Reduced DIDS-sensitive chloride conductance in Ae1-/- mouse erythrocytes

The resting membrane potential of the human erythrocyte is largely determined by a constitutive Cl⁻ conductance  100-fold greater than the resting cation conductance. The 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive electroneutral Cl⁻ transport mediated by the human erythroid Cl⁻/HCO₃⁻ exchanger, AE1 (SLC4A1, band 3) is > 10,000-fold greater than can be accounted for by the Cl⁻ conductance of the red cell. The molecular identities of conductive anion pathways across the red cell membrane remain poorly defined. We have examined red cell Cl⁻ conductance in the Ae1^−/− mouse as a genetic test of the hypothesis that Ae1 mediates DIDS-sensitive Cl⁻ conductance in mouse red cells. We report here that wildtype mouse red cell membrane potential resembles that of human red cells in the predominance of its Cl⁻ conductance. We show with four technical approaches that the DIDS-sensitive component of erythroid Cl⁻ conductance is reduced or absent from Ae1^−/− red cells. These results are consistent with the hypothesis that the Ae1 anion exchanger polypeptide can operate infrequently in a conductive mode. However, the fragile red cell membrane of the Ae1^−/− mouse red cell exhibits reduced abundance or loss of multiple polypeptides. Thus, loss of one or more distinct, DIDS-sensitive anion channel polypeptide(s) from the Ae1^−/− red cell membrane cannot be ruled out as an explanation for the reduced DIDS-sensitive anion conductance.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Attenuated expression of SECIS binding protein 2 causes loss of telomeric reserve without affecting telomerase

The family of selenoproteins have a broad range of functions, including protection against oxidative damage. Previous studies have shown that elevated levels of oxidative damage can induce accelerated loss of telomeric DNA during proliferation of mammalian cells. The incorporation of selenocysteine (Sec) into proteins in mammalian cells requires the Sec insertion sequence (SECIS) binding protein 2 (SBP2). Thus in the present study we have assessed the effect of knocking down the expression of SBP2 on telomere length. Following knock-down of SBP2 expression in two different human cell lines, the MSTO mesothelioma cell line (5 Kb average telomere length) and SY5Y neuroblastoma cell line (4.2 Kb average telomere length), we observed a significant reduction (−0.6 to −1.1 Kb; P  0.01) in telomere length as compared to control cells. This reduction in telomere length was independent of affects on telomerase, since both telomerase activity levels and Tert mRNA expression levels were not altered by knock-down of SBP2 expression. Furthermore, telomeres were particularly sensitive to S1 nuclease digestion following SBP2 knock-down, indicating an increased frequency of oxidative damage-induced lesions in the telomeric DNA in these cells. Together, these observations imply that selenoproteins may help protect telomeric reserve in mammalian cells.     

Involvement of Src in L-type Ca channel depression induced by macrophage migration inhibitory factor in atrial myocytes

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca²⁺ currents (I_Ca,L) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of I_Ca,L in human atrial myocytes and in HL-1 cells. Basal I_Ca,L was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming α_1C subunit of L-type Ca²⁺ channel (LCC α_1C) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y⁴¹⁶) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress I_Ca,L amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak I_Ca,L in HL-1 cells by  69% and  83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of I_Ca,L and down-regulated LCC α_1C subunit levels. The depression of I_Ca,L and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing I_Ca,L amplitudes through impairment of channel function, down-regulation of LCC α_1C subunit levels, and the activation of c-Src kinases in atrial myocytes.     

Thyrotropin receptor messenger ribonucleic acid is expressed in most brown and white adipose tissues in the guinea pig.

