Protein kinase C and Gi-protein mediated modulation of cAMP production in different stages of the rat seminiferous epithelium

The modulatory role of protein kinase C (PK-C)- and G_i-protein-mediated signal transduction systems was studied in the cyclic variation of follicle-stimulating hormone (FSH)-stimulated cAMP production of rat seminiferous tubules. FSH (Metrodin, Serono, 30 mg/1) stimulated cAMP production 10-fold (p < 0.01) in a 3 h incubation of 5 mm segments of seminiferous tubules of stages II–VI of the epithelial cycle, but only 2-fold (p < 0.01) in stages VII–VIII. The PK-C activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nmol/1) suppressed the FSH effect on cAMP output by 50–70% (p < 0.01) in stages II–VI, but had no effect in stages VII–VIII. If the tubular segments were preincubated for 3 h in the presence of pertussis toxin (PT, 100 μg/1), the FSH-stimulated cAMP production of stages VII-VIII increased by 100–200% (p < 0.01), and now they also became responsive to the TPA suppression. Conversely, no effect of PT was observed in stages II–VI. Cholera toxin (CT, 100 μg/1) and forskolin (Fk, 100 μmol/1) nearly similarly stimulated the cAMP production in both stages studied (about 10-fold, p < 0.01), and TPA and PT potentiated the effects in a non-additive fashion. In conclusion, both G_i-protein and PK-C-mediated mechanisms modulate cAMP production of rat seminiferous tubules. A clear cyclic variation can only be demonstrated in FSH-stimulated cAMP production, but not if the G_s-protein or adenylate cyclase are directly stimulated. Upon FSH stimulation, the low cAMP production in stages VII–VIII is mainly due to the G_i-protein-mediated inhibition. In contrast, the absence (or non-function) of this inhibition mechanism explains the brisk cAMP response to FSH in stages II–VI. PK-C activation suppresses FSH-stimulated cAMP production only if it is not inhibited by the G_i-protein-mediated mechanism (stages II–VI), probably by inhibiting the FSH-receptor-G_s-protein association. It also increases CT and Fk-stimulated cAMP production, in this case inactivating the G_i-protein.     

The Cardiac β -Adrenoceptor-G-protein(s)-adenylyl Cyclase System in Monocrotaline-treated Rats

In rats, injection of the alkaloid monocrotaline (MCT) causes right ventricular hypertrophy and cardiac failure. In order to study whether, in MCT-treated rats, changes in the cardiac β -adrenoceptor-G-protein(s)-adenylyl cyclase system might be comparable to those found in human primary pulmonary hypertension, we assessed in right and left ventricles from MCT-treated rats the components of the β -adrenoceptor system: the receptor number and subtype distribution (by (-)-[¹²⁵I]iodocyanopindolol binding), the G-proteins (by quantitative Western blotting), and the activity of adenylyl cyclase. A single injection of 60 mg/kg i.p. MCT caused in rats right ventricular hypertrophy (RVH); part of the rats developed cardiac failure (RVF). In these rats the cardiac β -adrenoceptor-G-protein(s)-adenylyl cyclase system was markedly changed β -adrenoceptors were desensitized due to a decrease in receptor number, an uncoupling of the receptor from the G_s-adenylyl cyclase system, a decrease in G_sand a decrease in the activity of the catalytic unit of adenylyl cyclase. In general, these changes were more pronounced in right ventricles v left ventricles, and in rats with RVF v rats with RVH. On the other hand, cardiac muscarinic receptors and G_iappeared not to be altered. We conclude that in MCT-treated rats changes in the cardiac β -adrenoceptor-G-protein(s)-adenylyl cyclase system occur that resemble those observed in human primary pulmonary hypertension. Thus, MCT-treated rat appears to be a suitable animal model to study in more detail the pathophysiology of the development of right heart failure, and to identify new therapeutic possibilities.     

