A study of effects of mouthwash on the human oral mucosae: with special references to sites, sex differences and smoking

Nitric oxide has been shown to decrease myocardial contractility and O2 consumption. This study was designed to evaluate the hypothesis that nitric oxide-mediated increases in cyclic GMP require elevated cyclic AMP to produce cardiac depression. Using isolated, Langendorff-perfused rat hearts, we determined the effects of intracoronary nitroprusside (NP, 1 and 10 mM) in the absence and presence of isoproterenol (ISO, 10(-8) M) on cardiac function, O2 consumption, cyclic GMP and cyclic AMP. ISO, with and without NP, increased cyclic AMP (from 287 +/- 21 to 477 +/- 33 pmol/g) without altering cyclic GMP. Left-ventricular pressure increased from 97 +/- 12 to 178 +/- 9 mm Hg and dP/dtmax from 1,786 +/- 275 to 4,049 +/- 354 mm Hg/s. NP increased cyclic GMP (from 4 to 30 pmol/g) in both the absence and presence of ISO, but NP did not alter cyclic AMP. Without ISO, NP insignificantly altered left-ventricular pressure; however, in the presence of ISO, NP significantly decreased left-ventricular pressure by -25 +/- 4 mm Hg and decreased dP/dtmax by -619 +/- 142 mm Hg/s. Isoproterenol increased O2 consumption, but the changes with NP were not significant. When this study was repeated in the presence of LY83583, a guanylate cyclase inhibitor, NP still produced cardiac depression in the presence of ISO. Therefore, cardiodepressant effects of NP were only observed against a background of inotropic stimulation with ISO. However, effects of NP on contractility were unrelated to increases in cyclic GMP or cyclic GMP-induced changes in cyclic AMP.     

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10(-1) M), NO (10(-10) - 10(-6) M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry. 2. Zaprinast (10(-4) M), increased NO-stimulated levels of cyclic GMP by 3-20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. 3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10(-5) M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10(-10) - 10(-8) M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10(-7) - 10(-6) M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10(-7) - 10(-5) M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. 4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.     

Hemodynamic effects of a calcium channel promoter, BAY y 5959, are preserved after chronic administration in ischemic heart failure in conscious dogs

BAY y 5959 is a dihydropyridine derivative that binds to L-type calcium channels in a voltage-dependent manner and promotes calcium entry into the cell during the plateau of the action potential by influencing mean open time. Because myofilament responsiveness to calcium is preserved in congestive heart failure (CHF), the inotropic responsiveness to this compound should be preserved in CHF, and tolerance should not develop despite long-term treatment. To test these hypotheses, CHF was induced in 14 chronically instrumented dogs by daily (30 +/- 5 days) intracoronary microsphere injections. The effects of BAY y 5959 (2-h i.v. infusions of 3 microg/kg/min and 10 microg/kg/min) were determined before heart failure, after heart failure was established and then 2 h after the end of a 5-day continuous BAY y 5959 intra-atrial infusion. Before CHF, the positive inotropic effect of BAY y 5959 at a dose of 10 microg/kg/min [left ventricular dP/dt (LVdP/dt) increased from 2955 +/- 132 mmHg to 4897 +/- 426 mmHg, P < .05] was associated with bradycardia (HR decreased from 92 +/- 4 to 78 +/- 6 b/min, P <.05), slight increases in mean arterial pressure (it increased from 100 +/- 2 mmHg to 113 +/- 5 mmHg, P <.05) and did not alter left ventricular end-diastolic pressure. In CHF, BAY y 5959 continued to induce dose-dependent increases in left ventricular systolic pressure, LVdP/dt and mean arterial pressure, as well as causing bradycardia and a significant decrease in left ventricular end-diastolic pressure. After a 5-day infusion of BAY y 5959, base-line LVdP/dt and left ventricular end-diastolic pressure improved. The responses of LVdP/dt and mean arterial pressure to BAY y 5959 were similar to those of the control state. The sustained responses in CHF and after long-term infusion suggest that BAY y 5959 may be an effective and potent inotropic agent for treatment of CHF that does not lead to tolerance to its positive inotropic effects.     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed. 2. NO (10(-7) M) induced only contraction while 10(-6) M NO induced contraction followed by relaxation. Methylene blue (up to 10(-4) M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10(-6) M NO. Administration of 8-bromo-cyclic GMP (10(-6)-10(-4) M) only induced relaxation. 3. Sodium nitroprusside (SNP; 10(-7)-10(-5) M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10(-8) M NO and 10(-6) M SNP; it was increased by a factor of 2 during contraction with 10(-7) M NO, and by a factor of 12 during relaxation with 3 x 10(-6) M NO. 4. The NO-induced contractions were not affected by ryanodine (3 x 10(-5) M) but were concentration-dependently reduced by nifedipine (10(-8)-10(-7) M) and apamin (3 x 10(-9)-3 x 10(-8) M). 5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca2+ influx through L-type Ca2+ channels, that might be activated in response to the closure of Ca(2+)-dependent K+ channels.     

