Krebs-Ringer Bicarbonate Buffer Research Articles

Novel method for isolation of adult porcine pancreatic islets with two-stage digestion procedure.

It is particularly difficult to isolate porcine islets (PI). Experience suggests that the success rate of porcine islet isolation (PII) is probably considerably influenced by the distension and digestion of the pancreas. In this study, we divided the digestion procedure into two stages and developed a new enzyme solution to improve both the distension and digestion procedures. As a result, we established a novel and stable method of large-scale adult porcine islet isolation (APII). The harvested pancreata of 2-year-old pigs weighing over 200 kg (n = 18) were distended by introducing our new enzyme solution gently and slowly through the pancreatic ducts. Two-stage digestion (cold, then warm) was then performed by first placing the distended pancreata on ice for 2 h to cause diffusion of the enzyme solution around the islets, and then by incubating the pancreata in a water bath at 37 degrees C for 45 min without shaking. The islets were purified by a COBE 2991 cell processor on dextran T70 discontinuous density gradients. Histological study was performed on porcine pancreata sampled after 0, 15, 30, and 45 min of the second stage, and stained with H&E stain. Next, islet equivalent was calculated. Static incubation study was performed by stimulating the islets with 3.3 and 16.7 mM glucose in Krebs' Ringer bicarbonate buffer (KRBB) solution at 37 degrees C for 1 h, and finally the insulin released was measured. The dilated acinar cells septa around the islets were observed at time 0. Destruction of the acinar cells around the islets by warm digestion was recognized at 15 and 30 min, and destroyed and separated acinar cells present around the islets at 45 min. During the entire course of the warm digestion, the islets remained intact. The number of isolated islets was 291,667 +/- 240,452 IEQ/pancreas (n = 14) and 3,294 +/- 2199 IEQ/g of pancreatic tissue. The purity of recovered porcine islets was over 90%. The concentration of the insulin secreted by 10,000 IEQ islets selected at random was 83.9 +/- 13.4 microU/dish/h in response to 3.3 mM glucose and 104.1 +/- 12.9 microU/dish/h in response to 16.7 mM glucose (n = 20). A success rate of approximately 80% was attained with APII. We demonstrated that this increase in the success rate was due to the improved distension and digestion provided by this method. This two-stage APII method with its new enzyme solution may facilitate the future use of porcine islets in clinical xenotransplantation trials.

Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes

Our laboratory has been routinely using suspended and cultured human hepatocytes for predicting drug metabolism and enzyme induction by drug candidates to aid drug discovery. Increasing limitation and irregular availability of human tissue has indicated the need for maximizing the use of this valuable resource. Cryopreservation of surplus hepatocytes after isolation would greatly increase the potential of this model. However, cryopreservation of hepatocytes by various methods has resulted in cells with poor metabolic activity and unacceptably low survival rates in culture. Recently Zaleski et al. (Biochem. Pharmacol. 46 (1993) 111–116) reported that cryopreserved rat hepatocytes retained metabolic capacity similar to fresh hepatocytes when the cells were preincubated for 30 min at 37°C in Krebs–Ringer bicarbonate buffer prior to freezing. To further explore this methodology, both the functional capacity of the cells in culture as well as their ability to retain CYP inducibility was investigated with thawed cryopreserved hepatocytes. Although human hepatocytes were used in this study the initial work focused on rat hepatocytes as a cell model. Our results showed that while the preincubation step did not appear to effect the initial viability of cryopreserved hepatocytes, survival of the cells in culture was greatly enhanced. Plating efficiencies for nonpreincubated cryopreserved hepatocytes were decreased to ≈15% of fresh cells after 48 h in culture. In contrast, cells that had been preincubated prior to freezing had an excellent plating efficiency (∼60%) and responded to classical CYP inducers dexamethasone, β-naphthoflavone and phenobarbital in a manner indistinguishable from that of fresh hepatocytes. Experiments with human hepatocytes has also demonstrated similar results. This is the first time to our knowledge that cryopreserved hepatocytes from both rat and human have been shown to reproducibly respond to CYP inducers in culture.

Sodium at high concentrations increases glucose-induced insulin secretion in the perfused rat pancreas.

To study the effects of supraphysiologically high sodium concentrations on insulin secretion, the rat pancreas was perfused with normal Krebs-Ringer bicarbonate buffer (KRBB) solution and with KRBB solutions containing an additional 25 and 50 mmol/l NaCl. The first phase of insulin secretion in response to 18 mmol/l glucose was enhanced in a dose-dependent manner, from 20.8+/-4.0 pmol for 4 min in normal KRBB to 36.2+/-10.3 pmol (P<0.05) and 60.3+/-14.6 pmol (P<0.01) with addition of 25 and 50 mmol/l NaCl, respectively. The second phase secretion was significantly increased by the addition of 50 mmol/l NaCl (259.4+/-38.6 vs 170.9+/-47.4 pmol for 16 min P<0.05). The addition of 50 mmol/l isethionate Na to normal KRBB solution also increased the first and second phases of insulin secretion in response to glucose. The increase in osmolarity by the addition of mannitol to normal KRBB solution did not affect the glucose-induced insulin secretion. A high sodium concentration affected neither tolbutamide-induced nor arginine-induced insulin secretion. It is suggested that sodium concentrations have an important role in insulin secretion in response to glucose, but not to other secretagogues.