High affinity TSH binding has been reported in a variety of tissues other than the thyroid, most commonly in adipocytes and lymphocytes. This extrathyroidal binding of TSH has been documented most carefully in the guinea pig epididymal fat pad, where it has been postulated to be due to the presence of the TSH receptor (TSH-R). Extrathyroidal TSH-R expression has also been theorized to account for the associated dermopathy and ophthalmopathy seen in some patients with Graves' disease. We have isolated a cDNA encoding a fragment of the guinea pig TSH-R and have used this as a probe to study the distribution of TSH-R mRNA in the guinea pig. We show here that TSH-R mRNA is expressed in most white adipose tissues and in all brown adipose tissues tested. However, no expression was detectable by Northern analysis or in most polymerase chain reaction experiments using guinea pig retroorbital tissues, bringing into question the proposed role of the TSH-R as an autoantigen in autoimmune ophthalmopathy. The presence of significant amounts of TSH-R mRNA in most adipose tissues suggests a more important role for TSH in lipolysis and thermogenesis than previously thought.     

Effects of mild calorie restriction and high-intensity interval walking in middle-aged and older overweight Japanese

We investigated whether a combination of mild calorie restriction (MCR) and high-intensity interval walking (HIW) improved physical fitness more than HIW alone in middle-aged and older overweight Japanese (40–69 years old, BMI  23.6 kg/m²). Forty-seven women and 16 men were divided into MCR + HIW and HIW groups. All subjects performed HIW: 5 sets of 3-min low-intensity walking (40% peak aerobic capacity for walking, VO_2peak) and 3-min high-intensity walking (70% VO_2peak) per day, 4 days per week, for 16 weeks while energy expenditure was monitored with a tri-axial accelerometer. The MCR + HIW group consumed meal replacement formula (240 kcal): a mixture of low-carbohydrates and -fat and high-protein, for either lunch or dinner everyday and therefore, had 87% of the energy intake of the HIW group during the intervention period. Although the HIW group showed improvements in BMI, blood pressure, and several blood chemicals, the MCR + HIW group had greater improvement. Moreover, the medical expenditure for the 6 months including the intervention period was 59% lower in the MCR + HIW group than in the HIW group. Our strategy of a short-term combination of MCR and HIW may thus prevent lifestyle-associated diseases and improve health in middle-aged and older overweight Japanese.     

Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.     

Kinetic evidence for modulation by glycophorin A of a conformational equilibrium between two states of band 3 (SLC4A1) bound reversibly by the competitive inhibitor DIDS

Recent evidence has suggested that erythrocytes naturally deficient in glycophorin A (GPA) have a reduced V_max for monovalent anion exchange. Unanswered is whether miss-folding of band 3 during biosynthesis, or the absence of GPA modulation of properly folded band 3 is responsible. Here, I determine the effect of selective depletion of GPA on the kinetics of reversible binding of the competitive transport inhibitor DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) to properly folded band 3. Reversible binding of DIDS follows biphasic kinetics: a fast phase {DIDS + band 3  (DIDS − band 3), k₁, k_− 1} and a slower phase {(DIDS − band 3)  (DIDS − band 3), k₂, k_− 2}. Selective depletion of GPA was accomplished by pretreating membranes with Triton X-100, over a range where erythrocyte hemolysis is inhibited by the detergent (0% to 0.03%, v/v). Pretreatment with sublytic Triton X-100: (a) virtually completely depleted GPA, (b) did not deplete membrane-bound band 3, and (c) slowed the overall rate of reversible binding of DIDS to band 3. Data analysis and model simulation studies indicated that the decrease in the rate of binding of DIDS was due exclusively to a decrease in k_− 2, with no change in the initial rate of binding. Thus, depletion of GPA does not alter the native conformation of band 3 at the DIDS binding site, but rather modulates a conformational equilibrium between two states of the binary complex formed by the competitive inhibitor DIDS, reversibly bound to properly folded band 3.     

