Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues.

1. Recent studies have suggested that the generation of nitric oxide (NO) and hydrogen peroxide (H2O2) by islet NO synthase and monoamine oxidase, respectively, may have a regulatory influence on insulin secretory processes. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presence of various pharmacological agents known to perturb the intracellular levels of NO and the oxidation state of SH-groups. 2. The NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) dose-dependently increased L-arginine-induced insulin release. D-Arginine did not influence L-arginine-induced insulin secretion. However, D-NAME which reportedly has no inhibitory action on NO synthase, modestly increased L-arginine-induced insulin release, but was less effective than L-NAME. High concentrations (10 mM) of D-arginine as well as L-NAME and D-NAME could enhance basal insulin release. 3. The intracellular NO donor, hydroxylamine, dose-dependently inhibited insulin secretion induced by L-arginine and L-arginine+L-NAME. 4. Glucose-induced insulin release was increased by NO synthase inhibition (L-NAME) and inhibited by the intracellular NO donor, hydroxylamine. Sydnonimine-1 (SIN-1), an extracellular donor of NO and superoxide, induced a modest suppression of glucose-stimulated insulin release. SIN-1 did not influence insulin secretion induced by L-arginine or the adenylate cyclase activator, forskolin. 5. The intracellular 'hydroperoxide donor' tert-butylhydroperoxide in the concentration range of 0.03-3 mM inhibited insulin release stimulated by the nutrient secretagogues glucose and L-arginine. Low concentrations (0.03-30 microM) of tert-butylhydroperoxide, however enhanced insulin secretion induced by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). 6. Islet guanosine 3':5'-cyclic monophosphate (cyclic GMP) content was not influenced by 10 mML-arginine or tert-butylhydroperoxide at 3 or 300 micro M but was markedly increased (14 fold) by a high hydroxylamine concentration (300 micro M). In contrast, islet adenosine 3':5'-cyclic monophosphate (cyclicAMP) content was increased (3 fold) by L-arginine (10 mM) and (2 fold) by tert-butylhydroperoxide(300 micro M).7. Our results strongly suggest that NO is a negative modulator of insulin release induced by the nutrient secretagogues L-arginine and glucose. This effect is probably not mediated to any major extent by the guanylate cyclase-cyclic GMP system but may rather be exerted by the S-nitrosylation of critical thiol groups involved in the secretory process. Similarly the inhibitory effect of tert-butylhydroperoxide is likely to be elicited through affecting critical thiol groups. The mechanism underlying the secretion promoting action of tert-butylhydroperoxide on IBMX-induced insulin release is probably linked to intracellular Ca2+-perturbations affecting exocytosis.8. Taken together with previous data the present results suggest that islet production of low physiological levels of free radicals such as NO and H202 may serve as important modulators of insulin secretory processes.

Protein phosphorylation substrates in normal and neoplastic squamous epithelia of the human upper aero-digestive tract.

Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriched preparations from squamous cell carcinomas and normal mucosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 52-55 kDa. Addition of Ca2+ or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca2+/phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heat-stable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors.

Tyrosine kinase activities in normal and neoplastic epithelia tissue of the human upper aero-digestive tract.