Formation and Retention of Cotinine during Placental Transfer of Nicotine in Human Placental Cotyledon

Maternal smoking during pregnancy causes reduction of fetal breathing movements, an effect attributed to nicotine in fetal blood. Nicotine is metabolized to cotinine which has a long plasma half-life and exhibits slow clearance across membrane barriers. It is also known to activate placental phospholipase-A₂-like enzymes, resulting in formation of prostaglandins. Therefore, we studied transport of nicotine in isolated perfused cotyledon of normal human term placenta. The placental cotyledon was perfused with aerated (21% O₂, 5% CO₂) Krebs-Ringer bicarbonate buffer (pH 7.4, 37°C) containing 2% albumin on both maternal (230 ml, 15 ml/min, 35 mm Hg) and fetal (93 ml, 1.75 ml/min, 70 mm Hg) sides in a closed recirculating system. Nicotine (2 mg) was added to the maternal perfusate; perfusate samples (1 ml) were collected from both sides at regular intervals and analyzed for nicotine and cotinine by high-pressure liquid chromatography. This study gave the following results: (1) In about 60–80 min, 18.6% of the nicotine added to the maternal perfusate was transferred to the fetal perfusate, and the maternal/fetal concentration ratio reached 1.0. These results show rapid placental transfer of nicotine, consistent with its high lipid solubility. (2) Less than 1% is metabolized to cotinine in placenta. The ratio of cotinine concentrations in maternal and fetal perfusates reached 1.0 in about 40 min. These studies were also verified using ¹⁴C-nicotine. (3) Maximal reduction in fetal breathing movements occurs at about 30 min, and recovery occurs at 90 min after tobacco smoking by the mother. These observations agree with the rate of placental transfer of nicotine. (4) When nicotine was added on the fetal side, part of it was metabolized to cotinine. However, the maximal concentration of cotinine was twice higher on fetal than on maternal side. These observations suggest that accumulation of cotinine on fetal side may activate prostaglandin formation and trigger spontaneous abortions in pregnant smokers.

Glucose augmentation of mastoparan-stimulated insulin secretion in rat and human pancreatic islets.

Mastoparan, a tetradecapeptide component of wasp venom, activates heterotrimeric G-proteins and stimulates exocytosis in several cell types, including the pancreatic beta-cell. In this study, its effects on insulin secretion were assessed in both rat and human pancreatic islets, along with the ability of glucose and alpha-ketoisocaproate (alpha-KIC) to augment mastoparan-stimulated release. In Ca2+-free Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose, 20 micromol/l mastoparan stimulated insulin secretion 12- and 14-fold in rat and human islets, respectively. The inactive analog mastoparan-17 had no effect on release. Under the same Ca2+-free conditions, 11.1 mmol/l glucose had no effect on insulin release alone, but augmented mastoparan-stimulated release by 74% in both rat and human islets. Stimulation of release by mastoparan and augmentation of release by glucose were unaffected by treatment with pertussis toxin. The effect of cellular GTP depletion on the mastoparan stimulation of release and augmentation by alpha-KIC was studied by culturing rat islets in the presence of 25 microg/ml mycophenolic acid for 20 h. In the control islets, alpha-KIC augmented mastoparan-stimulated insulin release by 80%. In the GTP-depleted rat islets, mastoparan-stimulated insulin release was not changed, while the augmentation by alpha-KIC was eliminated. Mannoheptulose completely blocked the augmentation by glucose. In conclusion, mastoparan stimulates insulin release by activation of a signal transduction pathway that can be augmented by nutrients such as glucose and alpha-KIC. Nutrient augmentation of this pathway is heavily dependent on GTP.

Inhibition of hepatic neoglucogenesis and glucose-6-phosphatase by aqueous extract ofBauhinia megalandra leaves

An aqueons extract of Bauhinia megalandra leaves causes a decrease of nearly 50% in the glucose production, from lactate or fructose, of rat liver slices incubated in Krebs-Ringer bicarbonate buffer supplemented with oleate-saturated albumin. In intact microsomes, the B. megalandra extract acts as a mixed non-competitive inhibitor of glucose-6-phosphatase. In the histone-disrupted microsomes the extract has no effect on glucose-6-phosphate hydrolysis. However in the presence of the extract, glucose-6-phosphatase activity was linear with time, as in the control, but at a lower level, reaching a plateau after 20 min. This suggests an inhibition of the catalytic subunit by accumulation of orthophosphate and/or glucose, as a consequence of the inhibition of its translocases (T2 and/or T3 respectively). Neither the intact nor the disrupted microsomal pyrophosphatase kinetics parameters were affected by the inclusion of the leaf extract. The above results strongly suggest that the main effect of the B. megalandra extract is the inhibition of the glucose-6-phosphate transporter (T1) of the microsomal glucose-6-phosphatase system; however, an inhibition of T3 was not ruled out. The glucose-6-phosphatase inhibition might explain the decrease in the liver slices neoglucogenic capacity and in turn could reduce glucose levels in diabetic patients. © 1998 John Wiley & Sons, Ltd.