Basal lipolysis, not the degree of insulin resistance, differentiates large from small isolated adipocytes in high-fat fed mice

Aims/hypothesis  Adipocytes in obesity are characterised by increased cell size and insulin resistance compared with adipocytes isolated from lean patients. However, it is not clear at present whether hypertrophy actually does drive adipocyte insulin resistance. Thus, the aim of the present study was to metabolically characterise small and large adipocytes isolated from epididymal fat pads of mice fed a high-fat diet (HFD). Methods  C57BL/6J mice were fed normal chow or HFD for 8 weeks. Adipocytes from epididymal fat pads were isolated by collagenase digestion and, in HFD-fed mice, separated into two fractions according to their size by filtration through a nylon mesh. Viability was assessed by lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium assays. Basal and insulin-stimulated d-[U-¹⁴C]glucose incorporation and lipolysis were measured. Protein levels and mRNA expression were determined by western blot and real-time RT-PCR, respectively. Results  Insulin-stimulated D-[U-¹⁴C]glucose incorporation into adipocytes isolated from HFD-fed mice was reduced by 50% compared with adipocytes from chow-fed mice. However, it was similar between small (average diameter 60.9 ± 3.1 μm) and large (average diameter 83.0 ± 6.6 μm) adipocytes. Similarly, insulin-stimulated phosphorylation of protein kinase B and AS160 were reduced to the same extent in small and large adipocytes isolated from HFD-mice. In addition, insulin failed to inhibit lipolysis in both adipocyte fractions, whereas it decreased lipolysis by 30% in adipocytes of chow-fed mice. In contrast, large and small adipocytes differed in basal lipolysis rate, which was twofold higher in the larger cells. The latter finding was associated with higher mRNA expression levels of Atgl (also known as Pnpla2) and Hsl (also known as Lipe) in larger adipocytes. Viability was not different between small and large adipocytes. Conclusions/interpretation  Rate of basal lipolysis but not insulin responsiveness is different between small and large adipocytes isolated from epididymal fat pads of HFD-fed mice.     

Thyroid-stimulating hormone stimulates lipolysis in adipocytes in culture and raises serum free fatty acid levels in vivo

Thyroid-stimulating hormone (TSH) stimulates adipocyte lipolysis, but signal transduction pathways activated by TSH for this response have not been directly studied. Using differentiated 3T3-L1 adipocytes as well as primary human adipocytes, we characterized the lipolytic action of TSH with dose-response and time-course studies, and compared it with isoproterenol. Thyroid-stimulating hormone stimulated phosphorylation of perilipin and hormone-sensitive lipase (HSL). Inhibition of protein kinase A with H89 blocked TSH-stimulated lipolysis as well as phosphorylation of perilipin and HSL. Thyroid-stimulating hormone stimulated lipolysis in vivo, as indicated by an elevation in serum free fatty acid (FFA) levels after recombinant human TSH administration to thyroidectomized patients (42% increase, n = 19, P < .05). For patients with a body mass index less than 30 kg/m², the TSH-induced increase in serum FFA levels was 53% (n = 11, P < .05), whereas levels in patients with a body mass index of at least 30 kg/m² (n = 8) did not change after TSH treatment. In summary, TSH stimulates lipolysis and phosphorylation of perilipin and HSL in a protein kinase A-dependent manner in differentiated adipocytes in culture and raises serum FFA levels in vivo.     

Some cellular characteristics of the epididymal adipose tissue in lean and obese-hyperglycaemic mice

Summary 1. The nitrogen content, total water content,in vivo ¹²⁵l-albumin space, the number and size of adipocytes per unit wet weight of epididymal fat pad and the plasma volume have been studied in lean and obese mice, and in obese mice on chronic restriction of their food intake. — 2. There were fewer, larger adipocytes per unit wet weight of tissue (in young obese mice) with a proportional decrease in the vascular space and water content, and the same nitrogen content as had epididymal adipose tissue from lean mice. — 3. There was a reduction in the nitrogen content of epididymal adipose tissue from obese mice on a restricted diet. — 4. These findings are discussed in relation to the reduced glucose metabolism and lipolysis that are apparent when metabolic data are expressed per unit wet weight.     

