Modified Krebs-Ringer Bicarbonate Buffer Research Articles

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

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

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

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

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

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

Acidosis prevents and alkalosis augments endothelium-dependent contractions in mouse arteries

Changes in pH modulate the responsiveness of vascular smooth muscle cells to vasoconstrictor stimuli, but their effect on endothelium-dependent responses is unknown. Therefore, the influence of moderate changes in pH on responses to endothelium-dependent and -independent agonists was determined in aortae and carotid arteries of 15- to 26-week-old male C57BL/6N mice. Isolated rings were suspended in Halpern-Mulvany myographs for isometric tension recording. The preparations were exposed to either acidic (pH 7), control (pH 7.4) or alkaline (pH 7.8) modified Krebs-Ringer bicarbonate buffer solutions and their contractions and relaxations compared. Endothelium-dependent relaxations to acetylcholine (in the presence of meclofenamate or of the thromboxane-prostanoid (TP) receptor antagonist S18886) were comparable at the three pH values tested in contracted aortic or carotid arterial rings. Endothelium-dependent contractions of quiescent carotid arteries were reduced in acidosis and potentiated in alkalosis compared to control; these effects were reversible. The carotid arteries produced equal amounts of 6-keto prostaglandin F1α and thromboxane B2 at the different pH values tested. Contractions to the full TP receptor agonist U46619 were similar in the three milieus, but after inducing partial TP receptor blockade (with low concentrations of the TP receptor antagonist S18886) they were depressed in acidosis compared to alkalosis. Prostacyclin as a partial TP receptor activator also induced weaker contractions at low than at high pH, whereas its vasodilator effect was not affected. These findings demonstrate that changes in pH modulate endothelium-dependent contractions in mouse arteries primarily by altering the sensitivity of TP receptors of vascular smooth muscle to endothelium-derived contracting factors.

Inhibition of Phosphoinositide 3-Kinase Potentiates Relaxation of Porcine Coronary Arteries Induced by Nitroglycerin by Decreasing Phosphodiesterase Type 5 Activity

Vessel tension can be modulated by phosphoinositide 3-kinase (PI3K) acting on l-type calcium channel, rho kinase and phosphodiesterase (PDE) type 3 in smooth muscle cells. Inhibition of PI3K could increase the relaxation of porcine coronary arteries to nitroglycerin independent of this pathway, and the aim of the present study was therefore to determine the underlying mechanisms. Isolated porcine coronary arteries were dissected from the heart and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The response of these vessels was studied by using the organ chamber technique; the content of cyclic guanosine monophosphate (cGMP) was determined by using enzyme-linked immunosorbent assay kit; and PI3K and Akt activity were determined by measuring the phosphorylation level of their downstream signaling molecule on Western blot. Inhibition of PI3K with 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) potentiated the relaxation of porcine coronary arteries to nitroglycerin and nitric oxide (NO), but not to 8-bromo-guanosine 3'5'-cyclic monophosphate, isoproterenol or (R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y27632). Increased relaxation induced by LY294002 was eliminated by Akt1/2 kinase inhibitor (Akt-I: 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate) or zaprinast, but was not affected by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, nifedipine or milrinone. Inhibition of Akt caused similar effects as LY294002. Incubation with LY294002 or Akt-I decreased the activity of PI3K and Akt but augmented the elevation of cGMP caused by NO. Enhanced cGMP elevation induced by LY294002 or Akt-I was also eliminated by zaprinast. PI3K-Akt signaling may affect vascular tone through a stimulatory effect on PDE type 5.