Tyrosine kinase activity was studied in the crude cytosolic and particulate fraction of normal mucosa and squamous cell carcinomas of the upper aero-digestive tract. In the presence of exogenously added phosphorylation substrate (Glu,Tyr4:1), the cytosolic tyrosine kinase activity was 6-fold higher in tumors compared to normal mucosa (p = 0.001), and in the particulate fraction the increase was 8-fold in tumors compared to normal mucosa. Different proposed tyrosine kinase inhibitors, including genistein, quercetin and the alpha-cyanocinnamide ST 638, were tested for their ability to inhibit phosphorylation of the synthetic tyrosine phosphorylation substrate. Phosphorylation of Glu,Tyr4:1 in tumors (cytosolic fraction) was reduced to 77.8 +/- 8.7% of the control value by 10 microM ST 638 (p less than 0.05), and to 50.7 +/- 10.4% by 100 microM quercetin (p less than 0.01). In normal mucosa (cytosolic fraction) the corresponding values were 41.7 +/- 16.6% in the presence of 10 microM ST 638 (p less than 0.05) and 32.1 +/- 5.8% in the presence of 100 microM quercetin (p less than 0.05). These inhibitors had no effect on the tyrosine kinase activity in the particulate fractions. Phosphorylation of endogenous proteins in the crude cytosolic fraction was evaluated by SDS-polyacrylamide gel electrophoresis after alkali treatment of the gels. Autoradiography of the gels treated in this manner revealed bands corresponding to phosphorylated proteins with apparent molecular weight of 18, 23, 37-38, 42-44 (double band), 53-55 (double band), 61 and 92-94 (double band) kD. Quercetin (100 microM) markedly reduced the phosphorylation of these proteins, while no effect of ST 638 could be seen. Heparin (20 micrograms/ml) stimulated the phosphorylation of three proteins with apparent molecular weight of 39 and about 72 kD, respectively, and inhibited the phosphorylation of 2 proteins with molecular weight of 92 and 53 kD in tumors. These features were observed in both tumors and normal tissue, with the exception that heparin only stimulated the 72 kD band in normal mucosa and that the phosphorylation was markedly higher in tumors. In summary, our results show an increased tyrosine phosphorylation in squamous cell carcinomas of the upper aero-digestive tract compared to normal oral mucosa. These differences and their origin might be of vital importance in the regulation of events leading to malignant transformation.

Protein kinase activities in neoplastic squamous epithelia and normal epithelia from the upper aero-digestive tract.

In the present study the activities of three different protein kinase were determined in squamous cell carcinoma from the upper aero-digestive tract, and compared with the activities in normal oral mucosa. The protein kinases investigated are: a) cAMP-dependent protein kinase; b) cGMP-dependent protein kinase, and c) casein kinase II. The basal protein kinase activity, when histone IIa was used as substrate, was about 3-fold higher in tumors, as compared to normal mucosa, in the soluble fraction (32.0 +/- 4.2 and 10.9 +/- 2.4 pmol 32P/mg prot. X min, respectively). In the particulate fraction the basal protein kinase activity was about 9 times higher in tumors as compared to normal mucosa (19.4 +/- 5.2 and 2.1 +/- 0.3 pmol 32P/mg prot X min, respectively). The protein kinase activity in the presence of cyclic nucleotide (cAMP/cGMP) minus the basal protein kinase activity was taken as the cAMP- and the cGMP-dependent protein kinase activity, respectively. Maximal protein kinase activity was obtained in the presence of 0.5 microM of cyclic nucleotide both in squamous cell carcinoma and normal mucosa. In the cytosolic fraction the cAMP-dependent protein kinase activity was 33.9 +/- 13.0 pmol 32P/mg prot. X min in tumors, and 28.2 +/- 5.8 pmol 32P/mg prot. X min in normal tissue, after stimulation with 0.5 microM cAMP. The cGMP-dependent protein kinase activity was 5-10% of the cAMP-dependent protein kinase activity, and no concentration-dependent stimulation with cGMP was seen. The cGMP-dependent protein kinase activity in the presence of 0.5 microM cGMP was 2.4 +/- 1.3 and 1.8 +/- 0.6 pmol 32P/mg prot. X min in tumors and normal mucosa, respectively. Casein kinase II activity was determined only in the cytosolic fraction and was found to be 3-fold higher in tumors as compared to normal mucosa (31.8 +/- 5.2 and 8.6 +/- 3.5 pmol 32P/mg prot X min, respectively). This study shows a general increase in histone phosphorylation and casein kinase activity in neoplastic squamous epithelia compared to normal epithelia. No evidence for an increase in cyclic nucleotide dependent protein kinase activities in neoplastic squamous epithelia was found. This study thus supports the idea that phosphorylation/dephosphorylation reactions may play an important role in the control of cell growth, differentiation and proliferation.

Protein kinase C activity in normal and neoplastic squamous epithelia from the upper aero-digestive tract.