Localization and Age Related Patterns of Nitric Oxide Synthase and Cyclic GMP in Rat Cervical Spinal Cord 1907

Recent studies demonstrated that there are age-related changes in atrial natriuretic factor (ANF) responsive particulate guanylyl cyclase (pGNC) and nitric oxide (NO) responsive soluble guanylyl cyclase (sGNC) activities in the central nervous system of the rat. To date no studies have appeared which described developmental aspects in ANF- or NO-responsive GNC activity in the rat cervical spinal cord. In view of the alleged roles of both ANF and NO during the maturation of neuronal networks, we studied the localization of pGNC and sGNC in the developing spinal cord in relation to other cell types at 2 weeks and 3 months after birth. Lewis rats were decapitated and the cervical spinal cord was removed and cut into 400 μm slices, which were placed immediately in aerated Krebs-Ringer bicarbonate buffer, kept at 25oC for 20 minutes. Subsequently the slices were placed in perfusion chambers and continuously perfused with the same buffer in the presence of 1 mM isobutylmethylxanthine to inhibit phosphodiesterase activity. ANF, sodium nitroprusside (SNP) as a NO-donor, NMDA, or NG-nitro-L-arginine methylester(L-NAME) were added to the perfusion medium as required. Slices were fixed with paraformaldehyde at appropriate times, cryostat sections were cut (10μm), and section were processed for immunocytochemistry. pGNC and sGNC activites were higher at 2 wks after birth compared to 3 months. At 2 wks ANF-responsive structures were astrocytes in the grey and white matter and neuronal cells in the grey matter. In both cases only subpopulations of cells were responsive. At the age of 3 months, ANF-responsive structures were isolated astrocytes in both grey and white matter, and in addition in fibers in lamina 1 and 2 which has a clear punctate staining pattern. NO-responsive GNC activity at 2 weeks after birth was observed in a large number of astrocytes, neuronal cells and fibers, and in oligodendrocytes. A colocalization of sGNC activity and NOS was observed in neuronal cells and fibers. At 3 month NO-responsive GNC was most found in neuronal fibers and occasionally in cell somata in all lamina of the grey matter. A frequent colocalization was observed between cGMP and NOS at 3 months. We conclude that both pGNC and sGNC have important roles during maturation of neuronal networks in the spinal cord. Considering the multitude of emerging target molecules for cGMP, we hypothesize that discrepancies in cGMP signal transduction may have important implication for the functioning of afferents and efferents in the spinal cord.

Evidence That Relaxin's Effects on Growth and Softening of the Cervix Are Not Mediated through Prostaglandins in the Rat

Relaxin plays a major role in promoting the growth and softening of the cervix that occurs during the second half of pregnancy in the rat. There is limited evidence that prostaglandins play a role in cervical softening in mammalian species. Accordingly, this study was conducted to determine if prostaglandins mediate relaxin’s effects on the rat cervix. To attain that objective, indomethacin was used to inhibit cyclooxygenase, the key enzyme in the conversion of arachidonic acid to prostaglandins. Twenty-six nonpregnant female rats were ovariectomized when they were 78 days old (day 1 of treatment). At ovariectomy (O), each rat was fitted with silicon tubing implants containing progesterone (P) and estrogen (E) in doses that provided blood levels similar to those during late pregnancy in rats. Rats were randomly assigned to three treatment groups. Group OPE controls (n = 8 rats) received 2 ml indomethacin vehicle (0.5% methyl cellulose, 0.025 Tween 80 in water) via gavage at 0900 h on days 8 and 9 and 0.5 ml relaxin vehicle (0.9% NaCl) sc at 6-h intervals from 1200 h on day 8 through 0600 h on day 10. Group OPER (n = 9 rats) was treated as group OPE except that 20 μg highly purified porcine relaxin was administered. Group OPERI (n = 9 rats) was treated as group OPER except that indomethacin was administered at a dose (20 mg/kg BW) that reduced cervical PGE2 levels by more than 90%. Between 0800 h and 1000 h on day 10, the cervices were removed, trimmed of fat, weighed, and placed in ice-cold Krebs-Ringer bicarbonate buffer, pH 7.5. Cervical extensibility (degree of softening) was determined within 4 h of tissue collection. Both the mean cervical wet weight and the mean cervical extensibility in the relaxin-treated group OPER rats were markedly greater (P < 0.01) than in the group OPE controls. Treatment with indomethacin did not diminish relaxin’s effects on either cervical wet weight or cervical extensibility. In conclusion, this study provides evidence that relaxin’s effects on cervical growth and softening in the rat are not mediated through prostaglandins.