Mechanistic insights into folic acid-dependent vascular protection: Dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: A novel HPLC-based fluorescent assay for DHFR activity

Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious. Expression of dihydrofolate reductase (DHFR) was markedly increased by folic acid (FA, 50 μmol/L, 24 h) treatment in endothelial cells. Tetrahydrofolate (THF) is formed after incubation of purified DHFR or cellular extracts with 50 μmol/L of substrate dihydrofolic acid. THF could then be detected and quantified by high performance liquid chromatography (HPLC) with a fluorescent detector (295/365 nm). Using this novel and sensitive assay, we found that DHFR activity was significantly increased by FA. Furthermore, FA improved redox status of Ang II treated cells by increasing H₄B and NO bioavailability while decreasing superoxide (O₂⁻) production. It however failed to restore NO levels in DHFR siRNA-transfected or methotrexate pre-treated cells, implicating a specific and intermediate role of DHFR. In mice orally administrated with FA (15 mg/kg/day, 16 days), endothelial upregulation of DHFR expression and activity occurred in correspondence to improved NO and H₄B bioavailability, and this was highly effective in reducing Ang II infusion (0.7 mg/kg/day, 14 days)-stimulated aortic O₂⁻ production. 5′-methyltetrahydrofolate (5′-MTHF) levels, GTPCH1 expression and activity remained unchanged in response to FA or Ang II treatment in vitro and in vivo. FA supplementation improves endothelial NO bioavailability via upregulation of DHFR expression and activity, and protects endothelial cells from Ang II-provoked oxidant stress both in vitro and in vivo. These observations likely represent a novel mechanism (intermediate role of DHFR) whereby FA induces vascular protection.     

Abdominal subcutaneous and omental adipocyte morphology and its relation to gene expression,lipolysis and adipocytokine levels in women

Objective

We tested the hypothesis that women with adipocyte hypertrophy in either omental (OM) or subcutaneous (SC) adipose tissue are characterized by alterations in adipocyte lipolysis and adipose tissue expression of genes coding for proteins involved in adipocyte metabolism or inflammation, independent of overall adiposity and fat distribution.

Methods

OM and SC fat samples were obtained surgically in 44 women (age: 47.1 ± 5.0 years, BMI: 27.7 ± 5.3 kg/m²). In a given depot, women with larger adipocytes than predicted by the regression of adipocyte size vs. total and regional adiposity measurements were considered as having adipocyte hypertrophy, whereas women with smaller adipocytes than predicted were considered as having adipocyte hyperplasia.

Results

Women with OM adipocyte hypertrophy had significantly lower SC GLUT4 mRNA abundance (p ≤ 0.05), higher SC CEBPB mRNA expression (p ≤ 0.05) as well as higher mRNA expression of OM PLIN (p ≤ 0.05), CD68 (p ≤ 0.10), CD14 (p ≤ 0.10), CD31 (p ≤ 0.05) and vWF (p ≤ 0.05) compared to women with OM adipocyte hyperplasia. OM adipocyte isoproterenol- (10^− 10 to 10^− 5 mol/L), forskolin- (10^− 5 mol/L) and dibutyryl cAMP- (10^− 3 mol/L) stimulated lipolysis was higher in women with hypertrophic OM adipocytes (p ≤ 0.05, for all). Women with SC adipocyte hypertrophy had lower SC mRNA expression of GLUT4 (p ≤ 0.10), higher SC mRNA expression of CEBPB (p ≤ 0.05), lower plasma adiponectin concentrations (p ≤ 0.05) and higher SC adipocyte isoproterenol- (10^− 9 to 10^− 5 mol/L) stimulated lipolysis (p ≤ 0.05) compared to women with SC adipocyte hyperplasia.

Conclusion

Hypertrophic adipocytes in both fat compartments are characterized by alterations in adipocyte lipolysis and adipose tissue expression of genes coding for proteins involved in adipocyte metabolism or inflammation.     

