Hydroxyurea enhances 3′-azido-3′-deoxythymidine (AZT) cytotoxicity in human chronic myeloid leukemia models*

Abstract: In this report we have evaluated the cytotoxic activity of 3′-azido-3′-deoxythymidine (AZT) used in combination with hydroxyurea (HU), an agent which disrupts de novo thymidylate synthesis. In 2 chronic myeloid leukemia (CML) cell lines, K.562 and RWLeu4, the IC50 of AZT was 8 (μmol/l and 28 μmol/l respectively, after a 5-day exposure, and the IC50 of HU was 80 μmol/l and 70 μmol/l respectively. In the presence of various concentrations of HU (1 μmol/l-100 μmol/l) the IC50 of AZT in both cell lines was significantly reduced and subsequent isobologram analysis revealed synergistic activity. Similarly, analysis of [3H]AZT incorporation into the DNA fraction of these cells indicated that exposure to AZT + HU resulted in an increased incorporation of AZT into DNA when compared to incubation in AZT alone. Biochemically, this effect appeared to be related to a decrease in dTTP pools caused by HU. The combination AZT + HU has also been demonstrated to exert a synergistic effect in inhibiting colony growth of bone marrow granulocyte-macrophage progenitors (CFU-GM) from patients affected by Ph1⁺ CML in chronic phase. These results are promising in view of a possible in vivo utilization of this drug combination.     

Hydroxyurea exerts bi-modal dose-dependent effects on erythropoiesis in human cultured erythroid cells via distinct pathways

Hydroxyurea (HU) has been shown to increase the proportion of fetal haemoglobin (HbF) in most sickle cell patients. A low-dosage regimen increased total haemoglobin (Hb) levels in some thalassaemia intermedia patients by preferentially increasing beta-globin biosynthesis. To further characterize these apparent dose-dependent effects of HU, we examined erythroid cells exposed to HU (5-100 micro mol/l) in two-phase liquid culture. Low doses (from 5 to 25 micro mol/l) increased Hb levels by up to 2.7-fold, and a high dose (100 micro mol/l) increased Hb levels when added at d 3-6 of phase II, with no significant changes in response to HU during the late stage of phase II culture (> or = 9 d). HU exposure during d 0-3 of phase II culture increased the number of erythroid colonies to a maximum of fivefold at 5 micro mol/l HU. GATA-1 mRNA was downregulated at a high dose and GATA-2 was dose dependently upregulated over a lower dosage range. Treatment with 100 micro mol/l HU dramatically upregulated the death receptor DR-5, caspase 3, as determined by cDNA microarray analysis. In contrast, 10 micro mol/l HU modestly upregulated mRNA levels of the early growth response gene. Our results suggest that HU exerts concentration-dependent effects on HbF production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms.     

Metformin potentiates the antigluconeogenic action of insulin

The interaction of metformin and insulin in the control of hepatic gluconeogenesis was examined in isolated hepatocytes from 48h starved rats using lactate (10(-2) mol/l) with pyruvate (10(-3) mol/l) as substrate. During 1 h incubations in the absence of added insulin, 10(-2) and 10(-3) mol/l metformin reduced gluconeogenesis by 65% and 59% respectively, but lower concentrations of metformin were not effective. Insulin alone (10(-6)-10(-8) mol/l) reduced (37%-16%) gluconeogenesis. The effect of insulin was enhanced (further reductions of 11%-24%) by 5 x 10(-4) mol/l metformin, although this concentration of metformin was not effective in the absence of insulin. At lower insulin concentrations (10(-9) and 10(-10) mol/l) which did not significantly affect gluconeogenesis alone, 5 x 10(-4) mol/l metformin decreased gluconeogenesis (32% and 28% respectively). At 10(-10) mol/l insulin, metformin concentrations of 10(-3)-10(-7) mol/l decreased gluconeogenesis by 60-20%. The results suggest that therapeutic concentrations of metformin may act synergistically with physiological concentrations of insulin to suppress hepatic gluconeogenesis.     

Involvement of prostaglandins in the inhibition of growth hormone production in cultured pituitary cells by insulin.