In the present study the protein kinase activity was determined in surgical specimens of squamous cell carcinoma from the upper aero-digestive tract, and the activity was compared with the activity in normal mucosa obtained from the same location in patients undergoing surgery for non-neoplastic diseases. The basal protein kinase activity in the soluble fraction was about 15-fold higher in tumors as compared to in normal mucosa. The difference between protein kinase activity in the presence of Ca2+ and presence/absence of phosphatidylserine/diolein was taken as the activity of protein kinase C. Protein kinase C activity in the presence of 0.5 mM Ca2+ was 10.0 +/- 2.1 pmol 32P/mg prot. x min in tumors (n = 19) and 2.4 +/- 0.7 pmol 32P/mg prot. x min. in normal mucosa (n = 6). A similar difference was obtained with 1 mM Ca2+. An even greater difference in protein kinase C activity was seen when the soluble enzyme had been partially purified by ion-exchange chromatography on DE-52 columns. In this case a 7-fold higher protein kinase activity was found in tumors as compared to in normal mucosa. In the particulate fraction the basal protein kinase activity was about 3-fold higher in tumors as compared to in normal mucosa. Protein kinase C activity in the particulate fraction, defined as described above, was 10.7 +/- 4.1 pmol 32P/mg prot. x min in tumors and 6.1 +/- 4.4 pmol 32P/mg prot. x min in normal mucosa. These results show a significantly higher activity in both total protein kinase activity and protein kinase C activity in epithelial tumors from the upper aero-digestive tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of tolerance to glyceryl trinitrate in an isolated human peripheral vein and its relation to cyclic GMP metabolism.

Human peripheral vein (v. saphena magna) was exposed in vitro to glyceryl trinitrate (GTN) 0.1 mM for 1 h. The subsequent relaxant effect of GTN (10nM-0.1 mM) on vessels contracted by serotonin (0.25 microM) was significantly reduced on vessels preexposed to GTN as compared to control vessels, indicating a development of partial tolerance as far as the vascular smooth muscle relaxant effect is concerned. The impaired relaxant effect was paralleled by a reduced increment of intracellular cyclic GMP. This is, in turn, probably a consequence of both a diminished guanylate cyclase activity and an elevated phosphodiesterase activity in the GTN-tolerant vessels. The relaxant activity as well as the level of intracellular cyclic GMP were restored in GTN-tolerant vessels by dipyridamole (5 microM), an agent with phosphodiesterase inhibiting properties.

The relaxant effect of glyceryltrinitrate on isolated human peripheral vein and its relation to cyclic GMP metabolism.

The relaxant effect of glyceryltrinitrate (GTN) on human vena saphena magna was studied in vitro. Vessels contracted by serotonin (0.25 microM) and phenylephrine (0.1 mM) were relaxed to the same extent (EC50 = 10 microM) by GTN, whereas in 100 mM K+-depolarized vessels the relaxation was significantly lower. The relaxant effect produced by GTN was preceded by an elevation of cyclic guanosine-3',5'-monophosphate (cGMP). For 0.1 mM GTN there was a 3-fold increase in cGMP after 3 min. A correlation between relaxation and increase in cGMP was established. When GTN was combined with dipyridamole (5 microM) the relaxant effect of GTN was significantly greater (EC50 = 0.1 microM). Phosphodiesterase inhibition, as a possible mechanism behind the observed better relaxation for the combination (GTN+dipyridamole), is briefly discussed. In conclusion, the relaxant effect of GTN on isolated human vena saphena magna seems to be dependent on the contractile stimuli used, increased by the addition of DIP and to be mediated via cGMP.

Guanylate cyclase activity in normal and neoplastic squamous epithelia from the upper aero-digestive tract.

Cyclic nucleotides (cAMP, cGMP) are suggested to participate in the regulation of cell growth and differentiation. Guanylate cyclase is the enzyme which catalyzes the synthesis of cGMP. The basal guanylate cyclase activity was slightly higher in well-differentiated squamous cell carcinomas than in normal mucosa, but was two-fold higher in papillomas. Poorly differentiated squamous cell carcinoma did not show any increased basal activity. Stimulation with nitroprusside (NP) resulted in a 20% increased activity for normal mucosa and a 30% increase for poorly differentiated carcinomas, whereas enzyme prepared from well-differentiated squamous carcinomas and papillomas showed a two-fold increase.

Effects of retinylacetate on the kinetics of rat liver guanylate cyclase: possible interaction with sulfhydryl groups.