DA1 Receptors Modulation in Rat Isolated Trachea

We have previously demonstrated that low dose of inhaled dopamine (0.5-2 microg kg(-1) min(-1)) induces broncodilatacion in patients with acute asthma attack, suggesting that this dopamine effect is mediated by dopaminergic rather than by adrenergic receptors. To understand better these dopamine effect, rat tracheal smooth muscle was used as a model to evaluate the responses of beta2-, alpha1-, alpha2-adrenergic and DA1 and DA2 dopaminergic antagonists. Tracheal rings from male Sprague-Dawley rats (n = 90) were excised and placed in an organ bath containing modified Krebs-Ringer bicarbonate buffer at 37 degrees C, and gassed with O2 (95%) and CO2 (5%). Contractile responses were recorded with an isometric transducer in a polygraph (Letica, Spain). Contraction was induced by accumulative doses of acetylcholine (0.1, 0.3, 1, 3, 10 mM) or by electric field stimulation (10 Hz at 2 milliseconds), and accumulative doses of dopamine were added to the bath. Low concentration (0.1-0.3 mM) elicited a small initial contraction, followed by a marked relaxation. Cholinergic contraction was completely reversed at 6 mM of dopamine. This biphasic dopaminergic response was not blocked by incubation with beta2-adrenergic antagonist propranolol (0.1 microM), alpha1-antagonist, terazosin (0.1 mM), alpha2-antagonist, yohimbine (0.1 mM), or by DA2 antagonist metoclopramide (1-8 mM); DA1 antagonist SCH23390 (0.1 microM) produced a sustained increase of basal tone but did not block initial dopaminergic contraction and partially inhibited bronchodilator effect of dopamine. Dopaminergic relaxation in rat trachea is mediated by DA1 rather than by DA2 receptors; and adrenergic receptors are not involved in such dopamine-induced response. Finally, DA1 antagonist SCH23390 exerts intrinsic contractile activity on airway smooth muscle that deserves further research.

Caerulein or taurocholate induced enzymatic and histologic alterations in the isolated perfused rat pancreas

Early events in the pathogenesis of experimental acute pancreatitis are intensively studied using isolated cells or animal models. However, the results and their interpretations are dependent on the complexity of biological structures. Therefore, we proposed that studies on isolated perfused pancreas can give additional information about processes leading to acinar cell injury. This hypothesis was examined adapting the well-established caerulein hyperstimulation model and the taurocholate model of acute pancreatitis to the extracorporeal perfused isolated rat pancreas. The pancreas was removed with the duodenum including the arterial supply. A continuous perfusion of the organ was performed with a modified Krebs-Ringer bicarbonate buffer. Intraarterial caerulein application or an intraductal taurocholate (3.5%) application were used to induce acinar cell injury which was determined as the release of amylase, lipase and lactate dehydrogenase into the portal outflow medium and into the transudation fluid and by examination of histological alterations. Trypsinogen release and activation was followed by analysis of trypsinogen activation peptide (TAP) in the transudation fluid and in pancreatic tissue. Perfusion of isolated rat pancreas with supramaximal concentrations of caerulein or retrograde injection of taurocholate (3.5%) resulted in acinar cell injury indicated by elevated levels of amylase and lipase into the perfusate and into the transudation fluid. TAP levels in the transudation fluid significantly increased after perfusion with caerulein or retrograde injection of taurocholate (3.5%). The histological alterations after taurocholate application include oedema and necrosis and show significant differences to the control perfusion. Extensive pancreatic necroses were not observed after caerulein hyperstimulation. The isolated perfused rat pancreas is a useful model to investigate pathophysiological mechanisms which are relevant for the early phase of acute pancreatitis. The caerulein and the taurocholate models are transferable to the isolated rat pancreas. Studies on isolated perfused rat pancreas enable pathophysiological investigations of the exocrine pancreas without influence of systemic components, but with preserved morphology.

Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.

Glucose-stimulated Cdc42 Signaling Is Essential for the Second Phase of Insulin Secretion

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.

The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis

Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [(32)P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of glucose stimulation in MIN6 beta cells. Tyrosine phosphorylation of Munc18c was also detected in 3T3L1 adipocytes and increased with insulin stimulation, suggesting that this may be a conserved mechanism. Syntaxin 4 binding to Munc18c decreased as Munc18c phosphorylation levels increased in pervanadate-treated cells, suggesting that phosphorylation dissociates the Munc18c-Syntaxin 4 complex. Munc18c phosphorylation was localized to the N-terminal 255 residues. Mutagenesis of one residue in this region, Y219F, significantly increased the affinity of Munc18c for Syntaxin 4, whereas mutation of three other candidate sites was without effect. Moreover, Munc18c-Y219F expression in MIN6 cells functionally inhibited glucose-stimulated SNARE complex formation and insulin granule exocytosis. These data support a novel and conserved mechanism for the dissociation of Munc18c-Syntaxin 4 complexes in a stimulus-dependent manner to facilitate the increase in Syntaxin 4-VAMP2 association and to promote vesicle/granule fusion.

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.

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.

Effect of epidermal growth factor on mouse sperm acrosome reaction induced by zona pellucida.