Postnatal Changes in the G-Proteins, Cyclic Nucleotides and Adenylyl Cyclase Activity in Rabbit Heart Cells

We have studied the postnatal changes in the levels of isoforms of stimulatory (G_s) and inhibitory (G_i) G-proteins, cAMP and cGMP in washed particulate membranes (WPM) from whole ventricles as well as from isolated ventricular myocytes and have also measured adenylyl cyclase (AC) activity in WPM prepared from isolated myocytes of adult (AD) and newborn (NB) rabbit heart. Immunoblot analysis for the levels of G_iα1, G_α2 G_iα3 and G_sα subunits showed that G_iα2 and G_iα3 were higher in WPM from whole ventricles of NB compared to AD. This ratio was much higher in WPM from isolated ventricular myocytes since G_iα2 and G_iα3 were either absent or present in extremely low immunodetectable levels in WPM from AD ventricular myocytes. G_iα1 levels were not different for AD compared to NB WPM, whether prepared from whole ventricle or from isolated myocytes. Two forms of G_sα, a small form (G_sα-S) and a large form (G_sα-L), were immunodetected at 43 and 48 kDa, respectively. The G_sα-S form was higher in AD WPM and the G_sα-L form was higher in NB WPM while the total G_sα(L+S) was not different. The G_sα results for WPM from isolated myocytes were not different from the results for WPM from whole ventricles. Basal levels of cAMP were 80% higher in NB compared to AD whole ventricles and were 200% higher in NB compared to AD isolated myocytes. Levels of cGMP were 4-5 fold higher in NB than in AD myocytes and ventricular tissue. Basal AC activity was higher in NB than in AD WPM from isolated myocytes and was enhanced by Gpp(NH)p pretreatment in AD but not in NB WPM. The isoproterenol-induced increase in AC activity was higher in AD compared to NB WPM and was completely abolished by Gpp(NH)p pretreatment in NB but not in AD WPM. Forskolin caused a greater increase in AC activity in NB than in AD WPM. The post-natal decrease in the levels of G_iα2 and G_iα3, particularly in isolated ventricular myocytes, may help to explain the smaller effects of isoproterenol and greater muscarinic influence on I_Ca+, as we previously showed, and the smaller effect of isoproterenol on AC activity in NB compared to AD WPM. It is not clear if the post-natal differences in the distribution of types of G_sα may also play a role in modulating the effect of isoproterenol. The postnatal decrease in the levels of cAMP and cGMP may be significant in regulating phosphodiesterases, protein phosphatases and kinases     

Pertussis toxin enhances follicle-stimulating hormone-stimulated cAMP production in rat seminiferous tubules in a stage-dependent manner

Cyclic (c) AMP production was measured in vitro in dissected pieces of adult rat seminiferous tubules of defined stages of the seminiferous epithelial cycle (VII-VIII and XIV-IV) in the absence and presence of human follicle-stimulating hormone (FSH) (Metrodin, Serono). The basal rate of cAMP production was stage dependent, being about 2-fold higher in stages XIV-IV. FSH stimulated cAMP output in a time- and dose-dependent (stimulation at doses greater than or equal to 3 mg/l) fashion, and also the stimulability of stages XIV-IV (on average 2-fold) was greater than that of stages VII-VIII. When the tubular pieces were incubated in the presence of pertussis toxin (PT, 100 micrograms/l), the FSH-stimulated cAMP production of stages VII-VIII was enhanced by about 2-fold (P less than 0.01) whereas the basal rate was unaffected. In contrast, neither the basal nor the FSH-stimulated cAMP production of stages XIV-IV were affected by PT. Presence of the Gi-protein in both stages studied was demonstrated with PT-induced ADP ribosylation. However, no release of a putative activator of the Gi-protein could be demonstrated into spent media of the seminiferous tubules when incubated with freshly separated tubules. It is concluded that the poor FSH stimulability in cAMP production of certain spermatogenic stages of adult seminiferous tubules is at least partly due to endogenous inhibitors acting through the inhibitory Gi-protein. This inhibition could be demonstrated in a stage-dependent manner, and was present in stages with the lowest production and least stimulability of cAMP production by FSH.     

