Dessislava Duridanova

Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.

Hydrogen sulfide (H2S) has been traditionally known for its toxic effects on living organisms. The role of H2S in the homeostatic regulation of pancreatic insulin metabolism has been unclear. The present study is aimed at elucidating the effect of endogenously produced H2S on pancreatic insulin release and its role in diabetes development. Diabetes development in Zucker diabetic fatty (ZDF) rats was evaluated in comparison with Zucker fatty (ZF) and Zucker lean (ZL) rats. Pancreatic H2S production and insulin release were also assayed. It was found that H2S was generated in rat pancreas islets, catalyzed predominantly by cystathionine γ-lyase (CSE). Pancreatic CSE expression and H2S production were greater in ZDF rats than in ZF or ZL rats. ZDF rats exhibited reduced serum insulin level, hyperglycemia, and insulin resistance. Inhibition of pancreatic H2S production in ZDF rats by intraperitoneal injection of DL-propargylglycine (PPG) for 4 weeks increased serum insulin level, lowered hyperglycemia, and reduced hemoglobin A1c level (P<0.05). Although in ZF rats it also reduced pancreatic H2S production and serum H2S level, PPG treatment did not alter serum insulin and glucose level. Finally, H2S significantly increased KATP channel activity in freshly isolated rat pancreatic β-cells. It appears that insulin release is impaired in ZDF because of abnormally high pancreatic production of H2S. New therapeutic approach for diabetes management can be devised based on our observation by inhibiting endogenous H2S production from pancreas.

Urocortin relaxes rat tail arteries by a PKA-mediated reduction of the sensitivity of the contractile apparatus for calcium

Urocortin is an endogenous vasodilator although the mechanism of vasorelaxation is not completely understood. The hypothesis that an alteration of smooth muscle calcium concentration is involved was tested using isometric tension recording and calcium fluorimetry. The relationship between contraction and intracellular calcium was also estimated. Urocortin produced a concentration dependent relaxation (pD2 8.59±0.06, n=6) of vessels pre‐contracted with a physiological salt solution containing 42 mM KCl (42 mM K‐PSS). Removal of the endothelium did not alter the effect of urocortin, pD2 was 8.49±0.11, n=5. Corticotropin‐releasing factor relaxed 42 mM K‐PSS pre‐contracted vessels with less potency compared to urocortin (pD2 6.99±0.28, n=5). Urocortin at 100 nM relaxed vessels pre‐contracted with 42 mM K‐PSS by 59.6±4.6% (n=8) and vessels pre‐contracted with 500 nM noradrenaline by 25.2±6.8% (n=6). Both effects were not accompanied by a change in the intracellular calcium concentration. Urocortin at 100 nM produced a significant rightward shift of 0.33±0.07 units of normalized intracellular calcium (n=5) of the relationship between tension and intracellular calcium. The urocortin‐induced relaxation was considerably reduced in the presence of 0.3 mM Rp‐8‐CPT‐cAMPS, a cyclic AMP‐dependent protein kinase (PKA) inhibitor. The PKA‐activator Sp‐5,6‐DCl‐cBIMPS relaxed 42 mM K‐PSS pre‐contracted vessels (pD2 4.98±0.07, n=6). Sp‐5,6‐DCl‐cBIMPS at 0.1 mM relaxed vessels by 85.3±2.5% (n=5), but did not change the intracellular calcium concentration. In conclusion, the data show that urocortin is a potent, endothelium‐independent dilator of rat tail arteries and suggest that this effect is mediated by PKA causing a reduction of the sensitivity of the contractile apparatus for calcium.

Effect of cisplatin and cobalt chloride on antioxidant enzymes in the livers of Lewis lung carcinoma-bearing mice: protective role of heme oxygenase

Changes in the activities of antioxidant enzymes superoxide dismutase, catalase (CAT), glutathione peroxidase and heme oxygenase (HO) and changes in lipid peroxidation and reduced glutathione (GSH) levels were measured in the livers of control and Lewis lung carcinoma (LLC)-bearing mice 24 h after a single injection of cisplatin or CoCl(2). Treatment with cisplatin induced the same degree of lipid peroxidation and GSH depletion as did CoCl(2) but the antioxidant enzymes were differently involved in cisplatin- and cobalt-induced oxidative stress responses. In cobalt-treated mice the activities of these enzymes were either inhibited or not changed significantly and only the HO activity was increased (5-fold) as a main protective enzyme. In cisplatin-treated animals the antioxidant enzymes were activated but the enhancement of HO and CAT was greater in LLC-inoculated mice. It is suggested that these two enzymes represent the protective response against cisplatin toxicity in the livers of tumor-bearing animals.