Evidence that relaxin's effects on growth and softening of the cervix are not mediated through prostaglandins in the rat.

Relaxin plays a major role in promoting the growth and softening of the cervix that occurs during the second half of pregnancy in the rat. There is limited evidence that prostaglandins play a role in cervical softening in mammalian species. Accordingly, this study was conducted to determine if prostaglandins mediate relaxin's effects on the rat cervix. To attain that objective, indomethacin was used to inhibit cyclooxygenase, the key enzyme in the conversion of arachidonic acid to prostaglandins. Twenty-six nonpregnant female rats were ovariectomized when they were 78 days old (day 1 of treatment). At ovariectomy (O), each rat was fitted with silicon tubing implants containing progesterone (P) and estrogen (E) in doses that provided blood levels similar to those during late pregnancy in rats. Rats were randomly assigned to three treatment groups. Group OPE controls (n = 8 rats) received 2 ml indomethacin vehicle (0.5% methyl cellulose, 0.025 Tween 80 in water) via gavage at 0900 h on days 8 and 9 and 0.5 ml relaxin vehicle (0.9% NaCl) s.c. at 6-h intervals from 1200 h on day 8 through 0600 h on day 10. Group OPER (n = 9 rats) was treated as group OPE except that 20 microg highly purified porcine relaxin was administered. Group OPERI (n = 9 rats) was treated as group OPER except that indomethacin was administered at a dose (20 mg/kg BW) that reduced cervical PGE2 levels by more than 90%. Between 0800 h and 1000 h on day 10, the cervices were removed, trimmed of fat, weighed, and placed in ice-cold Krebs-Ringer bicarbonate buffer, pH 7.5. Cervical extensibility (degree of softening) was determined within 4 h of tissue collection. Both the mean cervical wet weight and the mean cervical extensibility in the relaxin-treated group OPER rats were markedly greater (P < 0.01) than in the group OPE controls. Treatment with indomethacin did not diminish relaxin's effects on either cervical wet weight or cervical extensibility. In conclusion, this study provides evidence that relaxin's effects on cervical growth and softening in the rat are not mediated through prostaglandins.

Glycated IAPP shows a reduced inhibitory action on insulin secretion.

Islet amyloid polypeptide (IAPP) also called amylin is a 37 amino acid long peptide co-secreted with insulin in a regulated manner from pancreatic B-cell in response to nutrient ingestion. IAPP was originally isolated from post-mortem pancreatic amyloid deposits from human NIDDM cases (11 and has been associated with the pathogenesis of amyloidosis [2]. IAPP is structurally similar to calcitonin gene related peptide (CGRP) sharing approximately 46% sequence identity [l]. It is thought that these two peptide hormones may act via the same specific receptors on target tissues [3]. CGRP has an inhibitory action on insulin secretion in rat islets in vitro [4] and in vivo [5]. In addition IAPP has been demonstrated to have inhibitory actions on insulin secretion [6,7]. Hyperglycaemia resulting in glycation of proteins may contribute to the pathogenesis of diabetes. Glycation can result in alteration of biological activity of proteins and peptides including insulin [8,9]. Recent studies have demonstrated that pancreatic B-cell levels of glycated proinsulin and insulin are significantly raised in hyperglycaemic animal models of diabetes [lo]. The initial aim of the present study was firstly to investigate the action of rat IAPP on insulin secretion in glucose-sensitive pancreatic BRIN-BDll cells in culture. Secondly to structurally characterize a glycated IAPP analogue and examine its insulinotropic action in these cells. Rat IAPP fractions (1 mg/ml, 100 lg ) were prepared under hyperglycaemic conditions (220 mM glucose) at 37°C (pH 7.4, 10 mM sodium phosphate buffer, 887~1) with a 1000-fold molar excess (over IAPP) of NaBH3CN (12.5 PI). The reaction was stopped after 24 h by addition of 0.5M acetic acid (30~1) and samples frozen at -20’C prior to separation of glycated and non-glycated forms by HPLC. HPLC-purified glycated IAPP was characterized by plasma desorption mass spectrometry (Mr 4086.3 Da) demonstrating that it contained a single glucitol adduct (i.e. monoglycated). Automated peptide sequencing analysis utilizing on-line FTH derivatization yielded just 17 pmol PTH-Lys in cycle 1 compared to 1024 pmol PTHAsn in cycle 3 of operation. A strong signal was detected for PTH-derivatives in the remaining cycles through to FTH-Tyr37 at the C-terminus. These data indicated that the amino terminal Lysl was modified by glycation. BRIN-BD11 cells were cultured in RPMI-1640 tissue culture medium containing 10% (v/v) fetal calf serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose. Insulin release from BRIN-BDll cells was determined using cell monolayers. The cells were harvested with the aid of trypsin/EDTA (Gibco Life Technologies Ltd., Paisley, Strathclyde, UK), seeded into 24-multiwell plates (Nunc, Roskilde, Denmark) at a density of 2.5 x 105 per well, and allowed to attach during overnight culture. Acute studies of insulin release were preceded by 40 min pre-incubation at 37°C in 1.0 ml Krebs Ringer bicarbonate buffer (115 mM NaCI, 4.7 mM KCI, 1.28 mM CaC12.2H20, 1.2 mM KHzPO4, 1.2 mM MgS04.7Hz0,lO mM NaHC03,5 g/L bovine serum albumin, pH 7.4) supplemented with 1.1 mM glucose. Test incubations were performed at 37°C using the same buffer supplemented with glucose, non-glycated or glycated IAPP as indicated in the Table. After 20 min incubation, the buffer was removed from each well and aliquots were stored at -20°C for measurement of insulin by RIA (111. Results in Table 1 indicate that a range of IAPP concentrations (3x10-10 to 3x10-7 M) had no effect on acute (20 min) insulin release from pancreatic B-cells cultured at 5.6 mM glucose. However, under hyperglycaemic culture conditions (16.7 mM glucose) non-glycated IAPP (3x10-10 to 3x10-9 M) induced a significant 24-32% inhibition of insulin output compared to controls (glucose alone). Glycation of IAPP significantly reduces its inhibitory action on insulin secretion by 19-35% from 3x10-10 to 3x10-9 M at 16.7 mM glucose (Table 1). These results indicate that BRIN-BDll cells like normal pancreatic B-cells [7] are responsive to the inhibitory action of IAPP at least under hyperglycaemic conditions. These BRINBDll cells probably possess specific receptors for IAPP or CCRP which mediate the inhibition of insulin secretion. Furthermore structural modification of IAPP by glycation at the amino terminus significantly reduces its inhibitory action on pancreatic B-cell insulin release in vitro.