The effect of insulin was tested on the rate of synthesis and release of growth hormone in cultured rat anterior pituitary cells. Concentrations of insulin between 10(-9) and 10(-7) mol/l (6--600 ng/ml or 0.15--15 mu./ml) inhibited synthesis of growth hormone; 10(-8) mol insulin/l was most effective. The effect was observed after a time-lag of at least 1 h. Insulin at concentrations between 3 x 10(-9) mol/l and 3 x 10(-7) mol/l also inhibited growth hormone secretion in 30 min incubations. The most effective insulin concentration in this case was 3 x 10(-8) mol/l. Insulin (10(-9)-10(-7) mol/l) also decreased the intracellular content of prostaglandins E and F. The effect was rapid, reaching a maximum after 30 min. Indomethacin, an inhibitor of prostaglandin synthetase, dramatically lowered the concentration of prostaglandins in the cells within 30 min; growth hormone synthesis was also decreased, but not until after 2 h of incubation. The results suggest that an initial response to insulin treatment is a lowering of intracellular levels of prostaglandins, which may then mediate a decrease in growth hormone synthesis, after a 1--2 h delay.     

Effects of triiodothyronine, triiodothyroacetic acid, iopanoic acid and iodide on the thyrotropin-releasing hormone-induced thyrotropin release from superfused rat pituitary fragments

The effects of triiodothyronine, triiodothyroacetic acid, iopanoic acid and potassium iodide were investigated on basal and stimulated thyrotropin release in an in vitro experimental model. Rat pituitary fragments were superfused by Medium-199 with or without T3 (10(-7) mol/l), triiodothyroacetic acid (10(-8)-10(-6) mol/l), iopanoic acid (10(-7)-10(-5) mol/l) or potassium iodide (10(-7)-10(-4) mol/l). This was followed by a 6-min pulse of thyrotropin-releasing hormone (10(-8) mol/l). TSH was measured in 3-min fractions. The TRH-induced TSH release from the pituitary fragments was inhibited by T3 (10(-7) mol/l), by triiodothyroacetic acid (10(-7)-10(-6) mol/l), and by high concentrations of iodide (10(-4) or 10(-5) mol/l). Iopanoic acid had no significant effect at the concentrations tested. It is assumed that in vitro, and at similar concentrations, the inhibitory effect of triiodothyroacetic acid on the TRH-induced TSH secretion is comparable to that of T3, whereas iopanoic acid may have no direct detectable effect. In contrast, a direct inhibitory effect of inorganic iodide, at least in pharmacological concentrations in vivo, cannot be excluded.     

T3 stimulation of 2-deoxy-D-glucose uptake in cultured chick embryo carcass derived cells, requires neosynthesis of proteins

T3 stimulated, in a dose dependent manner, the uptake of 2-deoxy-D-glucose (2-DOG) into cultured chick embryo carcass derived cells. A significant increase in sugar uptake was seen already at a T3 concentration of 1 pmol/l. The stimulation of 2-DOG uptake increased with time during the 6 h of exposure to T3. The hormone also stimulated, within 45 min, the incorporation of [3H]leucine and [3H]uridine into the trichloroacetic acid precipitable material of these cells. Actinomycin-D (100 micrograms/l) and cycloheximide (1 mg/l) each were capable of blocking the stimulatory effect of 10(-8) mol/l T3 on sugar uptake and on uridine and leucine incorporation. Thus, T3 in these cultured chick embryo cells stimulated sugar transport through processes dependent on neosynthesis of proteins. In this respect the effect of T3 is different from that seen in cultured chick embryo cardiomyocytes.     