Enhanced heme oxygenase activity increases the antioxidant defense capacity of guinea pig liver upon acute cobalt chloride loading: comparison with rat liver.

Changes in the activity of so-called oxidative stress defensive enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and heme oxygenase, as well as changes in lipid peroxidation and reduced glutathione levels, were measured in guinea pig and rat liver after acute cobalt loading. Cobalt chloride administration produced a much higher degree of lipid peroxidation in guinea pig than in rat liver compared with the control animals. The intrahepatic reduced glutathione content in control guinea pig was higher than that in rat, but was equally decreased in both species after cobalt administration. The enzymatic scavengers of free radicals, superoxide dismutase, catalase and glutathione peroxidase, were significantly decreased in rat liver after acute cobalt loading, and as a compensatory reaction, the heme oxygenase activity was increased (seven-fold). In guinea pig liver, only superoxide dismutase activity was depleted in response to cobalt-induced oxidative stress, while catalase and glutathione peroxidase were highly activated and the heme oxygenase activity was dramatically increased (13-fold). It is assumed that enhanced heme oxygenase activity may have important antioxidant significance by increasing the liver oxidative-stress defense capacity.

4-Aminopyridine affects rat arterial smooth muscle BKCa currents by changing intracellular pH

The hypothesis whether or not 4‐AP can affect vascular smooth muscle BKCa currents was tested using the patch‐clamp technique, pH‐ and calcium‐fluorimetry, and freshly isolated rat arterial smooth muscle cells. Application of 4‐AP reversibly inhibited BKCa currents at an intracellular calcium ([Ca]i) of 250 nM with a half‐block of 2.5 mM at +50 mV. The presence of 2 μM thapsigargin, 10 μM heparin, and 10 μM ryanodine did not alter the effect of 4‐AP on BKCa currents at [Ca]i 250 nM. At [Ca]i<100 nM 4‐AP did not inhibit BKCa currents. Application of 4‐AP to the intracellular or extracellular side of excised BKCa channels did not alter channel activity or channel amplitude. Replacement of the pH‐sensitive calcium buffer EGTA by the pH‐insensitive calcium buffer BAPTA in the intracellular solution turned the 4‐AP‐induced inhibition of BKCa currents into a stimulation at [Ca]i 250 nM. Application of 4‐AP to single cells increased intracellular pH, which was accompanied by a reduction of [Ca]i in EGTA‐loaded cells and a stable [Ca]i in BAPTA‐loaded cells. Thus, these results suggest that in isolated vascular smooth muscle cells at [Ca]i>100 nM 4‐AP affects BKCa currents via an alteration of intracellular pH.

Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones

During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non‐specific ion channel in the synaptic vesicle membrane, in the context of the post‐fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non‐specific ion channel was examined using the fused vesicle‐attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non‐specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.

Urocortin hyperpolarizes stomach smooth muscle via activation of Ca2+-sensitive K+ currents

The effect of urocortin (Uro), a recently discovered neuropeptide with selectivity towards corticotropin-releasing hormone type 2 receptor, was tested on whole cell currents expressed by guinea-pig gastric antrum smooth muscle cells. Uro (1 pmol/l − 1 nmol/l) caused a concentration-dependent increase of Ca2+-sensitive K currents (IK) up to 500% as compared to control currents and did not affect the kinetics and voltage-dependence of inward Ca2+ currents. The IK-increasing effect of Uro was fully antagonized by preliminary emptying of intracellular Ca2+ stores with ryanodine and cyclopiazonic acid, as well as by bath application of selective blockers of adenylyl cyclase and cAMP-dependent protein kinase (PKA), but not by inhibitors of guanylyl cyclase, cGMP-dependent protein kinase, and protein kinase C. Comparable IK increase was obtained by forskolin (activator of adenylyl cyclase), Sp-cAMPS (activator of PKA), or by intracellular application of the catalytic subunit of PKA. It was concluded that Uro binds to a selective receptor in antral smooth muscle cells where it stimulates IK via PKA-dependent increase of Ca2+ concentration near the plasma membrane due to enhanced release from intracellular calcium stores.