Effects of PAF on cardiac function and eicosanoid release in the isolated perfused rat heart: comparison between normotensive and spontaneously hypertensive rats

The aim of this study was (a) in isolated perfused rat heart to characterize the effects of platelet-activating factor (PAF) on coronary flow, ventricular contractility, and eicosanoid release and (b) to determine whether PAF effects are altered in hearts from spontaneously hypertensive rats (SHR). PAF (10^–10–10^–7 mol) dose-dependently decreased coronary flow and ventricular contractility; concomitantly, coronary effluent concentrations of thromboxane (TX)B₂ and prostaglandin F₂ (PGF₂) were elevated but not those of prostacyclin. The PAF receptor antagonist WEB 2086 (10^–7–10^–5 mol/l) concentration-dependently antagonized these PAF effects. In addition, the cyclo-oxygenase inhibitor indomethacin (5×10^–5 mol/l) prevented PAF (10^–9–10^–7 mol) induced eicosanoid release; in the presence of indomethacin PAF caused coronary constriction and ventricular depression only at the highest dose (10^–7 mol) but had no effect at 10^–9 or 10^–8 mol. Moreover, the TXA₂ antagonist SQ 29,548 (10^–6 mol/l) completely inhibited 10^–8 mol PAF induced ventricular depression but did not effect coronary constriction. In SHR PAF (10^–9–10^–7 mol) evoked decreases in coronary flow and ventricular contractility did not differ from those in normotensive Wistar-Kyoto rats while PAF induced TXA₂ and PGF₂ release was markedly enhanced. In addition, decreases in coronary flow and ventricular contractility induced by the TXA₂ agonist U 46619 (10^–7 mol/l) were markedly depressed in SHR. We conclude that in isolated perfused rat heart PAF causes coronary constriction and depression of ventricular function mainly indirectly through released TXA₂ and/or PGF₂. Moreover, the fact that in SHR the PAF effects on coronary flow and ventricular function are not altered despite markedly enhanced TXA₂ and PGF₂ release supports the view that in the SHR the receptors mediating TXA₂ and/or PGF₂ effects are desensitized.     

Vaccination of European badgers (Meles meles) with BCG by the subcutaneous and mucosal routes induces protective immunity against endobronchial challenge with Mycobacterium bovis

Mycobacterium bovis is endemic in badger (Meles meles) populations of Ireland and the United Kingdom and infected badgers are a potential source of infection for cattle. In domestic livestock tuberculosis causes economic losses from lost production and the costs associated with eradication programmes, and in addition there is a risk of zoonotic infection. Whereas culling is currently used to control tuberculous badger populations in Ireland, vaccination, if it were available, would be preferred. A study was undertaken to examine the protective responses of badgers vaccinated either by the subcutaneous or mucosal (intranasal and conjunctival) routes with bacille Calmette-Guérin (BCG), when challenged with M. bovis by the endobronchial route. Three groups of badgers were used. The first group (n = 4) was vaccinated with 5 × 10⁵ colony forming units (cfu) of BCG by subcutaneous injection. In the second group (n = 5) badgers were vaccinated via the mucosal route by instilling 1.0 × 10⁵ cfu into each conjunctival sac and spraying 1.0 × 10⁵ cfu into each nostril (final vaccine dose of 4 × 10⁵ cfu). The control (n = 5) badgers served as a non-vaccinated group. Twelve weeks post-vaccination all badgers in the three groups were challenged with 10⁴ cfu of M. bovis by endobronchial inoculation. At 12 weeks post-infection all badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. Gross and histological lesions of tuberculosis were seen in all challenged badgers and M. bovis was recovered from all challenged badgers. However, across six of the eight parameters used to measure disease severity, the infection in the vaccinated badgers was significantly less severe than in the control group. The BCG vaccine induced a significant protective effect in the badgers and the protective immunity was generated by subcutaneous and mucosal vaccination.     

Role of carbonic anhydrase in basal and stimulated bicarbonate secretion by the guinea pig duodenum