Inhibition of rat granulosa cell differentiation by overexpression of Galphaq

Activation of FSH and LH receptors in undifferentiated granulosa cells (i.e., no prior exposure to FSH) results in comparable induction of progesterone production, but activation of the LH receptor is less effective than FSH in inducing aromatase and the native LH receptor. Because the LH receptor can also activate the Gαq signaling pathway, we investigated whether activation of this pathway could be responsible for these differences. Overexpression of Gαq inhibited FSH induction of both the estradiol and progesterone biosynthetic pathways as well as mRNA levels for cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), LH receptor (LHr), and P450aromatase (aromatase). This suppression was associated with a reduction (P < 0.05) in FSH-stimulated cAMP production. Lower cAMP levels were not due to reduced FSH receptor (FSHr) mRNA levels or reduced levels of Gαs. Phosphodiesterase (PDE) activity and regulator of G-protein signaling 2 (RGS2) mRNA levels were significantly (P < 0.05) increased by Gαq, both of which could account for diminished cAMP levels. We conclude that Gαq signaling pathway inhibits both estradiol and progesterone production comparably and thus activation of this pathway does not seem to account for differences between FSH and LH in the regulation of aromatase and the LH receptor.     

Long-Lasting Binding of IT-066 to Human Histamine H2 Receptor

Based on animal models, IT-066, a histamine H₂-receptor antagonist, is reported to possess potent and long-lasting antagonisms on histamine H₂ receptor (H₂R) -mediated effects. However, no reports have been published concerning its interaction with the human H₂R. The aim of this study is to characterize its interaction with human H₂R. Chinese hamster ovary cell lines stably expressing human H₂Rs were obtained. The effects of IT-066, famotidine, and ranitidine on tiotidine binding and histamine-stimulated cAMP production were analyzed. IT-066 inhibited [³H]tiotidine binding and histamine-stimulated cAMP production more potently than famotidine or ranitidine. In addition, preincubation with 10^–5 M IT-066, but not with 10^–5 M famotidine or 10^–4 M ranitidine, had marked inhibitory effects long after extensive washing. Paraformaldehyde fixation of the cells blunted inhibition of [³H]tiotidine binding induced by preincubation with IT-066, but not that by preincubation with famotidine or ranitidine. IT-066 has potent and long-lasting antagonisms on human H₂R. At least one of the IT-066 binding sites is not shared by famotidine, ranitidine, or tiotidine and is affected by paraformaldehyde fixation.     

Uptake of pentamidine in Trypanosoma brucei brucei is mediated by the P2 adenosine transporter and at least one novel, unrelated transporter

Diamidine drugs such as pentamidine and berenil (diminazene aceturate) are vital drugs for the treatment of early stage human African trypanosomiasis and the corresponding veterinary condition, respectively. The action of diamidines on trypanosomes is critically dependent on their efficient uptake by the parasite. We have therefore investigated the mode of uptake of pentamidine by Trypanosoma brucei brucei, using [¹²⁵I]iodopentamidine as a permeant. [¹²⁵I]Iodopentamidine uptake was linear for up to 15 min and inhibited by adenosine with a K_i value of 0.64±0.03 μM, to a maximum of 50–70%. The adenosine-sensitive flux was also inhibited by adenine with a K_i value of 0.44±0.04 μM. Iodopentamidine uptake was saturable, with the adenosine-insensitive flux displaying a K_m of 22±2 μM and a V_max of 2.2±0.9 pmol(10⁷ cells)⁻¹ s⁻¹, whereas the adenosine-sensitive flux was inhibited by much lower iodopentamidine concentrations. These results clearly demonstrate that iodopentamidine is taken up by at least two different T. b. brucei transporters, an adenosine-sensitive pentamidine transporter (ASPT1) and a low-affinity pentamidine transporter (LAPT1). The identity of these transporters was investigated, and their significance for drug uptake and resistance in African trypanosomes is discussed.     