The effect of retinylacetate (RA) was investigated on rat liver guanylate cyclase stimulated by nitroprusside (NP). The stimulated enzyme seemed to adhere to Michaëlis-Menten kinetics with an apparent Km for MnGTP of 0.10 mM and a Vmax of 410 pmol cGMP/min. X mg prot. RA (0.1-1mM) dose-dependently inhibited the enzyme stimulated by NP (0.1-10 mM). In the presence of 1 mM RA the activity was only 10% of the control activity. The inhibitory action of RA seemed to be non-competitive since it depressed Vmax without affecting the Km for MnGTP. In contrast, however, RA was instead found to stimulate the enzyme when low concentrations of NP (0.01-0.1 mM) were used. The concentration-activity curve for NP was bell-shaped showing an optimum at 0.5 mM. The inhibition induced by RA could not be surmounted by increasing the NP concentration indicating that RA did not compete with NP. A bell-shaped activity curve was also seen when the enzyme activity was measured in the presence of increasing Mn2+ concentrations and during these conditions RA also caused inhibition. In the presence of the sulphydryl reductant dithiothreitol (DTT), the NP concentration needed for optimal enzyme activation was about 100-fold less than in the absence of DTT. The maximal enzyme activity was also slightly increased. In the presence of DTT, RA was much less effective to induce inhibition of the stimulated enzyme, than when DDT was absent. (In the presence of DTT 1 mM RA caused only 30% inhibition compared to 90% inhibition in its absence).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenaline toxicity in mice: sensitization of alpha 1 adrenoreceptors by nitroglycerin.

In mice, injected subcutaneously with nitroglycerin (GTN) for 12 days, adrenaline exhibited an increased toxicity. The LD50 value for adrenaline in control animals was 11.1 mg/kg b.wt. In GTN-treated animals the LD50 value for adrenaline, measured 3 days after the last injection of GTN, was 9.1 mg/kg b.wt. (P = 0.05). In the animals sensitized with GTN, the adrenergic alpha-receptor blocker phentolamine (1 or 10 mg/kg b.wt.) protected from the lethal action of adrenaline (P = 0.06 and P = 0.001, respectively). A low dose (1 mg/kg b.wt.) of the adrenergic beta receptor blocker propranolol, was without effect while a higher dose (10 mg/kg b.wt.) potentiated the toxicity of adrenaline (P = 0.007). The alpha 1 adrenoreceptor antagonist, prazosin, (1 or 10 mg/kg b.wt.) was found to be highly effective in protecting the GTN-sensitized mice towards adrenaline (P = 0.003 and P = 0.001, respectively). By contrast, the alpha 2 adrenoreceptor antagonist, yohimbine, (1 or 10 mg/kg b.wt.) was much less effective (P = 0.988 and P = 0.111, respectively). It is concluded that the lethal action of adrenaline was caused by stimulation of alpha 1 adrenoreceptors, and that long term treatment with GTN caused a sensitization of these receptors in mice. The possible relevance of this finding for the reported withdrawal symptoms and sudden death phenomenon in nitroglycerin-exposed industrial workers is discussed.

Inhibitory effects of different retinoids (vitamin A analogues) on the stimulated rat liver guanylate cyclase activity.

Vitamin A and its analogues show the ability to prevent malignant cell transformation on induction with different carcinogens, such as nitrosamines. Cyclic GMP has been proposed as a positive modulator of malignant cell growth and the cGMP-forming enzyme guanylate cyclase is strongly stimulated by e.g. nitrosamines. In this study, we found that retinylacetate and retinal were very potent inhibitors of the stimulated guanylate cyclase. When a series of structurally different retinoids were tested in the same system, a wide range of inhibitory activity on guanylate cyclase was found for the different retinoids with some being completely ineffective. The most potent inhibitor was retinylacetate. Furthermore, the inhibitory profile of the retinoids on the guanylate cyclase did not seem to correlate to their in vivo activity as antineoplastic agents, as described in the literature. We therefore conclude that there does not exist a general connection between the anticancer activity and the guanylate cyclase inhibition of the retinoids. However, it can not be excluded that the guanylate cyclase inhibition might be of importance for the antineoplastic activity for some of the retinoids.