Metabolic Changes Induced by Cold Stress in Rat Liver Mitochondria

The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force-flux relationships showed net variation in flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H+ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The flux control coefficient of the H+ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn2+, Fe3+, ATP, and ADP/O ratios; the content of cytochromes c + c1 and b oscillated. The contents of Ca2+, Na+, Pi, and cytochromes a + a3 were not affected, whereas matrix ADP, AMP, K+, and Mg2+ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

Interaction Between Adrenergic and Peptide Stimulation in the Rat Pineal: Pituitary Adenylate Cyclase-Activating Peptide

Abstract: The 27 amino acid peptide, pituitary adenylate cyclase-activating polypeptide (PACAP-27), and its 38 amino acid analogue, PACAP-38, stimulate serotonin- N -acetyltransferase (NAT) activity and N -acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP-38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N-terminal histidine C-terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP-38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP-38 reduces the magnitude of subsequent stimulation with PACAP-38 or forskolin. Concurrent stimulation of α-receptors or treatment with active phorbol ester augments rat pineal response to PACAP-38 stimulation just as it increases the response to VIP, PHI, and β-receptor stimulation. Pineals from newborn rats respond to PACAP-38 with an increase in NAT activity and the increase is augmented by concomitant α₁-adrenergic stimulation. The putative PACAP inhibitor PACAP (6–38) and the putative VIP inhibitor (Ac-Tyr, d -Phe)-GRF 1–29 amide, in 100–1,000-fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.     

Age-Associated Changes in Pineal Adrenergic Receptors and Melatonin Synthesizing Enzymes in the Wistar Rat

The nocturnal stimulation of pineal melatonin synthesis and elevation of serum melatonin is known to be reduced in old age in several species. In Wistar rats the capacity of the beta-adrenoceptor to develop supersensitivity (increase in Bmax) during the light period of the diurnal light/dark cycle is lost during maturation (3-6 months) rather than old age. Further, the present study shows that neither the alpha 1- nor beta-adrenoceptor density of the pineal declines as rats age. Pineal hydroxyindole-O-methyltransferase activity does fall (17-55%) in rats after 18 months of age, but nocturnal pineal arylalkylamine N-acetyltransferase activity is not significantly altered. Thus, from examination of these parameters across the life span of the rat, it seems likely that the reported reduction in serum melatonin in old animals is related to a reduced capacity of the pineal to synthesize melatonin, rather than an altered responsiveness of the gland to neural stimulation.     

Adrenergic Stimulation of Cyclic GMP Formation Requires NO-Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α₁-adrenergic receptors. The effects of N -monomethyl- l -arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α₁-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l -arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.     

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.     

Induction of Prostanoid Biosynthesis by Bacterial Lipopolysaccharide and Isoproterenol in Rat Microglial Cultures

Abstract: We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E₂, prostaglandin D₂, and thromboxane A₂ was low in basal conditions and increased in a dose- and time-dependent way upon lipopolysaccharide treatment (1–100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100-fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B₄; although lipopolysaccharide failed to stimulate leukotriene B₄ release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [³H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of β-adrenergic receptor activation. The β-agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide. The drug appeared to act mainly through the inducible cyclooxygenase; because it did not stimulate arachidonic acid release, it enhanced the lipopolysaccharide-evoked prostanoid production observed after aspirin pretreatment and induced de novo synthesis of cyclooxygenase detectable by western blot analysis. We suggest that during cerebral inflammatory processes microglia can contribute to the establishment of high prostanoid levels, which can be further elevated by β-adrenergic activation.     

Intermitochondrial Contacts in the Mitochondriome of Cardiomyocytes under Normal Conditions,in Response to Physiological Stress,and in Pathology

Intermitochondrial contacts are universal and indispensable structures participating in the formation of mitochondriome in cardiomyocytes of invertebrate and vertebrate animals; they participate in the maintenance of the energy pool required for rhythmic contractions of the heart. At the ultrastructural level, a direct relationship is demonstrated between the functional load on the heart and the number of intermitochondrial contacts in the cardiomyocytes of swifts and rats: there was a twofold increase in the number of these contacts associated in actively flying adult birds as compared with chicks; a similar effect was observed in response to pressure overload associated with aortal stenosis; hypokinesia and hypoxia led to a twofold or even threefold decrease in their number.     

The Compensating Function of Submandibular Salivary Glands in Diabetes and Their Possible Stimulation by Isoproterenol

A brief review is presented of experimental studies conducted over several years (1970s–1990s) on the incretory potential of salivary glands, specifically on their role in the maintenance of carbohydrate homeostasis; a review is also presented of data on the stimulation of this potential by isoproterenol, a -adrenergic agonist.     

Ultrastructural and Functional Changes in Liver Mitochondria in a Rat Model of Type I Diabetes Mellitus

Biophysics - Abstract—The characteristics of the ultrastructure and functioning of mitochondria in the liver of Sprague Dawley rats in the experimental model of type I diabetes mellitus have...     

Infection by Trypanosoma cruzi Enhances Anion Conductance in Rat Neonatal Ventricular Cardiomyocytes

Recent studies on malaria-infected erythrocytes have shown increased anion channel activity in the host cell membrane, increasing the exchange of solutes between the cytoplasm and exterior. In the present work, we addressed the question of whether another intracellular protozoan parasite, Trypanosoma cruzi, alters membrane transport systems in the host cardiac cell. Neonatal rat cardiomyocytes were cultured and infected with T. cruzi in vitro. Ion currents were measured by patch-clamp technique in the whole-cell configuration. Two small-magnitude instantaneous anion currents, outward- and inward-rectifying, were recorded in all noninfected cardiomyocytes. In addition, ~10% of cardiomyocytes expressed a large anion-preferable, time-dependent current activated at positive membrane potentials. Hypotonic (230?mOsm) treatment resulted in the disappearance of the time-dependent current but provoked a dramatic increase of the instantaneous outward-rectifying one. Both instantaneous currents were suppressed by intracellular Mg(2+). T. cruzi infection did not provoke new anion currents in the host cells but caused an increase of the density of intrinsic swelling-activated outward current, up to twice in heavily infected cells. The occurrence of a time-dependent current dramatically increased in infected cells in the presence of Mg(2+) in the intracellular solution, from ~10 to ~80%, without a significant change of the current density. Our findings represent one further, besides the known Plasmodium falciparum, example of an intracellular parasite which upregulates the anionic currents expressed in the host cell.     