Chronic Effects of Inhaled Albuterol on β-Adrenoceptor System Function in Human Respiratory Cells

The in vivo effects of β-adrenergic receptor (βAR) agonists given chronically by metered-dose inhaler (MDI) on the molecular components of the β-adreno-ceptor system expressed by human respiratory cells are poorly understood. This study examined the effects of inhaled albuterol (180 μg four times daily for 7 days) on βAR function of airway epithelial cells (AECs) and alveolar macrophages (AMs) freshly isolated from 10 normal subjects. Responses were related to β₂AR genotype in codons 16 and 27, regions which affect chronic responses to β₂-agonists. In AEC, βAR density and adenosine cyclic 3′,5′-phosphate (cAMP) production in response to isoproterenol (ISO) were significantly lower in the albuterol versus placebo treatment arm (p < 0.01 for both). Moreover, in AEC, albuterol treatment increased βAR-kinase (βARK) protein immunoreactivity. In contrast, in AM, albuterol tended to decrease βAR density and cAMP production but changes did not achieve statistical significance (p > 0.20 for both) and had no effect on βARK immunoreactivity. Changes in (JAR density occurred in all subjects but tended to be greater in subjects with the glycine 16 genotype. In cultured cells exposed to equal concentrations of β-agonist in vitro, the magnitude of βAR down-regulation (p < 0.05) and cAMP densensiti-zation (p < 0.05) was greater in AEC than AM. These results indicate that albuterol taken by inhalation in a therapeutically relevant dose for 1 week produces pAR down-regulation, densensitizes the cAMP response of airway epithelial cells to a β₂-adrenergic agonist, and increases PARK immunoreactivity. Greater densensitization of AEC than AM in response to chronic albuterol inhalation likely reflects cell type-specific responses.     

In vivo and in vitro responses of the bovine corpus luteum after exposure to exogenous gonadotropin-releasing hormone and prostaglandin F(2α)

Administration of gonadotropin-releasing hormone (GnRH) early in the estrous cycle has been shown to cause subsequent altered luteal function. To determine whether membrane-related events may be involved in GnRH-attenuated luteal function, corpora lutea (CL) were removed from beef heifers on day seven of the estrous cycle after iv injection of GnRH or saline on day two of the cycle (n=5/group). Luteal slices were incubated with saline (control), luteinizing hormone (LH), or 8-bromo-cAMP for 2h. In vivo administration of GnRH reduced LH and cAMP-stimulated progesterone production by tissue (p<0.01), but basal progesterone production was not affected (p>0.05). Luteal adenylyl cyclase activity did not differ between saline and GnRH-treated animals (p>0.05). Then to examine if early administration of GnRH alters response of the CL to prostaglandin (PG) F_2α, beefheifers were injected with GnRH as described above (n=4/group), and then injected with PGF_2α on day eight and the CL removed 60 min later. Blood samples were collected for oxytocin (OT) analysis at frequent intervals after PGF_2α injection and for progesterone at 0 and 60 min. Induction of the early response gene c-jun or release of OT by PGF_2α was not altered by GnRH injection (p>0.05). Injection of PGF_2α decreased serum progesterone by 60 min postinjection (p<0.05), but concentrations of this steroid were unaffected by GnRH (p>0.05). Collectively, these data suggest that GnRH-induced alteration of bovine luteal function may be owing to events distal to cAMP synthesis that do not interfere with PGF_2α-induced expression of c-jun or OT release, cellular phenomena involved in luteolysis.     