Ca2+-induced Ca2+ release activates K+ currents by a cyclic GMP-dependent mechanism in single gastric smooth muscle cells

Abstract The participation of sarcoplasmic reticulum Ca 2+ release channels in the activation of Ca 2+ -sensitive K + currents ( I K(Ca) ) by cyclic dibutyryl GMP was investigated in smooth muscle cells from the circular layer of guinea-pig gastric fundus. All experiments were performed in the presence of 3 μM nicardipine into the bath and low Ca 2+ buffering capacity of the pipette-filling solution (pCa 7.4). Ruthenium red (10 μM) as well as its combination with 10 μM heparin abolished the cyclic GMP-induced activation of I K(Ca) , while 10 μM heparin remained ineffective. Ryanodine (10 μM) and the subsequently added 1 μM thapsigargin induced a relatively small increase in I K(Ca) amplitudes. The addition of 10 μM ryanodine to 1 μM thapsigargin-containing bath solution caused a vast increase in I K(Ca) . It is hypothesyzed that protein kinase G-induced vectorial Ca 2+ flux from the cell bulk and sarcoplasmic reticulum Ca 2+ stores toward the plasma membrane is realized by a spontaneous Ca 2+ -induced Ca 2+ release from a superficially situated Ca 2+ store.

Corticotropin-releasing hormone acts on guinea pig ileal smooth muscle via protein kinase A

Abstract In contraction studies corticotropin-releasing hormone (CRH) was found to relax ileal but not gastric and jejunal smooth muscles of the guinea-pig, precontracted with BaCl2. Under whole-cell patch-clamp conditions, CRH concentration-dependently activated Ca2+-sensitive K+ currents (IK) with ED50=20 pM at 100 nM and ED50=0.13 pM at 500 nM intracellular Ca2+ respectively. This increase was accompanied by significant hyperpolarization of the cell membranes. CRH 9–41 peptide fragment did not affect IK amplitude, membrane potential or contraction. The CRH-induced increase of IK densities was accelerated in the presence of high intracellular Ca2+ concentrations (500 nM) and was abolished by pretreatment of cells with either ryanodine or thapsigargin, which cause depletion of intracellular Ca2+ stores, as well as in cells treated under conditions prohibiting intracellular Ca2+ store refilling. The effect of CRH on IK was not affected by bath application of various selective inhibitors of membrane-bound phospholipases, protein kinase C, cGMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase II, but was effectively antagonized by blockers of protein kinase A (PKA) or adenylyl cyclase. Neither forskolin nor the catalytic subunit of PKA could mimic the effect of CRH on IK. Thus, it was suggested that CRH exerts its relaxing activity on ileal smooth muscle cells via PKA-dependent phosphorylation of some intracellular target coupled to sarcoplasmic reticulum Ca2+ storage machinery.

Inhibition of Ca2+ current in ileal cells by cyclopiazonic acid and ryanodine

The effects of cyclopiazonic acid and its combination with ryanodine on the inward Ca2+ current (ICa) were investigated in smooth muscle cells isolated from the circular layer of guinea-pig ileum. The ICa of these cells exhibited two components: a low-threshold, nicardipine (5 microM)-resistant, fast-inactivating component and a high-threshold, nicardipine-blockable and slowly inactivating component. Neither cyclopiazonic acid (up to 10 microM) nor ryanodine (10 microM) was able to affect both these components of ICa, when applied separately. Cyclopiazonic acid and ryanodine combination led to total abolishment of the high-threshold component, leaving the low-threshold component unaffected. The data presented suggest a process of Ca(2+)-dependent inactivation of the high-threshold component, elicited by an increase in the subplasmalemmal Ca2+ concentration due to Ca2+ released from the sarcoplasmic reticulum. It is considered that the combination of cyclopiazonic acid and ryanodine can be used as a valuable method to study the calcium sensitivity of both components of the ICa.