S-nitroso-N-acetylpenicillamine reduces leukocyte adhesion to type I collagen.

The initial step in the migration of neutrophils to the extravascular space is adhesion to the endothelium. We examined the effect of nitric oxide on this process by treating human neutrophils with S-nitroso-N-acetylpenicillamine (SNAP), a NO-producing compound. Since NO has been shown to increase the level of cGMP in other cell types, we used 8-Br-cGMP in order to mimic the effects of NO. Indeed, both these treatments resulted in a reduced adhesion of neutrophils to type I collagen coated surfaces. After a prolonged incubation with SNAP, the adhesion was the same as for untreated cells. SNAP incubation reduced the F-actin content in the cells whereas 8-Br-cGMP increased it, demonstrating different mechanisms of action on F-actin. These data suggest that endothelium-derived nitric oxide is an important endogenous modulator of neutrophil adhesion, but the effect is not mediated by a cGMP-dependent regulation of F-actin levels.

Effect of wall tension on DNA and protein synthesis in bovine mesenteric arteries in vitro.

The aim of this investigation was to study the effect of wall tension and calcium antagonists on DNA and protein synthesis in bovine mesenteric arteries in vitro. The wall tension of the bovine mesenteric arteries was raised by stretching the vessel wall perpendicular to the length axis of the vessel. DNA and protein synthesis were determined by measuring incorporation of 3H-thymidine into DNA and incorporation of 14C-leucine into protein respectively. Elevating the wall tension from 0.05 N to 0.5 N significantly increased 3H-thymidine incorporation and 14C-leucine incorporation after an incubation period of 3 hr. Stretch had no effect on the distribution of 3H-thymidine. The distribution of 14C-leucine was increased by stretch in regular medium and to a less extent in calcium-free medium, which suggest that stretch stimulates the membrane transport of 14C-leucine. When the tension was increased from 0.05 N to 0.5 N for 10 min. before the incubation with 3H-thymidine, no effect was found. One microM nifedipine or felodipine inhibited the increase in 3H-thymidine incorporation caused by stretching, while no effect was found on 14C-leucine incorporation. In calcium-free medium, stretch-induced DNA synthesis was completely abolished. 14C-Leucine incorporation was impaired in calcium-free medium but the stretch-induced increase still remained. The results suggest that mechanical force may play an important role in DNA synthesis and protein metabolism of vascular smooth muscle.

The cGMP modulator, LY83583 alters oxygen metabolites differently in cultured endothelial cells and isolated neutrophilic granulocytes.

The present study was designed to assess the effect of LY83583 on H2O2/O2- production from endothelial cells and neutrophils, as determined by chemiluminiscence generation in vitro. We found that LY83583 increased H2O2/O2-production from endothelial cells, but inhibited the H2O2/O2- production from phorbol myristate acetate-stimulated neutrophils. Furthermore, LY83583 consumed NADPH under certain conditions. Since neutrophils generate superoxide anion radicals via an NADPH-oxidase, we suggest that the reduction of chemiluminiscence, seen after addition of LY83583 to phorbol myristate acetate-stimulated neutrophils, is due to increased consumption of NADPH. In endothelial cells, NADPH is required as a co-factor in the generation of nitric oxide, which may interact with superoxide anion. A consumption in NAPDH would therefore be expected to decrease the production of nitric oxide and increase H2O2/O2-generation. The consumption of NADPH in endothelial cells could also cause reduced scavenger functions of the glutathion system, resulting in a further increase in H2O2 release.

Involvement of nitric oxide and peptides in the inhibitory non-adrenergic, non-cholinergic (NANC) response in bovine mesenteric artery.