Mechanism of anti-lipolytic action of acipimox in isolated rat adipocytes

Summary Acipimox is commonly used to treat hyper-triglyceridaemia in non-insulin-dependent diabetic patients, but its precise mechanism of action has yet to be elucidated. We examined the in vitro effects of acipimox on the lipolytic regulatory cascade in epididymal adipocytes isolated from Wistar rats. Acipimox inhibited the lipolytic rate stimulated by adenosine deaminase (1 U/ml) in a concentration-dependent manner, reaching a near-basal value at 10 mol/l acipimox. Lipolysis activated by sub-maximal levels of isoproterenol in combination with adenosine deaminase (20 mU/ml) was significantly (p<0.05) decreased by 100 mol/l acipimox, whereas, in the absence of adenosine deaminase, 100 mol/l acipimox showed no significant (p>0.05) inhibition. These findings suggested that the anti-lipolytic mechanism regulated by adenosine may also be regulated by acipimox. Acipimox diminished the intracellular cyclic AMP level produced by 25 nmol/l isoproterenol in the presence of adenosine deaminase (20 mU/ml) in a concentration-dependent manner. At the same level of stimulation, acipimox inhibited the cyclic AMP-dependent protein kinase activity ratio and lipolytic rate over the same concentration range, with significant (p<0.05) reductions occurring at and above, 0.5 mol/l and 10 mol/l acipimox, respectively. Western blotting showed that upon lipolytic stimulation (1 U/ml adenosine deaminase; 100 nmol/l isoproterenol) a threefold increase in the lipolytic rate was accompanied by a significant (p<0.05) rise in hormone-sensitive lipase associated with the lipid fraction. Acipimox (1 mmol/l) and insulin (1 nmol/l) re-distributed hormone-sensitive lipase back to the cytosol, with a corresponding significant (p<0.05) loss from the fat cake fraction of adipocyte homogenates. In conclusion, the anti-lipolytic action of acipimox is mediated through suppression of intracellular cyclic AMP levels, with the subsequent decrease in cyclic AMP-dependent protein kinase activity, leading to the reduced association of hormone-sensitive lipase with triacylglycerol substrate in the lipid droplet of adipocytes.Abbreviations NIDDM Non-insulin-dependent diabetes mel-litus - HSL hormone-sensitive lipase - cAMP adenosine 35-cyclic monophosphate - A-kinase cyclic AMP-dependent protein kinase - DTT dithiothreitol - PIA N⁶-[R-(–)-1-methyl-2-phenethyl]-adenosine - Hepes 4-(2-hydroxyethyl)-1-piperazi-neethanesulphonic acid - BSA bovine serum albumin - PCV packed cell volume - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - MOPS 3-(N-morpholino) propanesulphonic acid - ADA adenosine deaminase - NEFA non-esterified fatty acids - KRH Krebs-Ringer-Hepes - TCA trichloroacetic acid - PBS phosphate buffered saline - IC₅₀ half-maximal inhibition     

A novel synthetic,nonpsychoactive cannabinoid acid (HU‐320) with antiinflammatory properties in murine collagen‐induced arthritis

Objective

To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University–320 (HU‐320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU‐320 on macrophages and lymphocytes.

Methods

DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU‐320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU‐320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU‐320 on production of tumor necrosis factor α (TNFα) and reactive oxygen intermediates (ROIs). The effect of HU‐320 administration on lipopolysaccharide‐induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU‐320 psychoactivity was performed using established laboratory tests on Sabra mice.

Results

Systemic daily administration of 1 and 2 mg/kg HU‐320 ameliorated established CII‐induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII‐specific and polyclonal responses of murine and human lymphocytes were down‐modulated. HU‐320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU‐320 administration yielded no adverse psychotropic effects in mice.

Conclusion

Our studies show that the novel synthetic cannabinoid acid HU‐320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product.

     

Effects of rosiglitazone and oleic acid on UCP-3 expression in L6 myotubes

Aims: The regulation of uncoupling protein-3 (UCP-3) expression in muscle remains unclear, specifically in relation to dietary and drug treatments. The present study evaluated the effects of oleic acid and rosiglitazone on UCP-3 mRNA expression in differentiated L6 myotubes.
Methods: L6 myocytes were cultured and differentiated prior to exposure to rosiglitazone 10 µmol/l, oleic acid 100 µmol/l, or the combination, for 24 h, prior to semiquantitative evaluation of UCP-3 mRNA relative to GAPDH mRNA by RT-PCR.
Results: Exposure to oleic acid produced a significant increase in UCP-3 mRNA (0.012 ± 0.007 vs. 0.0011 ± 0.0006 for untreated cells, relative to GAPDH mRNA, p < 0.001). Rosiglitazone alone had no effect on UCP-3 expression and nor did the glitazone affect oleic-acid-induced upregulation of UCP-3.
Conclusions: In L6 myotubes, 24-h exposure to oleic acid produced a 10-fold increase in UCP-3 mRNA expression, but rosiglitazone had no effect. Oleic-acid-induced upregulation of UCP-3 was not affected (positively or negatively) by glitazone exposure.     