The Ratio of Thromboxane to Prostacyclin is Increased by Peroxide in a Dose-Dependent Manner, Along with Increased Vasoconstriction in the Human Placenta

Objective: To determine if perfusion of the human placenta with increasing doses of exogenous peroxide will result in a dose-dependent increase in the ratio of thromboxane to prostacyclin, and a progressive increase in placental vasoconstriction.Methods: Isolated human placental cotyledons (n = 7) were perfused serially for 20-min intervals with control Krebs-Ringer-bicarbonate (KRB) buffer gassed with 95% O2 and 5% CO2, and then KRB buffer containing progressively increasing concentrations of f-butyl hydroperoxide (10, 25, 50, and 100 μmol/L). Control KRB buffer was perfused between each dose of t-butyl hydroperoxide. Perfusion pressure was continually monitored and maternal and fetal effluent samples were collected and analyzed for thromboxane and prostacyclin by their stable metabolites, TXB2 and 6-keto PGF1α.Results: Compared to control KRB buffer, perfusion with t-butyl hydroperoxide at concentrations of 25, 50, and 100 μmol/L resulted in a progressive and significant increase in the fetal secretion rate ratio of thromboxane to prostacyclin (22.9 ± 7.9 vs. 29.1 ± 7.3, 38.8 ± 2.3, and 52.4 ± 8.9, respectively). Peroxide significantly increased the thromboxane secretion rate on the fetal side (0.52 ± 0.2 ng/min vs. 1.3 ± 0.38 ng/min, 1.4 ± 0.31 ng/min, and 1.9 ± 0.24 ng/min), as well as on the maternal side (3.3 ± 0.75 ng/min vs. 5.4 ± 0.82 ng/min, 6.1 ± 0.96 ng/min, and 7.5 ± 1.2 ng/min) for the 25, 50, and 100 μmol/L concentrations, respectively. Peroxide perfusion did not have a significant effect on the fetal secretion rate of prostacyclin, and maternal secretion of prostacyclin was nondetectable. Coincident with the increase in the thromboxane to prostacyclin ratio, peroxide also increased the perfusion pressure (PP) and vascular resistance (VR) in a dose-dependent manner for the 25, 50, and 100 μmol/L concentrations (PP: 32.9 ± 2.5 mm Hg for control vs. 39.0 ± 5.6 mm Hg, 39.7 ± 2.5 mm Hg, and 50.4 ± 2.9 mm Hg; VR: 13.8 ± 1.9 mm Hg±min/mL vs. 16.2 ± 2mm Hg±min/mL, 16.6 ± 1.8mm Hg-min/mL, and 21.7 ± 2.2 mm Hg±min/mL, respectively). The increases in perfusion pressure and vascular resistance were highly correlated with the increase in the thromboxane to prostacyclin ratio (r = 0.826 and r = 0.848, respectively).Conclusions: Perfusion of the isolated human placental cotyledon with progressively increasing concentrations of peroxide resulted in a progressive increase in the ratio of thromboxane to prostacyclin. The increase in the ratio was due to the stimulation of thromboxane. There were also progressive increases in perfusion pressure and vascular resistance that were highly correlated with the increase in the thromboxane to prostacyclin ratio.