The cyclic GMP mediated non-adrenergic non-cholinergic (NANC) relaxation in field stimulated bovine mesenteric artery and its modulation by various factors was studied. Electrical field stimulation of precontracted (Phe 2.5 microM or histamine 5 microM) bovine mesenteric arteries resulted in relaxations varying between 10-70% in different preparations. Tetrodotoxin (3 microM) completely blocked the inhibitory NANC response. Preincubation with high concentrations (100 microM-1 mM) of NG-nitro-L-arginine for 15 min. significantly reduced the relaxation induced by electrical field stimulation. Blockade of cyclooxygenases and prostaglandin synthesis by indomethacin had no effect on the relaxatory response to electrical field stimulation. Neither the alpha 2-adrenoceptor selective antagonist yohimbine (1 microM) nor the alpha 2-adrenoceptor selective agonist UK 14,304 (1 microM) had any significant effect on the electrical field stimulation-induced relaxation. Pertussis toxin (100 ng/ml) was without effect on relaxations elicited by electrical field stimulation. GTP in the concentration range 10 microM-1 mM slightly potentiated the relaxant response. N-carboxymethyl-Phe-Leu (an inhibitor of enkephalinase) or aprotinin (an inhibitor of several proteases) had no significant effect on the electrical field stimulation response. Addition of trypsin (100 U/ml) in combination with chymotrypsin (20 U/ml) significantly reduced the electrical field stimulation-induced relaxation. In the present study we have found indications for the involvement of nitric oxide and possibly also peptides in mediating the inhibitory NANC response (relaxation) in bovine mesenteric arteries.

Lactate accumulation in isolated hypoxic rat ventricular myocardium: effect of different modulators of the cyclic GMP system.

Different agents which are known to increase tissue levels of cyclic guanosine 3':5'-monophosphate (cGMP), were found to decrease the lactate accumulation induced by hypoxia in isolated, non-beating rat myocardium from the right ventricle. One microM sodium nitroprusside increased the intracellular cGMP content 4 times during hypoxic conditions, and after 5 min. of hypoxia the intracellular lactate accumulation decreased by about 20%. 0.1 microM atrial natriuretic peptide increased cGMP 10 times during hypoxic conditions and decreased the lactate accumulation by about 40%. The reduction in lactate accumulation was mimicked by 1 mM 8-Br-cGMP and by Zaprinast (10 microM), a selective inhibitor of cGMP phosphodiesterase, which reduced lactate accumulation by 60% and 45%, respectively. Glyceryl trinitrate (1 nM and 1 microM) caused a slight increase in lactate accumulation both during normooxic and hypoxic conditions, but had no effect on tissue levels of cGMP. In conclusion, the results indicate that cyclic GMP reduces lactate accumulation in hypoxic, non-beating rat heart ventricular muscle and suggests that atrial natriuretic peptide, which is released from atrial tissue, may have beneficial metabolic effects on the heart.

Tissue disposition of glyceryl trinitrate, 1,2-glyceryl dinitrate, and 1,3-glyceryl dinitrate in tolerant and nontolerant rats.

The tissue distribution of glyceryl trinitrate (GTN) and its two dinitrate metabolites 1,2-glyceryl dinitrate (1,2-GDN) and 1,3-glyceryl trinitrate (1,3-GDN), was studied in GTN-tolerant and nontolerant male Sprague-Dawley rats. The concentrations of GTN, 1,2-GDN, and 1,3-GDN were measured in plasma, heart, brain, liver, aortic tissue, and adipose tissue at various time points after a subcutaneous dose of GTN (50 mg/kg). At the first time point (5 hr), concentrations of GTN, 1,2-GDN, and 1,3-GDN in plasma were equal for tolerant and nontolerant rats, but the elimination rate was altered for the tolerant rats as compared with nontolerant rats. In adipose tissue, the concentration of GTN was significantly higher as compared with concentrations of the dinitrate metabolites. In contrast, the other tissues studied showed significantly higher concentrations of the GDNs when compared with GTN. The 1,3-GDN/1,2-GDN ratio decreased with time for both tolerant and nontolerant rats. This study indicates that long-term GTN administration results not only in tolerance development, but also in altered pharmacokinetics of GTN, 1,2-GDN, and 1,3-GDN. The results also show that the 1,3-GDN/1,2-GDN ratio is dependent on the GTN concentration.

Comparative in vitro study of a series of organic nitroesters: unique biphasic concentration-effect curves for glyceryl trinitrate in isolated bovine arterial smooth muscle and lack of stereoselectivity for some glyceryl trinitrate analogues.