Field stimulation-induced noradrenaline release from guineapig atria is modulated by prejunctional α2-adrenoceptors and protein kinase C

Summary Guinea-pig left atria were loaded with 10 Ci 7-[³H]noradrenaline, and noradrenaline release from sympathetic nerve endings was then elicited by refractory period field stimulation. When one pulse of 0.2 ms duration was applied during each refractory period, the resulting transmitter release was halved by 3×10^–7 mol/l of the ₂-adrenoceptor agonist clonidine and increased about 2.5-fold by either 3×10^–7 mol/l of the ₂-adrenoceptor antagonist idazoxan, 5×10^–3 mol/l of the potassium channel blocker tetraethylammonium chloride (TEA) or 3×10^–7 mol/l phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). Phorbol-12-myristate-13-acetate-4-O-methylether, a compound which does not stimulate PKC, was ineffective. The stimulatory effect of PMA was antagonized by 7×10^–5 mol/l of the PKC inhibitor polymyxin B. No significant transmitter release was observed when either PMA or TEA was applied together with 10^–7 mol/l of the sodium channel blocker tetrodotoxin. Combinations of either idazoxan and TEA or PMA and TEA caused greater increased of the noradrenaline release than any individual drug given alone. Thus, different mechanisms of action seem to mediate the increase of noradrenaline release by action potential prolongation on the one hand and activation of PKC or inhibition of ₂-adrenoceptors on the other hand. In contrast, the effects of idazoxan and PMA were not additive which suggests a common mechanism of action. In atria pretreated for 10 min with 10^–4 mol/l N-ethylmaleimide, an alkylating agent which inactivates G_i-proteins, neither idazoxan nor PMA caused a significant increase of the stimulation-induced transmitter release, while TEA was still effective. When a train of four pulses, lasting 0.05 ms each, was applied during each refractory period, the resulting transmitter release was not modified by idazoxan or PMA, but was significantly increased by TEA. From these results, a scheme is proposed which, links the regulation of noradrenaline release by prejunctional ₂-adrenoceptors and protein kinase C via an influence on a common inhibitory G_i-protein.     

Influence of rat seminiferous tubules on Leydig cell testosterone production in vitro

The interaction of seminiferous tubules and Leydig cells was investigated in the rat in vitro. Crude collagenase-dispersed, or Percoll-purified, Leydig cells were incubated together with seminiferous tubules at different stages of the cycle, isolated by transillumination-assisted microdissection. The testosterone production was measured. Seminiferous tubules inhibited testosterone production of crude Leydig cell preparations, but induced a clear stimulation (30–100%) in Percoll-purified cells. The stimulation was maximal at stages VII and VIII of the cycle, significantly higher than at stages II–VI (P < 0.05). The stimulation by seminiferous tubules was observed both in basal and hCG-stimulated testosterone production. The effect was independent of FSH or GnRH action. These results demonstrate the presence of paracrine regulatory interaction between seminiferous tubules and Leydig cells, and are in agreement with the concept of a perferential androgen requirement of stages VII and VIII of the cycle.     

Effect of somatostatin-14 on duodenal mucosal bicarbonate secretion in guinea pigs

The role of somatostatin-14 in duodenal mucosal HCO ₃ ^– secretion was investigated in anesthetized, indomethacin-treated guinea pigs. Net HCO ₃ ^– output from the isolated, perfused (24 mM NaHCO₃ + 130 mM NaCl) proximal duodenum was measured during intravenous infusion (alone or in combination) of somatostatin-14, carbachol, vasoactive intestinal peptide (VIP), and prostaglandin E₂ (PGE₂). In homogenates of duodenal enterocytes, the effect of these agents on adenylate cyclase activity was studied. Basal duodenal HCO ₃ ^– secretion (3.5±0.2µmol/cm/10 min) was reduced dose dependently by somatostatin-14 (10^–11 mol/kg, 10^–9 mol/kg, and 10^–7 mol/kg). Carbachol, VIP, and PGE₂ (all 10^–8 mol/kg) increased basal duodenal HCO ₃ ^– secretion two- to threefold. Somatostatin-14 (10^–7 mol/kg) abolished the stimulatory effect of carbachol and VIP, but not that of PGE₂. Basal adenylate cyclase activity in isolated duodenal enterocytes (9.4±1.0 pmol cAMP/mg protein/min) was unaltered by somatostatin (10^–6 mol/liter) or carbachol (10^–3 mol/liter). VIP (10^–8 mol/liter) and PGE₂ (10^–7 mol/liter) increased adenylate cyclase activity two- to threefold, and these effects were unchanged by somatostatin-14 (10^–6 mol/liter). In conclusion, somatostatin-14 inhibits basal and carbachol- and VIP-stimulated duodenal HCO ₃ ^– secretion, and its mechanism of action is not via inhibition of adenylate cyclase activity in duodenal enterocytes.This study was supported by grants from the German-Israel Foundation for Scientific Research and Development (grant I-78-054.2/88), and the Israeli Ministry of Health.     