Effect of islet amyloid polypeptide on somatostatin inhibition of insulin secretion from isolated rat pancreatic islets

In this study, we investigated the presence of islet amyloid polypeptide (IAPP) in somatostatin cells of rat endocrine pancreas and the effect of exogenous IAPP and somatostatin, separate or combined, on in vitro insulin secretion. By immunocytochemistry, IAPP was found in both B and D cells of rat pancreatic islets. Furthermore, the labeling density of IAPP in D cells was nearly four times higher than in B cells. After a 2-day preincubation in RPMI 1640 (11.1 mM glucose), isolated rat pancreatic islets were exposed to IAPP and/or somatostatin for 90 min in modified Krebs-Ringer bicarbonate (KRB) buffers containing 11.1 or 22.2 mM glucose, or 11.1 mM glucose+10 mM l-arginine, respectively. At 11.1 mM glucose, insulin secretion was not affected by IAPP and/or somatostatin at concentrations investigated. Insulin response to 22.2 mM glucose was inhibited by exogenous somatostatin. Arginine-stimulated insulin secretion was also inhibited by somatostatin, and the effect was significantly potentiated with additional 10 −5 M IAPP. The study shows that rat pancreatic D cells have higher IAPP density than B cells in the same islets and that IAPP and somatostatin may cooperate on rat pancreatic B cells to regulate the insulin secretion in response to potent stimulation.

MPTP toxicity in rat striatal slices: dopamine uptake alteration does not appear to be related to lipid peroxidation

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is used to create experimental models of parkinsonism, induces both dopaminergic neurotoxicity and peroxidation reactions. The present work investigated the interaction between the dopamine (DA) uptake system, lipid peroxidation and MPTP in a rat striatum slice model. [ 3H]DA uptake was decreased and the concentration of thiobarbituric acid reactive substances (TBARS) increased after a plain preincubation in Krebs—Ringer bicarbonate buffer for 150 min. The decrease in [ 3H]DA uptake and the increase in TBARS were suppressed by the iron-chelating agent desferrioxamine. Inhibition of [ 3H]DA uptake was intensified, [ 3H]GBR 12 935 binding to DA uptake sites was decreased and TBARS production was inhibited in slices after preincubation with MPTP. MPTP effects were inhibited by l-deprenyl, a MAO-B inhibitor. These results suggest that the spontaneous decrease in DA uptake during simple preincubation in pure Krebs—Ringer solution was related to spontaneous TBARS generation. During MPTP preincubation, alteration of the DA uptake mechanism was not due to additional lipid peroxidation since TBARS production was decreased. MPTP effects could have resulted from other events which are discussed.

Increased myocardial oxygen consumption and resting heat production, as measured by microcalorimetry, after propranolol and carvedilol treatment: Is there a partial agonistic effect in the rat?

This study investigated the influence of β-blockade on the resting heat production of myocardial tissue by microcalorimetry. During one week, propranolol (β1β2-adrenoceptor antagonist) was orally given to 14 rats − 5 mg kg−1 once daily, and carvedilol (β1β2- and α1-antagonist) to eight rats − 3 mg kg−1 once daily; 36 rats were controls. Thin slices of cardiac tissue, ≈10 mg, were removed from the apex. Carbogen-saturated Krebs-Ringer bicarbonate buffer with glucose as substrate was pumped through the microcalorimetric ampoule during the measurement at 37°C. Unexpectedly, the mean resting heat production was higher after both propranolol, 1.25 mW g−1 wet tissue (p < 0.01, ANOVA) and carvedilol, 1.19 mW g−1 (p < 0.05) treatments, than in the control group, 1.01 mW g−1. The same applied to oxygen consumption. The calculated anaerobic fractions were 16, 8 and 24% in the respective groups, but differences were not significant. Also, when added in vitro, propranolol caused an enhanced myocardial resting heat production by an average of 23%. As resting myocardial metabolism contributes to the overall cardiac energetics to a relatively minor extent, the net result of treatment will probably be of only marginal physiological importance. The experimental outcome is indicative of a stimulation of resting myocardial metabolic activity after propranolol and carvedilol, rather than a predicted decrease. We hypothesize that the absence of anything to depress in the non-beating heart tissue, reveals a small degree of partial β-agonist activity, possibly via the newly discovered β3-adrenoceptor.

Interleukin-1 reduces the glycolytic utilization of glucose by pancreatic islets and reduces glucokinase mRNA content and protein synthesis by a nitric oxide-dependent mechanism.