Four different organic nitroesters, constituting a homologous series based on unbranched polyalcohols, were compared with regard to in vitro relaxation of isolated bovine mesenteric arteries contracted with 2.5 microM phenylephrine. The organic nitroesters included ethylene glycol dinitrate (EGDN), dinitratopropane (DPN), glyceryl trinitrate (GTN), and tetranitratobutane. Glyceryl trinitrate exhibited a biphasic concentration-effect relationship, with pD2 values of 11.5 +/- 0.5 and 7.2 +/- 0.2 for the high-pD2 and low-pD2 component of the relaxation curve, respectively. The high-pD2 and low-pD2 component contributed 28 and 72% of the maximal response, respectively. EGDN, DPN, and tetranitratobutane induced monophasic concentration-effect curves with pD2 values of 7.4 +/- 0.1, 7.8 +/- 0.2, and 6.9 +/- 0.6, respectively. Stereoisomeric forms of DPN and tetranitratobutane showed no difference with regard to relaxing potency in bovine mesenteric artery. GTN has a partly unique mechanism for vascular smooth muscle relaxation that distinguishes this compound from other related organic nitroesters.

Epithelial modulation of allergen and drug effects in guinea pig airways.

The effect of egg albumin (EA) challenge on tracheal tube preparations from sensitized guinea pigs was studied with regard to EA permeability, histamine release and penetrability, and the contractile response of the preparation. We used a plethysmographic method that allowed simultaneous measurement of changes in smooth muscle tension and collection of samples for determination of mediators. Our results clearly show that epithelial damage potentiates the contractile response to histamine, potassium ions, and acetylcholine. Epithelial damage did not alter the maximal contractile response in preparations challenged with high antigen concentrations (EA, 1 mg/ml), but histamine release measured in the perfusate increased substantially. The permeability of the preparations to EA was greater when the epithelium was damaged. No increase in the permeability in response to the EA challenge was observed. The present study has demonstrated that guinea pig airway epithelium constitutes a barrier for both antigen and drugs. We also present a method for recording contractile responses from intact whole tracheal preparations, in which the epithelium can still act as a barrier, as is the case in vivo.

Heterogeneity in human soluble guanylate cyclase due to alternative splicing.

Two forms of the smaller subunit of the human soluble guanylate cyclase enzyme have been cloned by using PCR. One of the clones (HSGC-1) is identical to bovine and rat lung smaller subunit cyclase. However, the other (HSGC-2) is lacking 33 amino acids. Comparison of its sequence with published partial genomic sequences of bovine guanylate cyclase indicates that HSGC-2 is formed due to alternative splicing.

Tissue levels of glyceryl trinitrate and cGMP after in vivo administration in rat, and the effect of tolerance development.

The present study compares the tissue distribution of glyceryl trinitrate (GTN) in plasma, heart, brain, aortic tissue, and adipose tissue from GTN tolerant and GTN nontolerant rats at various time points. Furthermore, the cGMP levels in brain, heart, and aortic tissue were studied at various time points as well as the concentration-effect relationship for GTN in aorta isolated at different time points after the last exposure to GTN. Concentrations of GTN were found to be higher in all tissues studied as compared with plasma, and the concentrations of GTN were higher in tissues from tolerant rats as compared with nontolerant rats, except for aortic tissue. Concentration-effect curves obtained in vitro showed that aortic smooth muscle was still tolerant 24 h after the last dose of GTN. The cGMP level in brain was significantly increased by 40% 2 h after a single dose of GTN (50 mg/kg) and in aortic tissue by 50% at 15 min and at 2 h after a single dose of GTN (50 mg/kg). There was no effect on cGMP in brain, while an increase was seen in aortic tissue 15 min after the last dose in tolerant animals. No change in cGMP level was seen in heart neither in nontolerant nor in tolerant animals at 15 min and at 2 h. No effect on cGMP levels in brain, heart, and aortic tissue was seen 8, 16, and 24 h after exposure to GTN in either tolerant or nontolerant rats. In conclusion, GTN does not involve the cGMP system in heart, and tolerance development caused a less pronounced GTN-induced cGMP increase in aortic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of nitric oxide and cyclic GMP as mediators of endothelium-independent neurogenic relaxation in bovine mesenteric artery.