Modulation of myocardial economy and efficiency in mammalian failing and non-failing myocardium by calcium channel activation and beta-adrenergic stimulation

OBJECTIVE: We investigated the energy-metabolic consequences of positive inotropic stimulation by the calcium channel activator, BAY K 8644, in comparison with isoprenaline, focussing both on the economy of force development and the efficiency of external work. METHODS: In the first instance, heat liberation was measured in isometrically contracting right ventricular papillary muscles from guinea pigs by means of antimony-bismuth thermopiles; in the second instance, external work and myocardial oxygen consumption were analyzed in isolated failing and non-failing working rat hearts. RESULTS: In the guinea pig muscle strip preparations BAY K 8644 (10(-5) M) and isoprenaline (10(-8 M) increased peak developed force from 13.7 +/- 2.7 to 37.6 +/- 14.9 mN/mm2 and from 13.6 +/- 5.2 to 38.8 +/- 3.3 mN/mm2, respectively (P < 0.01). Stress-time integral was increased from 10.3 +/- 3.0 to 34.7 +/- 19.2 mN.s/mm2 by BAY K 8644 and from 9.5 +/- 2.4 to 23.0 +/- 1.6 mN.s/mm2 by isoprenaline. Whereas a significant decrease in the ratio between stress-time integral and initial heat (integral of Pdt/IH) (i.e., economy contraction) was observed for isoprenaline (5.26 +/- 1.91 before and 3.11 +/- 0.72 N.m.s.J-1 after treatment (P < 0.01), BAY K 8644 did not significantly alter this index (5.26 +/- 2.39 before and 6.22 +/- 2.63 N.m.s.J-1 after treatment). Similar results were obtained for the ratio between stress-time integral and tension-dependent heat. Significantly more calcium ions were required for equieffective activation of the contractile proteins with isoprenaline as compared to BAY K 8644. In working preparations of sham-operated and infarcted rat hearts, the increase in myocardial oxygen consumption per minute (delta MVO2) for a given increase in external work per minute (delta P) was significantly higher with isoprenaline than with equipotent concentrations of BAY K 8644 or high calcium. CONCLUSIONS: Inotropic mycardial stimulation by BAY K 8644 is associated with higher economy and efficiency than stimulation by isoprenaline when analyzed both by heat measurements in isometric preparations and by myocardial oxygen consumption in working heart preparations.     

Characterization of a constitutive type III nitric oxide synthase in human U937 monocytic cells: stimulation by soluble CD23

1. Angiotensin II had a bimodal effect on human neutrophil migration. Low concentrations of angiotensin II stimulated random migration. At a concentration of 10(-10) M it caused a maximal increase of migration; migration increased from 47.2 +/- 2.1 microns in the absence of angiotensin II, to 73.1 +/- 2.2 microns with 10(-10) M angiotensin II present in the lower compartment of the Boyden chamber (n = 5, P < 0.001). Stimulation of migration by angiotensin II was partly chemotactic and partly chemokinetic. Angiotensin II concentrations of 10(-8) M and higher inhibited chemotactic peptide-stimulated chemotaxis. 2. The stimulant effect of angiotensin II on migration was completely dependent on extracellular Ca2+. In the presence of 1 mM Ca2+, angiotensin II stimulated migration to 76.1 +/- 1.7 microns, while migration in the absence of Ca2+ was 42.2 +/- 1.9 microns (n = 4, P < 0.001). Different types of calcium channel blockers either moderately or strongly inhibited angiotensin II-activated migration. Stimulation of migration by angiotensin II in intact cells required higher concentrations of Ca2+ than in electroporated cells. This supports the view that there is an influx of Ca2+ through the plasma membrane, and a requirement of calcium for an intracellular target. 3. Angiotensin II-stimulated migration was inhibited by pertussis toxin; from 71.6 +/- 2.0 microns in the absence, to 43.6 +/- 1.5 microns in the presence of pertussis toxin (n = 4, P < 0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTP gamma S. This suggests that one or more G-proteins are involved in the activating effect of angiotensin II. 4. Inhibitors of soluble guanylate cyclase and antagonists of cyclic GMP-dependent kinase strongly inhibited the activating effect of angiotensin II. The results suggest that the activating effect of angiotensin II is mediated by cyclic GMP and by cyclic GMP-dependent kinase.     