The effect of epidermal growth factor (EGF) on the acid-solubilized zona pellucida (ZP)-induced acrosome reaction was investigated in mouse sperm. Mouse epididymal sperm were capacitated in modified Krebs-Ringer bicarbonate buffer (m-KRB) for 120 min and further treated with acid-solubilized ZP(4 zona/microliters) for an additional 60 min to induce the acrosome reaction. The chlortetracycline fluorescence assay was used to monitor the acrosome reaction. The acrosome reacted sperm featured the acrosome reacted (AR) pattern, which demonstrates the lack of fluorescence on the head and bright midpiece. EGF caused an early increase in the AR pattern in response to the acid-solubilized ZP in a dose-dependent manner. The EGF-dependent stimulation of the ZP-induced acrosome reaction was inhibited by an inhibitor of protein tyrosine kinases, genistein or activators of Ca++ and phospholipid-dependent protein kinase (protein kinase C). Furthermore, the stimulatory effect of EGF was not attenuated when sperm were capacitated in the presence of islet-activating protein, an inactivator of inhibitory guanine nucleotide-binding regulatory protein (Gi protein). EGF stimulates the ZP-induced acrosome reaction in a manner that is independent of the Gi protein. The EGF action is regulated by protein tyrosine kinase and protein kinase C.

Nuclear Maturation of Domestic Cat Ovarian Oocytes in Vitro1

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 micrograms/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degrees C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MII); metaphase I (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p less than 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of amino acids on the absorption of trivalent chromium and its retention by regions of the rat small intestine

This study utilized the double perfusion technique to measure the effects of dietary amino acids on the absorption of trivalent chromium (Cr 3+) and its retention by regions of the rat small intestine. A nutrient-rich tissue culture medium (TCM) with Cr 3+ was perfused through the intestinal lumen as a test meal. Simultaneously, a modified Krebs-Ringer Bicarbonate buffer was perfused through the intestinal vasculature to replace the intestinal blood supply. In two experimental groups, amino acids were omitted from the test meal; one group received a buffered salt solution, the other a specially formulated TCM without amino acids. With no amino acids in the test meal, chromium transport into the vascular perfusate was less than 50% of control, while significantly more chromium (>200%) was retained by the intestinal mucosa. The retention of chromium was greatest in the proximal small intestine when amino acids were present; in their absence, the region of maximal retention shifted to the distal segment. We postulate that dietary amino acids provide ligands necessary for chromium uptake and its transport into the vasculature. The reasons for the regional specificity of the retained chromium are unknown but may reflect the active transport of ligands, such as amino acids or bile salts, that ligate chromium and other minerals.

Effect of luminal perfusion on glucose production by isolated proximal tubules

The effects of luminal perfusion on glucose production by the proximal tubule were examined by use of the technique of in vitro microperfusion with an ultramicroassay for glucose to measure the net glucose production rates in isolated mouse midproximal tubule segments. Tubules bathed in Krebs-Ringer bicarbonate (KRB) buffer containing L-glutamine and acetate and perfused with KRB buffer at a high flow rate produced glucose at a lower rate (0.12 +/- 0.02 pmol.min-1.mm-1) than unperfused segments (0.40 +/- 0.03) or segments perfused at a lower flow rate (0.24 +/- 0.03). In contrast, the estimated rates of glucose utilization were not affected by luminal perfusion. The inhibition of net fluid reabsorption by perfusion with a modified KRB buffer containing amiloride or by addition of ouabain to the bath medium raised glucose production rates to levels equaling or exceeding those observed in unperfused tubules. The inhibition of glucose production by luminal perfusion occurred in the presence of multiple substrates (i.e., glutamine, acetate, lactate, pyruvate, alanine, and valerate) or nonammoniagenic substrates (i.e., lactate and pyruvate) in the bath medium. Thus net glucose production is inhibited by luminal perfusion and the inhibitory effect is dependent on intact fluid reabsorption. The reduction in net glucose production observed with perfusion does not result from increased glucose utilization and is not dependent on the presence of specific substrates.