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.     

Growth Hormone Plus Resistance Exercise Attenuate Structural Changes in Rat Myotendinous Junctions Resulting from Chronic Unloading

Myotendinous junctions (MTJs) are specialized sites on the muscle surface where forces generated by myofibrils are transmitted across the sarcolemma to the extracellular matrix. At the ultrastructural level, the interface between the sarcolemma and extracellular matrix is highly folded and interdigitated at these junctions. In this study, the effect of exercise and growth hormone (GH) treatments on the changes in MTJ structure that occur during muscle unloading, has been analyzed. Twenty hypophysectomized rats were assigned randomly to one of five groups: ambulatory control, hindlimb unloaded, hindlimb unloaded plus exercise (3 daily bouts of 10 climbs up a ladder with 50% body wt attached to the tail), hindlimb unloaded plus GH (2 daily injections of 1 mg/kg body wt, i.p.), and hindlimb unloaded plus exercise plus GH. MTJs of the plantaris muscle were analyzed by electron microscopy and the contact between muscle and tendon was evaluated using an IL/B ratio, where B is the base and IL is the interface length of MTJ’s digit-like processes. After 10 days of unloading, the mean IL/B ratio was significantly lower in unloaded (3.92), unloaded plus exercise (4.18), and unloaded plus GH (5.25) groups than in the ambulatory control (6.39) group. On the opposite, the mean IL/B ratio in the group treated with both exercise and GH (7.3) was similar to control. These findings indicate that the interaction between exercise and GH treatments attenuates the changes in MTJ structure that result from chronic unloading and thus can be used as a countermeasure to these adaptations.Key words: Myotendinous junction, ultrastructure, exercise, growth hormone, atrophy, disuse, unloading     

Eating or Drinking Elicited by Direct Adrenergic or Cholinergic Stimulation of Hypothalamus

A double cannula system, allowing repeated stimulation of central structures with crystalline chemicals, was developed. This technique was employed to study the effects of adrenergic and cholinergic stimulation of the lateral hypothalamus of rats. Drug-specific effects on the feeding and drinking mechanisms, respectively, were observed.     

Mechanical Stimulation of Gap Junctions in Bone Osteocytes is Mediated by Prostaglandin E2

Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factors) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin E₂ (PGE₂) release and increase cyclooxygenase 2 (COX-2) expression. When PGE₂ was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE₂. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.     

Character of Viscero- and Somatomotor Interactions in Rat Pups at Changes of the Level of Activity of Adrenergic Structures

In the 5–10-day old rat pups, in the course of simultaneous recording of ECG, respiration rate (RR), and spontaneous periodic motor activity (SPMA) there were determined parameters of the studied processes and their correlations at changes of levels of activity of adrenergic structures. As adrenoactive agents, the sympathomimetic amphetamine, - and -adrenolytics phentolamine and propranolol, as well as the dopamine receptor blocker haloperidol were used. It has been established that injection of amphetamine significantly increases intensity of the decasecond rhythm in the activity pattern of excitable structures. However, the action both on the - and -adrenoreceptors and on dopamine receptors produces no significant changes of this rhythm parameters. Based on this, it can be suggested that an increase of the decasecond rhythm is due to the ability of amphetamine to inhibit the pentose phosphate cycle activity. Administration of adrenolytics decreases RR and heart rate (HR). Amphetamine restores them by a simultaneous increase of pathological disturbances of the respiration rhythm, which appear at injections of sympatholytics. Blockade of dopamine receptors leads to replacement of motor activity fires by jerks following in the rhythm close to the previous one. As a rule, at the blockade of -adrenoreceptors the SPMA pattern is deprived of obvious rhythmic components, whereas at the blockade of -adrenoreceptors, they are preserved. Analysis of the obtained data indicates that the respiration frequency modulation that increases at action on adrenoreactive structures correlates with the motor activity changes. However, there are several peculiarities due to action on various types of adrenoreceptors. Of the leading significance in the normal intersystem interaction is preservation of balance between individual adrenergic structures.     

Changes in Cellular Distribution of Connexins 32 and 26 during Formation of Gap Junctions in Primary Cultures of Rat Hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sectionsin vivo.Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.     

大鼠肝线粒体中的脂肪酸分析

用双 2 乙基己基酚酞酸酯 (DEHP)诱导大鼠肝过氧化物酶体增殖 ,然后用蔗糖密度梯度离心法分离大鼠肝线粒体 ,用毛细管气相色谱法测定肝线粒体中的脂肪酸含量。测定结果 :所测 1 4种脂肪酸的总量 ,青年正常组大于青年诱导组 (P <0 .0 1 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。不饱和脂肪酸与脂肪酸总量的比例 ,老年诱导组大于老年正常组 (P <0 .0 5 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。长链脂肪酸与脂肪酸总量的比例 ,老年正常组小于老年诱导组 (P <0 .0 5 )。结果表明 ,用DEHP诱导大鼠肝过氧化物酶体增值 ,影响肝线粒体脂肪酸正常代谢 ,使线粒体膜结构发生变化 ,这种变化 ,青年鼠与老年鼠不同