Hydroxyurea exerts a cytostatic but not immunosuppressive effect on T lymphocytes

OBJECTIVE: To demonstrate that, despite a dose-dependent cytostatic effect, hydroxyurea (HU) does not have immunosuppressive effects. METHODS: The effects of HU on T lymphocyte proliferation parameters, activation phenotype and cytokine production were examined in vitro after exposure to clinically relevant concentrations of HU (10, 50, and 100 micromol/l). The effects of HU in vivo on CD4 T cell counts, viral load, activation phenotype and virus-specific response were examined in 17 Rhesus macaques infected with SIV(mac251) and randomized into three groups: untreated controls; treated with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) and didanosine (ddI) only; and treated with PMPA, didanosine, and HU. RESULTS: The in vitro inhibition of T lymphocyte proliferation confirmed the cytostatic effect of HU, with a linear dose-dependent effect; however, no relevant differences were found in the expression of activation markers between treated and untreated controls. Both T helper type 1 and type 2 cytokine production were enhanced by HU. Consistent with the in vitro results, a blunted increase of peripheral CD4 T cells was observed in vivo in the HU group, without relevant effects on the expression of activation markers, and SIV-specific T cell responses were not affected by HU. CONCLUSIONS: Hyper-proliferation of T-lymphocytes is a major factor contributing to HIV pathogenesis. HU exerts a cytostatic effect on T lymphocytes, without altering their activation and apparently without having an immunosuppressive effect. The increase in cytokine production at the single cell level might compensate for the decrease in the percentage of activated CD4 T lymphocytes, without overall impairment of HIV-specific immune responses.     

The effect of theophylline and cyclic adenosine 3',5'-monophosphate on renin release by afferent arterioles

The effects of theophylline and cyclic adenosine 3',5'-monophosphate (cAMP) on renin release by afferent arterioles were studied. Rabbit afferent arterioles (seven to 10), obtained by a microdissection technique, were incubated for three consecutive 20 min periods in 100 microliters of Medium 199 with 0.1% bovine serum albumin (BSA). Afferent arterioles exposed to theophylline, 1 x 10(-4) mol/l, produced a greater than 100% increase in renin release (0.18 +/- 0.04 to 0.42 +/- 0.05 ng angiotensin l/h per arteriole per h incubation). The renin release stimulated by theophylline was completely abolished by indomethacin and meclofenamate. The stimulation of renin secretion was also blocked when extracellular calcium concentration was decreased to 10(-7) mol/l and the arterioles were permeabilized with calcium ionophore. Dibutyryl cAMP (db-cAMP), 1 x 10(-4) mol/l, and forskolin, 1 mumol/l or 100 mumol/l, failed to stimulate renin release by afferent arterioles. The results of this study therefore suggest that theophylline-stimulated renin release may be mediated through a prostaglandin pathway. The stimulation of renin secretion is also dependent on the extracellular concentration of calcium.     

Inhibitory effects of metformin on insulin and glucagon action in rat hepatocytes involve post-receptor alterations