Insulin effect on thyroid hormone uptake in rat skeletal muscle

Previous work has indicated that thyroid hormone entry into cells includes an energy-dependent, saturable process. In this study we investigated the effect of insulin on T 3 uptake in rat skeletal muscle. Intact soleus muscles were preincubated for 30 minuted at 37°C, pH 7.4, in modified Krebs-Ringer bicarbonate buffer in the presence or absence of insulin and then for 60 minutes after adding 50 pmol/L [ 125I] T 3. The results showed a stimulatory effect of insulin that was half maximal at 33 nmol/L and maximal at 100 nmol/L. Addition of 10 μmol/L T 3 to the incubation medium completely blocked the effect, showing that the action of insulin is exerted only on the specific component of T 3 uptake. The substitution of extracellular sodium with an equimolar amount of lithium also prevented the insulin effect. [ 125I] T 4 uptake was unaffected by insulin. These results indicate that T 3 uptake in skeletal muscle is sensitive to insulin, that insulin stimulates the specific component of T 3 uptake in a dose-dependent manner, and that its action requires extracellular sodium. In contrast, T 4 uptake is insensitive to insulin action in accordance with previous results that indicated passive diffusion as its major pathway for cell entry.

Luminal secretion of ammonia in the mouse proximal tubule perfused in vitro.

A major portion of the total ammonia (tNH3 = NH3 + NH+4) produced by the isolated perfused mouse proximal tubule is secreted into the luminal fluid. To assess the role of Na+-H+ exchange in net tNH3 secretion, rates of net tNH3 secretion and tNH3 production were measured in proximal tubule segments perfused with control pH 7.4 Krebs-Ringer bicarbonate (KRB) buffer or with modified KRB buffers containing 10 mM sodium and 0.1 mM amiloride. Net tNH3 secretion was inhibited by 90% in proximal tubule segments perfused with the pH 7.4 modified KRB buffer while tNH3 production remained unaffected. The inhibition of net tNH3 secretion by perfusion with the modified KRB buffer was only partially reversed by acidifying the modified KRB luminal perfusate from 7.4 to as low as 6.2. These data indicate that the Na+-H+ exchanger facilitates a major portion of net tNH3 secretion by the proximal tubule and that luminal acidification may play only a partial role in the mechanism by which the Na+-H+ exchanger mediates net tNH3 secretion.

Meiosis in Bovine Oocytes Matured in Vitro and in Vivo

Meiosis in bovine oocytes has; been studied after maturation in vitro or in vivo. Oocytes for in vitro maturation were collected from the ovaries of slaughtered cattle without regard to the phase of the estrous cycle while in vivo maturation was studied in oocytes from gonadotrophin-stimulated heifers at times varying between 6 and 36 h after the beginning of behavioural estrus. Oocytes from slaughtered cattle were classified according to their cumulus complex and ooplasm and were cultured for 6, 12, 18, 24, 36 or 48 h in modified Krebs-Ringer bicarbonate buffer before fixation) for cytogenetic analysis. Oocytes from stimulated heifers were aspirated from follicles or flushed from the oviducts, classified according to cumulus and ooplasm, and fixed within 6 h of collection. Nuclear maturation was more rapid in vitro than in vivo. The largest proportion of oocytes reached maturity (Mil) after 12 to 18 h in culture or 30 to 36 h after the onset of behavioural estrus. Oocytes devoid of cumulus cells or showing signs of vacuolation or degeneration had virtually no capacity for nuclear maturation.

Relationship between coronary flow and adenosine release in reactive hyperemia

A linear relationship was found between coronary flow and adenosine release during the course of reactive hyperemia. Isolated guinea pig heart was perfused with a modified Krebs Ringer bicarbonate buffer containing 2.0 mM pyruvate. Hyperemia was induced with 30, 60 and 90-second coronary occlusions. The hyperemic response was divided into three consecutive 13-second intervals (I, II and III), and perfusate efflux from coronary circulation was collected during the last 10 seconds of each interval for adenosine assay using the HPLC. The data show a control flow of 3.13 ± 0.4 ml/min/g and adenosine release of 66 ± 4 pmoles/min/g. Flow increased by 99, 38 and 23% at I, II and III, respectively following 30-second occlusion, whereas adenosine release increased by 241, 132 and 91% for I, II and III. A 60-second occlusion increased the flow by 125, 64 and 34% with a simultaneous increase in the release of adenosine by 464, 155 and 133%, respectively, for I, II and III. Marked elevations in flow (165, 92 and 59%) and in adenosine release (659, 194 and 176%) for I, II and III were observed following 90-second occlusion. The linear relationship between coronary flow and adenosine release had r values of 0.84, 0.74 and 0.88 for 30, 60 and 90-second occlusions, respectively. This study quantifies the relationship between coronary flow and adenosine release during the course of reactive hyperemia. It also suggests that on a percent basis, adenosine contributes equally to the hyperemia at I, II and III.