The role of carbonic anhydrase in the process of proximal duodenal mucosal bicarbonate secretion was investigated in the guinea pig. In a series of experimentsin vivo, the duodenum was perfused with 24 mmol/liter NaHCO₃ solution (+ NaCl for isotonicity) to ensure that active duodenal HCO ₃ ^– secretion against a concentration gradient was measured. Acetazolamide (80 mg/kg) was infused intravenously to examine the role of carbonic anhydrase on basal and agonist-stimulated HCO ₃ ^– secretion. Acetazolamide abolished basal HCO ₃ ^– secretion and significantly decreased HCO ₃ ^– secretion after stimulation with dibutyryl 5-cyclic adenosine monophosphate (dBcAMP, 10^–5 mol/kg), dibutyryl 5-cyclic guanosine monophosphate (dBcGMP, 10^–5 mol/kg), prostaglandin E₂ (PGE₂, 10^–6 mol/kg), PGF₂ (10^–6 mol/kg), tetradecanoyl-phorbol-acetate (TPA, 10^–7 mol/kg), glucagon (10^–7 mol/kg), vasoactive intestinal polypeptide (VIP, 10^–8 mol/kg), and carbachol (10^–8 mol/kg). Utilizing a fluorescence technique, we could detect the enzyme carbonic anhydrase in equal amounts in villous and crypt cells of the proximal duodenal epithelium; no activity was demonstrated in tissues pretreated with acetazolamide. In conclusion, carbonic anhydrase is required for both basal and stimulated duodenal HCO ₃ ^– secretion.This research was supported by the German-Israel Foundation for Scientific Research and Development, Grant Number I-78-054.2/88.     

A study of the effects of human blood derivatives and individual growth factors on [3H]thymidine uptake in bovine retinal pericytes and endothelial cells

Pericytes disappear early, selectively and specifically from retinal capillaries in diabetic microangiopathy, but little is known of their growth and turnover in health and disease. We have studied the effects of human blood derivatives and of a panel of individual growth factors on [³H]thymidine incorporation in bovine retinal pericytes and endothelial cells. Human serum and platelet-rich plasma stimulated incorporation of the nucleotide in a dose-dependent manner in both cell types, and did so more potently than platelet-free plasma. Consistent and significant stimulation of DNA synthesis in pericytes was observed with basic fibroblast growth factor (ED₅₀= 1.8×10^–13 mol/l), acidic fibroblast growth factor (7.4× 10^–12 mol/l), insulin-like growth factor 1 (8.6×10^–10 mol/l), insulin (158 U/ml) and endothelin-1 (6.1×10^–10 mol/l). Transforming growth factor ₁ inhibited DNA synthesis (ID₅₀=3.6×10^–10 mol/l) and so did heparin (1.4×10^–6 mol/l) and low molecular weight heparin (2.9×10^–6 mol/l). Retinal endothelial cells were stimulated by basic fibroblast growth factor (3.2×10^–13 mol/l) and acidic fibroblast growth factor (1.3×10^–9 mol/l), and inhibited by transforming growth factor ₁, (1.6×10^–12 mol/l). Neither cell type was stimulated by platelet-derived growth factor (A+B chain heterodimer), epidermal growth factor, growth hormone, or nerve growth factor (7S complex). The characteristics and active concentrations of the above growth factors suggest that none is solely responsible for the pericyte mitogenic activity of platelets, serum or plasma. Some, though, may play a role in the regulation of pericyte turnover through paracrine mechanisms which should be further investigated.     

Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na/HCO3 cotransport

The sarcolemmal Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pH_i was measured in single cells in a medium with HCO₃⁻ using the fluorescent pH indicator BCECF. The NH₄⁺ pulse method was used to induce an intracellular acid load and the acid efflux (J_H) in the presence of the Na⁺/H⁺ exchanger blocker HOE642 (10 μM) was calculated as indicator of NBC activity. The following J_H data are presented at pH_i of 6.8 ( and ^# indicate p < 0.05 after ANOVA vs. control and Ang II, respectively). The basal J_H (1.03 ± 0.12 mM/min, n = 11) was significantly increased in the presence of 100 nM Ang II (1.70 ± 0.15 mM/min, n = 8). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80 ± 0.08 mM/min, n = 11^#), 300 μM apocynin (NADPH oxidase blocker; 0.80 ± 0.13 mM/min, n = 6^#), 500 μM 5-hydroxidecanoate (mitochondrial ATP dependent K⁺ channel, mK_ATP, blocker; 0.97 ± 0.21 mM/min, n = 9^#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 μM; 0.56 ± 0.18 mM/min, n = 6^#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mK_ATP opening and mitochondrial ROS production (“ROS-induced ROS-release mechanism”). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.