The effect of the hypoglycaemic biguanide, metformin, on insulin binding and insulin action was investigated in rat hepatocyte monolayers. The binding of insulin was not modified in cultured cells exposed for 24 or 48 h to metformin at concentrations ranging from 1 mumol/l to 1 mmol/l, and no effect could be detected on insulin-induced down regulation. Metformin did not alter insulin stimulation of amino acid transport but the stimulatory effect of insulin on glycogen synthesis was reduced by 20%, 30% and 63% for metformin at 0.01, 0.1 and 1 mmol/l, respectively. Both responsiveness and sensitivity were altered by the biguanide. Metformin also inhibited basal glycogen synthesis and cellular glycogen contents were markedly decreased after exposure of cells to metformin (0.01-1 mmol/l). We also investigated the effect of metformin on glucagon action and metformin (0.1-1 mmol/l) was found to decrease the stimulatory effect of glucagon on amino acid uptake and on gluconeogenesis from alanine. These inhibitory effects of the biguanide were still observed when glucagon was replaced by dibutyryl cAMP. These in vitro studies demonstrate that: 1) metformin has no direct effect on insulin binding in hepatocytes, indicating that alteration of insulin stimulation of glycogen synthesis is due to modifications at the post receptor level. 2) metformin alters the action of glucagon in hepatocytes at a post AMP cyclase step. They also suggest that one of the mechanism of action of metformin may be to antagonize the effect of glucagon rather than to potentiate the action of insulin.     

Adrenomedullin and membrane fluidity of erythrocytes in mild essential hypertension

OBJECTIVE: Adrenomedullin is a newly discovered 52 amino acid peptide that has a potent vasodilating action. The present study was undertaken to investigate the role of adrenomedullin in the regulation of membrane fluidity of erythrocytes in patients with essential hypertension. METHODS AND RESULTS: We used an electron paramagnetic resonance and spin-labeling method. Adrenomedullin significantly decreased the order parameter for 5-nitroxide stearate and peak height ratio for 16-nitroxide stearate obtained from electron paramagnetic resonance spectra of erythrocyte membranes in normotensive volunteers (mean +/- SEM order parameter value: control, 0.718 +/- 0.003, n = 16; adrenomedullin at 10(-9) mol/l, 0.692 +/- 0.004, n = 16, P < 0.05; adrenomedullin at 10(-8) mol/l, 0.690 +/- 0.004, n = 16, P < 0.05; adrenomedullin at 10(-7) mol/l, 0.683 +/- 0.004, n = 16, P < 0.05). The findings showed that adrenomedullin increased the membrane fluidity of erythrocytes. In addition, the effect of adrenomedullin was significantly potentiated by prostaglandin E1 and dibutyryl cyclic AMP. In contrast, the calcium ionophore A23187 counteracted the actions of adrenomedullin. In patients with essential hypertension, who had higher order parameter values, the membrane fluidity of erythrocytes was significantly lower than in the normotensive control subjects (order parameter: 0.728 +/- 0.004 in hypertensives, n = 20; 0.692 +/- 0.002 in normotensives, n = 36, P < 0.01). The effect of adrenomedullin on membrane fluidity was more pronounced in the erythrocytes of essential hypertensive than in the erythrocytes of normotensive subjects (change in the order parameter with adrenomedullin at 10(-9) mol/l: -4.2 +/- 0.3% in hypertensives, n = 20; -1.8 +/- 0.2% in normotensives, n = 20, P < 0.05; adrenomedullin at 10(-8) mol/l: -4.5 +/- 0.3% in hypertensives, n = 20; -1.8 +/- 0.2% in normotensives, n = 36, P < 0.05). CONCLUSIONS: The results of the present study demonstrate that adrenomedullin significantly increased the membrane fluidity of erythrocytes. The mechanisms were partially mediated by a prostaglandin E1- and cyclic AMP-dependent pathway which might be linked to changes in intracellular calcium kinetics. The greater effect of adrenomedullin in patients with essential hypertension suggests that the peptide might actively participate in the regulation of membrane functions in hypertension.     

Effects of omeprazole and cimetidine on gastric acid secretion and right atrial beating frequency in isolated organ preparations from the guinea pig

The effects of omeprazole , a substituted benzimidazole, on gastric acid secretion and on right atrial beating frequency have been investigated in guinea pig preparations in vitro. Cimetidine was used as a reference compound. Omeprazole , at 10(-6) mol/l, inhibited basal acid secretion, whereas cimetidine, at 10(-5) mol/l, did not. There were also differences in the effects on stimulated secretion. Cimetidine (10(-5) mol/l) competitively inhibited histamine-stimulated acid secretion without affecting the maximal histamine response. In contrast, omeprazole concentration dependently depressed (EC50 approximately 5 X 10(-7) mol/l) the maximal histamine response without any effect on the histamine sensitivity. Furthermore, dibutyryl-cAMP-stimulated acid secretion was inhibited by omeprazole but not by cimetidine. The inhibitory effect of omeprazole (2 X 10(-6) mol/l) on histamine-stimulated acid secretion was reversed by repeated washing of the serosal side of the mucosa. Omeprazole was devoid of histamine H2 receptor antagonistic activity, since it had no effect on the chronotropic response to histamine in the guinea pig right atrium. It is concluded that omeprazole inhibits gastric acid secretion by a non-histaminergic, reversible mechanism and that the site of action is beyond the cAMP step within the parietal cell.     