Effects of luteinizing hormone, follicle stimulating hormone, prostaglandin E1 and other hormones on adenosine-3':5'-cyclic monophosphate and testosterone production in rat testis tissues

The site and specificity of LH action on cAMP and testosterone production in rat testis have been investigated. In tissue from hypophysectomized rats LH added in vitro specifically stimulated cAMP production in interstitial tissue, whereas FSH added in vitro specifically stimulated cAMP production in seminiferous tubules. No synergistic effect of FSH and LH was found in either tissue. LH markedly stimulated testosterone production in the interstitial tissue whereas only a very small increase was obtained in the seminiferous tubules using tissue from hypophysectomized rats. These results suggest that seminiferous tubules do not significantly contribute to testis testosterone biosynthesis. No stimulatory effect of testosterone production attributable to FSH could be demonstrated in either tissue or in whole testis tissue when this hormone was added alone or with LH in vitro.LH-stimulated testosterone production in total testis tissue from intact rats, when expressed in terms of mg of interstitial tissue present, was two times higher compared with interstitial tissue obtained by dissection. Addition of seminiferous tubules to the interstitial tissue did not increase the testosterone production. In contrast cAMP production per mg interstitial tissue present was the same in all three preparations.Prostaglandin E₁ caused a small but significant increase in cAMP production in interstitial tissue but had no effect on testosterone biosynthesis. None of the other hormones tested (oestradiol-17β, ACTH, GH, prolactin, testosterone) had any effect on cAMP or testosterone production by rat testes preparations in vitro.     

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y₁₂ and EP3 receptors, act as antagonists at G_i-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G_i-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y₁₂ antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA₂ mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI₂, iloprost, PGD₂, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE₁ and PGE₂ as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y₁₂ antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.     

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1 −/− mice

Aims/hypothesis Islets or beta cells from Sur1^–/– mice were used to determine whether changes in plasma membrane potential (V_m) remain coupled to changes in cytosolic Ca²⁺ ([Ca²⁺]_i) in the absence of K_ATP channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca²⁺]_i influences oscillations in V_m in sur1^–/– beta cells.Methods Plasma membrane potential and ion currents were measured with microelectrodes and the patch–clamp technique. [Ca²⁺]_i was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations.Results Membrane depolarisation of Sur1^–/– islets by arginine or increased extracellular K⁺, elevated [Ca²⁺]_i and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1^–/– islets. Oscillations in V_m were influenced by [Ca²⁺]_i as follows: (1) elevation of extracellular Ca²⁺ lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca²⁺-dependent outward current that was augmented by increased Ca²⁺ influx through L-type Ca²⁺ channels; (3) Ca²⁺ depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1^–/– islets, elevating [Ca²⁺]_i and insulin secretion; (4) store depletion activated a Ca²⁺ influx that was not inhibitable by the L-type Ca²⁺ channel blocker D600.Conclusions/interpretation Although V_m is largely uncoupled from glucose metabolism in the absence of K_ATP channels, increased electrical activity leads to elevations of [Ca²⁺]_i that are sufficient to stimulate insulin secretion. In Sur1^–/– beta cells, [Ca²⁺]_i exerts feedback mechanisms on V_m by activating a hyperpolarising outward current and by depolarising V_m via store-operated ion channels.     