Electrical field stimulation (EFS) of phenylephrine-contracted bovine mesenteric arteries pretreated with guanethidine elicited a relaxation that amounted to roughly 40%. This relaxation was sensitive to tetrodotoxin pretreatment, suggesting a neurogenic origin. The EFS-induced relaxation was correlated to an increase in cGMP level, from 14.2 +/- 2.5 pmol/g wet wt in nonstimulated arteries to 31.6 +/- 3.4 pmol/g wet wt after 1 minute of EFS. cAMP values were not affected by EFS. Methylene blue (5 microM) and the compound LY 83583 (10 microM), inhibitors of soluble guanylate cyclase, inhibited the EFS-induced relaxation by 60% and 50%, respectively. Zaprinast (1 microM), a selective inhibitor of cGMP degradation, significantly (p = 0.005) potentiated the EFS-induced relaxation. The relaxation induced by EFS in bovine mesenteric arteries exhibits characteristics similar to the relaxations evoked by organic nitroesters and endothelium-dependent vasodilators, both of which are suggested to be mediated by cGMP and probably with nitric oxide as the common activator of the cGMP system. The possible involvement of nitric oxide as a mediator of EFS-induced relaxations was investigated with the use of known modulators of endogenous nitric oxide production. Preincubation of the arteries with 1 mM arginine or 1 mM N-alpha-benzoyl-L-arginine, both reported to potentiate endogenous nitric oxide production, or 5 mM L-canavanine, 0.25 mM NG-monomethyl-L-arginine, or 0.1 mM NG-nitro-L-arginine, alleged inhibitors of endogenous nitric oxide production, were without effect on the relaxation induced by EFS. However, pyrogallol, a generator of superoxide anions, was a potent inhibitor of relaxations induced by EFS in bovine mesenteric arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Antinociceptive actions of alpha 2-adrenoceptor agonists in the rat spinal cord: evidence for antinociceptive alpha 2-adrenoceptor subtypes and dissociation of antinociceptive alpha 2-adrenoceptors from cyclic AMP.

The antinociceptive actions of intrathecal injections of two alpha 2-adrenergic agonists, UK-14,304 and guanfacine, were investigated in rats after pretreatment of the animals with the noradrenaline neurotoxin N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP4) 14 days in advance. The chronic noradrenaline depletion induced by DSP4 caused a marked increase in sensitivity of the antinociceptive action of UK-14,304 in the tail-flick test. By contrast, the antinociceptive effect of guanfacine was not appreciably affected by the DSP4 treatment. The antinociceptive effects of both UK-14,304 and guanfacine were blocked by intraperitoneal injections of yohimbine, a result indicating that both drugs induced their actions by activating alpha 2-adrenoceptors. Both UK-14,304 and guanfacine were found to reduce the production of cyclic AMP (cAMP) in the spinal cord, as determined using an in vitro radioisotopic method. The cAMP inhibitory effects of both agonists were effectively blocked by yohimbine, but not by prazosin, a finding indicating the alpha 2-adrenergic nature of the response. However, the cAMP inhibitory effect of UK-14,304 was not potentiated by pretreatment with DSP4, a finding in marked contrast with the strong potentiation of the antinociceptive action of UK-14,304 induced by the chronic depletion of endogenous noradrenaline. Moreover, intrathecal injections of forskolin, which increased the endogenous levels of spinal cord cAMP fivefold, did not modify the antinociceptive effects of UK-14,304 or guanfacine in neither normal nor DSP4-treated animals. It is suggested that there exist pharmacologically differing alpha 2-adrenergic receptor pathways capable of mediating antinociceptive effects at the level of the spinal cord. The cAMP inhibitory actions of spinal cord alpha 2-adrenoceptors appear not to be directly linked with the antinociceptive actions of these receptors.

Relaxation of bovine mesenteric arteries by glyceryl trinitrate and other nitro-compounds: evidence for partly different mechanisms of action.

The effect of pertussis toxin (PTX) and the cyclic GMP lowering agent LY83583 on the relaxatory response induced by glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN), isosorbide-5-mononitrate (IS-5-MN) and sodium nitroprusside (SNP) in bovine mesenteric artery (BMA) was investigated. Pretreatment with PTX (100 ng/ml; 2 hr induced a 100-fold right shift of the concentration-effect curve for GTN, while no effect on the relaxatory response elicited by ISDN, IS-5-MN or SNP was seen. The relaxatory effect of all the substances tested was markedly reduced by LY83583 (10 microM). The basal cGMP level as well as the GTN induced increase in cGMP were markedly reduced when BMA was exposed to LY83583. The substance also reduced the activation of soluble guanylate cyclase by SNP. Based on the different sensitivity towards PTX it is suggested that GTN induces vascular smooth muscle relaxation by a partly different mechanism than ISDN, IS-5-MN and SNP. As far as the GTN induced relaxation is concerned the sensitivity towards PTX indicates the involvement of regulatory component, possibly a G-protein. However, cyclic GMP seems to play a crucial role in mediating the relaxatory response of all the substances tested since the cGMP-lowering agent LY83583 markedly inhibited the relaxant response induced by all the vascular relaxant agents investigated.