Culture of rat pancreatic islets with interleukin-1 (IL-1) results in up-regulation of the inducible isoform of nitric oxide synthase and overproduction of nitric oxide (NO). This is associated with reversible inhibition of both glucose-induced insulin secretion and islet glucose oxidation, and these effects are prevented by the inducible nitric oxide synthase inhibitor NG-monomethylarginine. IL-1 also induces accumulation of nonesterified arachidonic acid in islets by an NO-dependent mechanism, and one potential explanation for that effect would involve an IL-1-induced enhancement of islet glycolytic flux. We have therefore examined effects of IL-1 on islet glycolytic utilization of glucose and find that culture of islets with IL-1 in medium containing 5.5 mM glucose results in suppression of islet glucose utilization subsequently measured at glucose concentrations between 6 and 18 mM. The IL-1-induced suppression of islet glucose utilization is associated with a decline in islet glucokinase mRNA content, as determined by competitive reverse transcriptase-polymerase chain reaction, and in glucokinase protein synthesis, as determined by immuoprecipitation experiments, and all of these effects are prevented by NG-monomethylarginine. These findings suggest that IL-1 can down-regulate islet glucokinase, which is the primary component of the islet glucose-sensor apparatus, by an NO-dependent mechanism. Because reductions in islet glucokinase levels are known to cause a form of type II diabetes mellitus, these observations raise the possibility that factors which increase islet NO levels might contribute to development of glucose intolerance.

Interaction between NO and oxytocin: influence on LHRH release.

Nitric oxide synthase (NOS)-containing neurons have been localized in various parts of the CNS. These neurons occur in the hypothalamus, mostly in the paraventricular and supraoptic nuclei and their axons project to the neural lobe of the pituitary gland. We have found that nitric oxide (NO) controls luteinizing hormone-releasing hormone (LHRH) release from the hypothalamus acting as a signal transducer in norepinephrine (NE)-induced LHRH release. LHRH not only releases LH from the pituitary but also induces sexual behavior. On the other hand, it is known that oxytocin also stimulates mating behavior and there is some evidence that oxytocin can increase NE release. Therefore, it occurred to us that oxytocin may also stimulate LHRH release via NE and NO. To test this hypothesis, we incubated medial basal hypothalamic (MBH) explants from adult male rats in vitro. Following a preincubation period of 30 min, MBH fragments were incubated in Krebs-Ringer bicarbonate buffer in the presence of various concentrations of oxytocin. Oxytocin released LHRH at concentrations ranging from 0.1 nM to 1 microM with a maximal stimulatory effect (P < 0.001) at 0.1 microM, but with no stimulatory effect at 10 microM. That these effects were mediated by NO was shown by the fact that incubation of the tissues with NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, blocked the stimulatory effects. Furthermore, the release of LHRH by oxytocin was also blocked by prazocin, an alpha 1-adrenergic receptor antagonist, indicating that NE mediated this effect. Oxytocin at the same concentrations also increased the activity of NOS (P < 0.01) as measured by the conversion of [14C]arginine to citrulline, which is produced in equimolar amounts with NO by the action of NOS. The release of LHRH induced by oxytocin was also accompanied by a significant (P < 0.02) increase in the release of prostaglandin E2 (PGE2), a mediator of LHRH release that is released by NO. On the other hand, incubation of neural lobes with various concentrations of sodium nitroprusside (NP) (300 or 600 microM), a releaser of NO, revealed that NO acts to suppress (P < 0.01) the release of oxytocin. Therefore, our results indicate that oxytocin releases LHRH by stimulating NOS via NE, resulting in an increased release of NO, which increases PGE2 release that in turn induces LHRH release. Furthermore, the released NO can act back on oxytocinergic terminals to suppress the release of oxytocin in an ultrashort-loop negative feedback.

Oxytocin stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamic explants by releasing nitric oxide.

Oxytocin induces mating behavior in rats of both sexes. Previous experiments revealed that progesterone-induced sex behavior in ovariectomized, estrogen-primed rats was caused by release of NO from NOergic neurons that stimulated the release of luteinizing hormone-releasing hormone (LHRH). The LHRH activated brain-stem neurons that initiated the lordosis reflex. We hypothesized that oxytocin might similarly release NO in the medial basal hypothalamic region that would stimulate release of LHRH into the hypophyseal portal vessels to release luteinizing hormone. To investigate this hypothesis, medial basal hypothalamic explants were preincubated in Krebs-Ringer bicarbonate buffer for 30 min, followed by a 30-min incubation in fresh Krebs-Ringer bicarbonate buffer containing the compounds to be tested. Oxytocin stimulated LHRH release 3- to 4-fold at the lowest concentration tested (10(-10) M). Values remained at a plateau as the concentration was increased to 10(-7) M and then declined in a concentration-dependent manner, so that there was no stimulation with a concentration of 10(-5) M. Oxytocin (10(-7) M) stimulated release of prostaglandin E2 into the medium, a finding consistent with a role of NO in the response. That NO indeed mediated the action of oxytocin was supported by blockade of the action of oxytocin by the competitive inhibitor of NO synthase (NOS), N(G)-monomethyl-L-arginine (300 microM). Furthermore, oxytocin (10(-9) to 10(-7) M) activated NOS as measured at the end of the experiments. Oxytocin appeared to act to stimulate norepinephrine terminals in the medial basal hypothalamus, which activated NOS by alpha1-adrenergic receptors, because prazocine, an alpha1 receptor blocker, inhibited the LHRH-releasing action of oxytocin. Finally, incubation of neural lobe explants with sodium nitroprusside, a NO releasor, revealed that nitroprusside (300-600 microM, but not 900 microM) inhibited oxytocin release. Therefore, the NO released by oxytocin also diffuses into the oxytocin neuronal endings and inhibits oxytocin release, forming a negative feedback loop. The results indicate that oxytocin is important not only in induction of mating, but also in stimulating LHRH release with subsequent luteinizing hormone discharge that plays a crucial role in reproduction.

