. The NO-induced decrease of ACh release is probably due to an NO-induced increase of GABAergic system inhibition.

Mechanisms of Myocardial Protection by Adenosine-Supplemented Cardioplegia: Differential Response of Calcium-Independent Protein Kinase C Isozymes

Background. Adenosine-supplemented cardioplegia improves myocardial function after cardioplegic arrest. However, the underlying cellular mechanism(s) responsible for adenosine's protective actions remains unclear. We tested the hypothesis that protection by adenosine-supplemented cardioplegia would be associated with selective activation of protein kinase C (PKC) isozymes δ and ϵ.Materials and methods. Isolated rat hearts were perfused (37°C, Krebs–Ringer bicarbonate buffer) for 30 min, after which baseline functional measurements were made. This was followed by 120 min of cold cardioplegic arrest at 4°C with either St. Thomas No. 2 (ST#2), ST#2 + adenosine (100 μM, ADO) or ST#2 + ADO + 8-sulfophenyltheophylline (50 μM, SPT). Hearts were reperfused for 60 min and functional measurements made. Distribution of PKC isoforms was determined (immunoblotting) after 30 min of warm perfusion (No-CDPL) or after 30 min of perfusion followed by 15 min of cardioplegic arrest.Results. ADO prevented myocardial dysfunction after cardioplegic arrest. PKC-δ did not differ in the cytosolic fraction among groups. However, ADO prevented increases in particulate fraction PKC-δ, but elicited a significant increase in the particulate fraction PKC-ϵ, while ST#2 or SPT significantly decreased the cytosolic fraction PKC-ϵ. Both functional and cellular changes associated with ADO were receptor mediated.Conclusion. This novel, dual action of adenosine-supplemented cardioplegia on PKC isoforms may be responsible for the associated functional improvements.

Storage and Microencapsulation of Islets for Transplantation

Microencapsulation is an effective means of immunoisolation for pancreatic islet transplants. However, the process of isolating, purifying, encapsulating, and transplanting islets in a single day is labor intensive and difficult for routine use. There is an apparent need for reliable methods of islet storage, and cryopreservation has emerged as an attractive system of islet banking. While studies have shown that cryopreserved islets are viable when tested unencapsulated after thawing, it is not clear if the combination of freezing and encapsulation would affect islet function. The purpose of the present study was to determine the in vitro function of cryopreserved islets following thawing and microencapsulation. Islets were isolated from the pancreata of Sprague-Dawley rats and cryopreserved under liquid nitrogen for either 1 week or 1 month, following an overnight culture at 37 degrees C. Upon thawing, the islets were tested either unencapsulated or after encapsulation in polylysine-alginate membrane. In all experiments islets were preperifused for 1 h at 37 degrees C with a modified Krebs-Ringer bicarbonate buffer containing 3.3 mM (60 mg/dl) glucose and maintained at pH 7.4 by continuous gassing with 95% air/5% CO2. Following basal effluent sample collection on ice, the glucose concentration was raised to 16.7 mM (300 mg/dl). It was found that, within 10 min of high glucose stimulation, an average of twofold increase in insulin secretion (p < 0.01) was obtained in islets within or without microcapsules. We conclude that islets cryopreserved for 1 month prior to thawing and microencapsulation retained functional viability as determined in in vitro experiments.

Elucidation of intestinal absorption of d, l-amino acid enantiomers and aging in rats

At the present time, the origin of protein bound d-amino acid (AA) has been fairly well elucidated, but that of free d-AA is still not well understood. To gain greater understanding of this, intestinal absorption in rats of free d, l-AA enantiomers (arginine, alanine and aspartic acid as models for basic, neutral and acidic AAs, respectively, in this study) and the relationship between age and absorption were investigated. The degree of rat intestinal absorption of free d, l-AAs was evaluated using apparent membrane permeability coefficients ( P app) which were obtained from an in situ intestinal single-pass perfusion method with Krebs-Ringer bicarbonate buffer (pH 7.4) solution containing d, l-AA enantiomers. Determinations of d, l-AA enantiomers in perfusion (in- and outflow) solutions were carried out by the in-capillary derivatization high-performance capillary electrophoretic methods (ICD-HPCE methods) that were previously developed by our group. Collectively, our observations suggest: (1) that the P app of l-AA is higher than that of the d-isomer; (2) that d-AA can be absorbed as well as l-AA using a sodium ion-dependent transporter that is located on the brush border membrane of rat intestinal epithelial cells; (3) that P app reached a maximum at 8 weeks of age, but were measured at decreased amounts at 52 and 104 weeks of age. These results suggest that free d-AA in a mammalian body originates from ‘exogenous sources’.