Effect of auranofin on resorption, prostaglandin synthesis and ultrastructure of bone cells in cultured mouse calvaria

Auranofin (AF) in concentrations between 3 x 10(-7) and 3 x 10(-6) mol/l stimulated bone resorption in cultured neonatal mouse calvariae significantly with 1 x 10(-6) mol/l being most potent. Complete inhibition by 5 x 10(-7) mol/l indomethacin and increased medium concentrations of prostaglandin (PG) E2 and 6-keto-PGF1 alpha after 72 h indicate a PG mediated mechanism. Morphology revealed active osteoclasts. Cytotoxic effects were observed with 3 x 10(-6) and 1 x 10(-5) mol/l AF with osteocytes and osteoblasts being considerably more sensitive than osteoclasts. The latter concentrations inhibited bone resorption stimulated by parathyroid hormone (PTH) 1,25-dihydroxyvitamin D3, PGE2, thrombin and interleukin 1. The stimulatory effect of AF on PG production and subsequent bone resorption could limit its therapeutic usefulness.     

Effects of bromocriptine on hormone production and cell growth in cultured rat pituitary cells

Rat pituitary adenoma cells (GH3) that spontaneously synthesize and secrete both prolactin (Prl) and growth hormone (GH) were used in this study. Bromocriptine (5 X 10(-5) mol/l), a dopamine (DA) agonist, induced a rapid reduction in Prl and GH secretion with maximum effect (approximately 60%) after 15 min of treatment. Bromocriptine also inhibited Prl and GH production in a time- and dose-dependent manner with ED50 at 4 X 10(-6) mol/l and 7 X 10(-6) mol/l, respectively. Maximum effect was obtained at 5 X 10(-5) mol/l of bromocriptine which after 24 h of treatment reduced the production of Prl and GH by approximately 70 and approximately 50%, respectively. After 9 days of treatment both Prl and GH production was reduced by more than 95%. Bromocriptine also reduced cellular growth rate. The ED50 was approximately 1 X 10(-5) mol/l and the maximum effect (greater than 50%) was observed at 5 X 10(-5) mol/l. All effects of bromocriptine were reversible upon cessation of treatment. The antiproliferative effect of bromocriptine was also observed using a rat hepatoma cell line (MH1C1) and a human epithelial cell line (HE), suggesting a non-receptor mediated growth inhibition at high concentrations of the drug. In conclusion, the inhibitory effect of bromocriptine on secretion and production of both Prl and GH in GH3 cells occurs at a lower concentration than its effect on cell proliferation. The pharmacological effects of bromocriptine in vivo on Prl and GH producing adenomas may be explained by an action directly at the pituitary level.     

Facilitation of sympathetic neurotransmission by phosphatidylinositol-4,5-bisphosphate-dependent regulation of KCNQ channels in rat mesenteric arteries