Nitric oxide mediates mucin secretion in endotoxin-induced otitis media with effusion.

The mechanisms that regulate mucin release in chronic otitis media with effusion, a leading cause of hearing loss in children, remain largely unknown. We developed an animal model using Sprague-Dawley rats to determine the factors responsible for mucin production in chronic otitis media with effusion. N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of nitric oxide synthase, was used to investigate the role of nitric oxide in mucin secretion by the middle ear epithelium. All rats underwent eustachian tube obstruction. In the first set of rats, the middle ear was then injected transtympanically with 35 microl of either 300 mOsm Krebs-Ringer bicarbonate buffer (control group) or 1 mg/ml lipopolysaccharide in Krebs-Ringer (experimental group 1). In a second set of rats, the middle ear space was injected with lipopolysaccharide and then infused at a continuous rate for 7 days with either Krebs-Ringer (experimental group 2) or 1 mmol/L L-NAME in Krebs-Ringer (experimental group 3) through an osmotic infusion pump. After 7 days the volume of effusion and the quantity of mucin collected were significantly greater in lipopolysaccharide-exposed ears than in controls. In addition, antimucin immunostaining demonstrated mucous cell hyperplasia in response to lipopolysaccharide. The lipopolysaccharide-induced production of mucin and mucous cell hyperplasia was inhibited in ears treated with lipopolysaccharide and L-NAME. These results suggest that nitric oxide is a mediator in the pathway of mucin secretion in chronic otitis media with effusion.

Nitric oxide mediates mucin secretion in endotoxin-induced otitis media with effusion: Student Research Award 1996

The mechanisms that regulate mucin release in chronic otitis media with effusion, a leading cause of hearing loss in children, remain largely unknown. We developed an animal model using Sprague-Dawley rats to determine the factors responsible for mucin production in chronic otitis media with effusion. N -nitro- l-arginine methyl ester ( l-NAME), a competitive inhibitor of nitric oxide synthase, was used to investigate the role of nitric oxide in mucin secretion by the middle ear epithelium. All rats underwent eustachian tube obstruction. In the first set of rats, the middle ear was then injected transtympanically with 35 μl of either 300 mOsm Krebs-Ringer bicarbonate buffer (control group) or 1 mg/ml lipopolysaccharide in Krebs-Ringer (experimental group 1). In a second set of rats, the middle ear space was injected with lipopolysaccharide and then infused at a continuous rate for 7 days with either Krebs-Ringer (experimental group 2) or 1 mmol/L l-NAME in Krebs-Ringer (experimental group 3) through an osmotic infusion pump. After 7 days the volume of effusion and the quantity of mucin collected were significantly greater in lipopolysaccharide-exposed ears than in controls. In addition, antimucin immunostaining demonstrated mucous cell hyperplasia in response to lipopolysaccharide. The lipopolysaccharide-induced production of mucin and mucous cell hyperplasia was inhibited in ears treated with lipopolysaccharide and l-NAME. These results suggest that nitric oxide is a mediator in the pathway of mucin secretion in chronic otitis media with effusion. (Otolaryngol Head Neck Surg 1997;116:308-16.)

Effects of azido-3'-deoxy-thymidine on luteinizing hormone, follicle-stimulating hormone and prolactin release by the pituitary-hypothalamus complex.

Azido-3'-deoxy-thymidine (AZT) is a drug extensively used in the treatment of AIDS. AZT was incubated in vitro either with the pituitary-hypothalamus complex (PHc) or the intact pituitary (PI) of male rats. The PHc is comprised of the hypothalamus and the attached pituitary gland. After a preincubation period, the PHc or PI was incubated for 1 or 2 h with Krebs-Ringer bicarbonate buffer or either of two different concentrations of AZT (1 and 10 microM). In the control incubations, the PHc released less prolactin (PRL) and more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) than the PI, indicating that hypothalamic control of the pituitary was exerted in vitro, presumably by diffusion of releasing and inhibiting hormones from the neurohypophysis to the anterior lobe of the hypophysis. Both concentrations of AZT evoked a significant increase in release of PRL and a decreased release of LH and FSH from the PHc. In the case of LH, the higher concentration of AZT partially suppressed LH release within 1 h. The other effects were not dose-related and were observed after incubating the tissue with AZT for 2 h. However, incubation of the PI with AZT failed to alter anterior pituitary hormone release, illustrating that the site of action of AZT is in the hypothalamus. We hypothesize that AZT blocks DNA synthesis resulting in suppression of synthesis and consequent release of hypothalamic hormones that control release of pituitary hormones in vitro. The results raise the possibility that AZT may alter hypothalamic-pituitary function in vivo.