Cultured adult rabbit myocytes: effect of adding supplements to the medium, and response to isoprenaline

INTRODUCTION: The aims of this study were to investigate: (1) the effect of supplementing the culture medium on preservation of L-type calcium channel current (1Ca,L) in adult rabbit ventricular myocytes cultured for 4 days; and (2) preservation of the ICa,L response in cultured myocytes to the beta-adrenergic agonist isoprenaline. METHODS AND RESULTS: Adult rabbit myocytes were cultured on laminin-pretreated glass coverslips. The basic, serum-free culture medium was supplemented with 2 mM L-carnitine, 5 mM creatine, and 5 mM taurine. Myocytes were whole cell patch-clamped, and the L-type Ca channel current was recorded selectively as Ba flux (IBa,L) via the channels. IBa,L density (i.e., IBa,L amplitude normalized to membrane capacitance) in myocytes maintained in supplemented medium did not change significantly during culture (P > 0.1). By comparison, IBa,L density in myocytes cultured in nonsupplemented medium declined by 36% after 24 hours in culture (day 1) and then recovered by the fourth day (day 4). There was no significant difference in the response to isoprenaline of acutely isolated myocytes and 4-day cultured myocytes. Isoprenaline 100 nM increased peak IBa,L by 149% +/- 32% (mean +/- SEM) in acutely isolated myocytes (n = 4 cells), and by 224% +/- 60% (n = 6 cells) and 159% +/- 24% (n = 8 cells) in day 1 and 4 cultured myocytes, respectively. CONCLUSIONS: Supplemented medium improved IBa,L density in cultured myocytes. beta-Adrenergic receptors and intracellular messenger pathways appear to remain intact in adult rabbit myocytes cultured for up to 4 days.     

Effects of flunixin and mefenamic acid on cardiac pacemaker cells. Structure-activity relationship and comparison with clonixin

1. The electrophysiological effects of flunixin and mefenamic acid, non-steroidal analgesics, on frog cardiac pacemaker cells, were studied by intracellular action potential recording. 2. Results show that flunixin (Flx) between 2 x 10(-6) and 1 x 10(-4) M, exerts a frequency, OS, APA and Vmax concentration-dependent decrease, similar to the effects of clonixin (Clx) reported elsewhere (Morales et al., Gen. Pharmac. 23, 515-521, 1992). 3. At 2.5 x 10(-4) M, Flx induces a complete cessation of the electrical activity of subsidiary pacemaker cells. 4. Mefenamic acid (Mef), in spite of its structural similarity with Clx and Flx, induces no appreciable electrophysiological changes. 5. Flx effects were partially reversed by increasing external calcium concentration and fully antagonized by BAY K-8644, a calcium L-type channel agonist. 6. By comparing the structures of the three fenamates studied, it is suggested that the nitrogen of the heterocycle and the electron-donor capacity of the bencenic ring are essential features for the calcium-antagonist activity of Clx and Flx.     