Effect of improved metabolic control on loss of kidney function in Type 1 (insulin-dependent) diabetic patients: an update of the Steno studies

Summary We re-examined 69 of the 70 patients entering the two independent Steno Studies of effects of improved metabolic control on progression of late diabetic complications. They were analysed according to an intent to treat after follow-up for 8 years (Steno Study 1) and 5 years (Steno Study 2). The glycaemic control had improved in the insulin infusion group compared with the conventional treatment group (mean HbA_1c) by 2.0±0.6% vs 0.7±1.2 in Steno Study 1 and by 1.8±1.2% vs 0.4±1.3 (p<0.01) in Steno Study 2. In the insulin infusion groups three patients had died during episodes of ketoacidosis. These were not caused by malfunction of the insulin infusion pumps. In the conventional treatment groups, three patients suffered five cardiovascular events causing two deaths. From the sixth month of Steno Study 1 the annual change of the glomerular filtration rate was –3.7 (–5.4 to –2.0) ml·min^–1·1.73 m^–2 vs –1.0 (–2.1 to –0.1) (conventional vs insulin infusion group, mean (95% confidence interval, p<0.01)). The change in urinary albumin excretion was associated with the glycaemic control (n=69, r=0.49, p<0.0002). No progression was observed among 32 patients with low range microalbuminuria (30 to 99 mg/24 h). Among the 19 patients with an initial albumin excretion between 100 and 300 mg/24 h, progression of complications was more frequent during conventional treatment (n=10) vs insulin infusion (n=9): Clinical nephropathy (10 of 10 vs 2 of 9, p<0.01) and arterial hypertension (7 of 10 vs 1 of 9, p<0.01). The glomerular filtration rate declined during conventional treatment by –23 (–42 to –4) ml·mm^–1·1.73 m^–2 (p<0.05) but not during insulin infusion (–13 (–31 to 5) NS). These results suggest that patients at risk of nephropathy should be offered near normal glycaemic control in order to preserve their kidney function.     

Effects of nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester on duodenal alkaline secretory and ulcerogenic responses induced by mepirizole in rats

The inhibition of nitric oxide (NO) production by NO synthase inhibitors stimulates HCO ₃ ^– secretion in the rat duodenal mucosa. Therefore, we examined the effects of N^G-nitro-l-arginine methyl ester (l-NAME, the NO synthase inhibitor) and nitroprusside (the exogenous NO donor) on the duodenal HCO ₃ ^– and ulcerogenic responses in anesthetized rats. Animals were administered mepirizole (200 mg/kg, subcutaneously) for induction of duodenal ulcers, and gastric acid and duodenal HCO ₃ ^– secretions were measured with or without pretreatment withl-NAME (5 mg/kg, intravenously) or nitroprusside (4 mg/kg, intravenously). Mepirizole increased acid secretion, decreased the acid-induced duodenal HCO ₃ ^– secretion, and induced hemorrhagic lesions in the proximal duodenum. The inhibition of NO production byl-NAME potentiated the acid secretory response, increased the duodenal HCO ₃ ^– secretion, and prevented the duodenal lesions, and these changes were all antagonized by simultaneous administration ofl-arginine (200 mg/kg, intravenously) but notd-arginine. On the other hand, nitroprusside slightly reduced the acid response but further decreased the HCO ₃ ^– output, resulting in aggravation of duodenal lesions induced by mepirizole. These data suggest that the inhibition of endogenous NO production by the NO synthase inhibitorl-NAME increases duodenal HCO ₃ ^– secretion and protects the duodenal mucosa against acid injury.     