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.

Effects of sodium nitrite on ultraviolet light-induced relaxation and ultraviolet light-dependent activation of guanylate cyclase in bovine mesenteric arteries.

It was demonstrated that precontracted strips from different bovine mesenteric arteries showed variation in sensitivity to ultraviolet radiation (366 nm). Some strips relaxed when they were exposed to ultraviolet light, others showed no sensitivity at all and, finally, some showed contraction. However, all arteries relaxed when they were irradiated with UV-light in the presence of 10 microM NaNO2. Ultraviolet radiation (366 nm) increased the activity of guanylate cyclase in crude homogenate from bovine mesenteric arteries by about 20-fold in the presence of NaNO2, while UV-light in the absence of sodium nitrite had no effect on the guanylate cyclase activation. These results support the notion that nitrite may be essential for vascular smooth muscle relaxation by UV-light, possibly through the release of nitric oxide.

Studies of the effect of glyceryl trinitrate and cyclic GMP on calcium turnover in bovine mesenteric artery.

It was recently observed that the relaxation induced by glyceryl trinitrate (GTN) showed a biphasic concentration-response curve; a high-sensitivity component represented by concentrations less than 1 nM and a low-sensitivity component represented by concentrations greater than 1 nM. The effect of two glyceryl trinitrate concentrations (0.1 nM and 1 microM) were tested on the uptake of 45Ca2+ to tissue pieces of bovine mesenteric arteries (BMA) as well as on the uptake of 45Ca2+ to a microsomal preparation of BMA. The effect of GTN and 8-Br-cGMP was also studied on the IP3-induced release of Ca2+ from the microsomal preparation preloaded with 45Ca2+. The influence of IP3 and GTN on the activity of Ca2(+)-ATPase in the microsomal preparation was tested as well. The phenylephrine-stimulated uptake of Ca2+ to tissue pieces of BMA was significantly reduced by the high GTN-concentration (1 microM) but not by the lower concentration. The uptake of Ca2+ to the microsomal preparation was significantly stimulated by the two GTN-concentrations tested, as well as by 8-Br-cGMP (0.1 mM). The calcium release induced by IP3 (1 microM) from the microsomal preparation was inhibited by both the low and the high GTN-concentration and by 8-Br-cGMP (0.1 mM). The Ca2(+)-ATPase activity was stimulated by both GTN-concentrations tested while it was inhibited by IP3. It is concluded that GTN is able to induce a reduction of the free intracellular Ca2+ by several mechanisms, which are of importance for the relaxation represented by the high-affinity component. The low-affinity component in addition reduces the inflow of Ca2+ over the plasma membrane.

Cyclic guanosine monophosphate metabolism in human amnion cells trisomic for chromosome 21.

An extra copy of chromosome 21, a small chromosome or a specific segment of it, is the cause of the disorder known as Down's syndrome (DS). Genes mapped to this chromosome include superoxide dismutase-1 (SOD-1) along with other enzymes. Gene dosage effects have been shown for some of these enzymes, including SOD-1. Increased SOD-1 has been suggested to stimulate the cGMP-forming enzyme, guanylate cyclase (GC). In the present study we have used amnion cells from DS subjects and normal subjects in order to indirectly test the effects of SOD-1 on the cGMP metabolism. We have measured the cAMP and cGMP content, SOD-1 activity, GC activity and cGMP phosphodiesterase (G-PDE) activity in amnion cells from DS subjects and normal subjects, respectively. The levels of cGMP in DS amnion cells were lower than in normal cells, although the SOD-1 activity was higher in DS amnion cells. Furthermore, the GC activity and the G-PDE activity were found to be lower in the trisomic cells. Our results do not support the suggestion that SOD-1 has a stimulatory effect on the GC activity.