Sympathetic nerves regulate vascular tone by releasing neurotransmitters into the vasculature. We previously demonstrated that bradykinin facilitates sympathetic neurotransmission in rat mesenteric arteries. Although little is known about the intracellular mechanism modulating this neurotransmission, recent cell line experiments have shown that the KCNQ channel, which is inhibited by the depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP(2)), participates in the control of neurotransmission by bradykinin. In the present study, we examined the mechanism regulating neurotransmitter release from rat perivascular sympathetic nerves. Excitatory junction potentials (EJPs) elicited by repetitive nerve stimulation (1?Hz, 11 pulses, 20?μs, 20-50?V), a measure of sympathetic purinergic neurotransmission, were recorded with a conventional microelectrode technique in rat mesenteric arteries. Bradykinin (10(-7)?mol?l(-1)) significantly enhanced the amplitude of EJPs (n=22, P<0.05). This enhancing effect was abolished by N-type calcium-channel inhibition with ω-conotoxin GVIA (2 × 10(-9)?mol?l(-1), n=8). The blockade of phospholipase C with U-73122 (10(-6)?mol?l(-1), n=17) also eliminated the facilitatory effect of bradykinin. In addition, the effects of bradykinin were diminished by the prevention of PIP(2) resynthesis with wortmannin (10(-5)?mol?l(-1) n=7) or KCNQ channel inhibition with XE-991 (10(-5)?mol?l(-1), n=7). On the other hand, depletion of intracellular calcium stores with cyclopiazonic acid (3 × 10(-6)?mol?l(-1), n=6) or the inhibition of protein kinase C with bisindolylmaleimide-I (10(-6)?mol?l(-1), n=9) did not alter the action of bradykinin. These data demonstrate that the hydrolysis of PIP(2) by phospholipase C, which is activated by G(q/11)-coupled receptors, and subsequent KCNQ channel inhibition enhance sympathetic purinergic neurotransmission presumably via the activation of N-type calcium channels in rat mesenteric arteries.     

The bimodal effect of interleukin 1 on rat pancreatic beta-cells--stimulation followed by inhibition--depends upon dose, duration of exposure, and ambient glucose concentration

To investigate the hypothesis that interleukin 1 initially stimulates and then suppresses beta-cell function and that this sequential effect is directly related to interleukin 1 dose, duration of exposure, and ambient glucose concentration, insulin release was measured from cultured newborn rat islets exposed for 6 h to 6 days to interleukin 1 at doses ranging from 20 to 2000 ng/l at glucose concentrations of 3.3, 5.5 and 11 mmol/l. After 6 h of exposure and at all three glucose levels, all doses of interleukin 1 stimulated insulin release, maximal stimulation (370% of control) being observed at 5.5 mmol/l glucose and 100 ng/l interleukin 1. In contrast, after 6 days, all doses of interleukin 1 were inhibitory irrespective of glucose level, maximal inhibition (90%) being observed at 11 mmol/l glucose and 2000 ng/l interleukin 1. At 24 and 48 h of exposure, the biphasic effect of interleukin 1 was observed: lower doses of interleukin 1 at lower glucose concentrations at 24 h being more stimulatory with transition to inhibition directly related to higher glucose levels, higher interleukin 1 doses, and longer exposure. After 48 h, 200 ng/l of interleukin 1 increased insulin release to 220% at 3.3 mmol/l glucose, but at 11 mmol/l glucose a 60% suppression was seen. On the basis of these data we suggest that interleukin 1's effect on beta-cells is bimodal: stimulation followed by inhibition. Increasing interleukin 1 dose and ambient glucose concentration shift this response to the left. Experimental results will, and in vivo effects may, depend upon these three variables.     

Hydroxyurea in thalassemia intermedia—a promising therapy

Pharmacological agents such as hydroxyurea (HU) have been known to cause induction of fetal hemoglobin and possibly may alleviate the symptoms in thalassemia intermedia patients. Thirty-seven patients with beta-thalassemia intermedia were enrolled to assess response to HU therapy. Major response was defined as transfusion independence or hemoglobin rise of more than 20 g/l and minor response as rise in hemoglobin of 10-20 g/l or reduction in transfusion frequency by 50%. The median age was 10 years (range: 4-50 years) and median follow-up was 12 months (range: 4-36 months). Twenty-six patients (70.2%) showed response to HU therapy. Seventeen patients (45.9%) were major responders, and nine patients (24.3%) showed minor response. There was no correlation of response with beta-thalassemia mutation or XmnI polymorphism; however, the presence of alpha(3.7) deletion was associated with major response in three patients. Mean fetal hemoglobin (HbF) levels rose on HU therapy. Older age, low baseline F cell percent, and low baseline HbF levels (below 10%) were predictors of poor response. Response was evident within 1 month of starting HU therapy in the majority of responders. Thus, a short trial of HU therapy can predict durable response.