High-Temperature Chemical Stability of Cr(III) Oxide Refractories in the Presence of Calcium Aluminate Cement

Al₂O₃-CaO-Cr₂O₃ castables are used in various furnaces due to excellent corrosion resistance and sufficient early strength, but toxic Cr(VI) generation during service remains a concern. Here, we investigated the relative reactivity of analogous Cr(III) phases such as Cr₂O₃, (Al_1−xCr_x)₂O₃ and in situ Cr(III) solid solution with the calcium aluminate cement under an oxidizing atmosphere at various temperatures. The aim is to comprehend the relative Cr(VI) generation in the low-cement castables (Al₂O₃-CaO-Cr₂O₃-O₂ system) and achieve an environment-friendly application. The solid-state reactions and Cr(VI) formation were investigated using powder XRD, SEM, and leaching tests. Compared to Cr₂O₃, the stability of (Al_1−xCr_x)₂O₃ against CAC was much higher, which improved gradually with the concentration of Al₂O₃ in (Al_1−xCr_x)₂O₃. The substitution of Cr₂O₃ with (Al_1−xCr_x)₂O₃ in the Al₂O₃-CaO-Cr₂O₃ castables could completely inhibit the formation of Cr(VI) compound CaCrO₄ at 500–1100 °C and could drastically suppress Ca₄Al₆CrO₁₆ generation at 900 to 1300 °C. The Cr(VI) reduction amounting up to 98.1% could be achieved by replacing Cr₂O₃ with (Al_1−xCr_x)₂O₃ solid solution. However, in situ stabilized Cr(III) phases as a mixture of (Al_1−xCr_x)₂O₃ and Ca(Al_12−xCr_x)O₁₉ solid solution hardly reveal any reoxidation. Moreover, the CA₆ was much more stable than CA and CA₂, and it did not participate in any chemical reaction with (Al_1−xCr_x)₂O₃ solid solution.     

Stepwise activation of the gonadotropic signal transduction pathway, and the ability of prostaglandin F2α to inhibit this activated pathwayto inhibit this activated pathway

Through selective activation of the gonadotropic signal transduction pathway, we have determined the probable site of the antigonadotropic effects of prostaglandin F_2α (PGF_2α) in the human granulosa-luteal cell (hGLC). The gonadotropic signal transduction pathway was activated at the level of the receptor (luteinizing hormone and β-adrenergic), stimulatory G protein (G_s), adenylate cyclase (AC), and protein kinase A (PKA) by human chorionic gonadotropin (hCG) and isoproterenol (Iso), cholera toxin (CTX), forskolin, and dibutryl cAMP (Db cAMP), respectively. Concomitantly, the ability of PGF_2α to inhibit progesterone production in response to the activation of this cascade at these different levels was examined. hGLCs were obtained from in vitro fertilization patients and were precultured for 8 d in Medium 199 supplemented with fetal bovine serum (M199; 10% FBS). Following the preculture period, cells were treated with either vehicle or one of the above activators of the gonadotropic pathway, either in the absence or presence of PGF_2α (in M199; No FBS). Following the treatment period, media were collected and assayed for progesterone by RIA. Prostaglandin F_2α (10⁻⁶ M) significantly inhibited hCG (1 IU/mL), Iso (10⁻⁵ M), CTX (1 μg/mL), and forskolin- (10⁻⁵ M) stimulated progesterone production. Conversely, PGF_2α did not inhibit progesterone production stimulated by a saturating concentration of Db cAMP (10⁻⁶ M). The ability of PGF_2α to inhibit hCG- or CTX-stimulated progesterone production was attenuated by pertussis toxin (PTX; 50 ng/mL). In conclusion, through a pertussis toxinsensitive G protein, PGF_2α inhibits progesterone production at a level below AC, and above the activation of PKA by cAMP.     

PGE2 reverses Gs-mediated inhibition of platelet aggregation by interaction with EP3 receptors,but adds to non-Gs-mediated inhibition of platelet aggregation by interaction with EP4 receptors

Prostaglandin E₂ (PGE₂) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3′5′-monophosphate (cAMP). PGE₂ reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a G_s-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE₂. Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE₂ were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through G_s-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE₂ reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE₂ is mediated via the EP3 receptor. In contrast, PGE₂ added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE₂ is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE₂ may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.