Nitric oxide increases stimulation-evoked acetylcholine release from rat hippocampal slices by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an endothelium-derived relaxing factor and its main mechanism of action is activation of soluble guanylyl cyclase. NO and NO-related compounds have been reported to affect several neuronal functions in the central nervous system. In this study, we investigated the effects of NO donors (sodium nitroprusside (SNP) and (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409)) on acetylcholine (ACh) release from rat hippocampal slices. SNP (10(-5) M) and FK409 (10(-4) M) increased electrical stimulation-evoked ACh release without affecting basal release. As dibutyryl cyclic GMP inhibited stimulation-evoked ACh release, the effects of these NO donors were not due to soluble guanylyl cyclase activation. Atropine increased stimulation-evoked ACh release by blocking presynaptic muscarinic autoreceptors, and SNP increased stimulation-evoked ACh release in the presence of atropine, suggesting that SNP and atropine increase stimulation-evoked ACh release by different mechanisms. The present results indicate that NO enhances some part of the excitation-secretion coupling pathway without inducing ACh release directly and these effects are mediated by cyclic GMP-independent mechanism.     

Regulation of guanosine 3':5'-cyclic monophosphate in ovine tracheal epithelial cells

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue. 2. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)). 3. Both 10(-7)-10(-3) M and 10(-7)-10(-3) M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10(-3) M 3-isobutyl-l -methylxanthine (IBMX) (both greater than 25 x baseline values with highest drug concentration). 4. The increase in production of cyclic GMP in response to 10(-6) M SNP and 10(-5) M SNAP was markedly inhibited by both 5 x 10(-5) M haemoglobin (102% and 92% inhibition) and 5 x 10(-5) M methylene blue (82% and 84% inhibition). 5. The increase in cyclic GMP in response to 10(-3) M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10(-7)-10(-3) M IBMX, 10(-7)-10(-4) M milrinone and 10(-7)-10(-4) M SKF 96231. Only 10(-4)-10(-3) M IBMX significantly increased cyclic GMP levels. 6. Cyclic GMP production was also significantly elevated from baseline by 10(-5) M ANP, 10(-5) M BNP, 10(-5) M CNP and 200 iu ml-3 of E. coli STa toxin in the presence of 10(-3) M IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (2-4 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed. 7. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.     

Analytical and clinical evaluation of new diagnostic tests for myocardial damage

We tested the hypothesis that preventing cyclic GMP degradation with zaprinast, (a selective cyclic GMP-phosphodiesterase inhibitor) would produce a blunted reduction in myocardial O2 consumption in renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy. Four groups of anesthetized open-chest New Zealand white rabbits (n = 26) were utilized. Either vehicle or zaprinast (3 x 10(-3) M) was applied topically to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. The 1K1C rabbits had a greater heart weight-to-body weight ratio (2.94 +/- 0.08 g/kg) than controls (2.58 +/- 0.17). Systolic blood pressure was higher in 1K1C (102 +/- 9 mm Hg) than in controls (86 +/- 3). Zaprinast significantly and similarly increased cyclic GMP in both control (3.90 +/- 0.47 to 4.66 +/- 0.89 pmol/g) subepicardium (EPI) and (5.08 +/- 0.69 to 7.06 +/- 1.36) subendocardium (ENDO) and 1K1C hearts (5.53 +/- 0.61 to 7.48 +/- 1.51 EPI and 6.48 +/- 0.42 to 8.88 +/- 1.08 ENDO). Myocardial O2 consumption (ml O2/min/ 100 g) was significantly lower in controls treated with zaprinast (EPI: 8.8 +/- 0.1; ENDO: 9.5 +/- 1.9) than in controls treated with vehicle (EPI: 13.6 +/- 1.3; ENDO: 16.2 +/- 2.9). This effect was diminished in 1K1C rabbits treated with zaprinast (EPI: 10.3 +/- 2.4; ENDO: 11.2 +/- 2.6) compared with the vehicle-treated 1K1C group (EPI: 13.3 +/- 1.2; ENDO: 14.5 +/- 2.4). There was a similar increase in myocardial cyclic GMP after treatment with zaprinast, but a greater depression of myocardial O2 consumption in control animals than in 1K1C after treatment with zaprinast. This suggested that the reduction in myocardial O2 consumption, related to increases in cyclic GMP caused by cyclic GMP-phosphodiesterase blockade, was less in 1K1C cardiac hypertrophy.     

Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle

BACKGROUND: The role of the L-type calcium channel in human heart failure is unclear, on the basis of previous whole-cell recordings. METHODS AND RESULTS: We investigated the properties of L-type calcium channels in left ventricular myocytes isolated from nonfailing donor hearts (n= 16 cells) or failing hearts of transplant recipients with dilated (n=9) or ischemic (n=7) cardiomyopathy. The single-channel recording technique was used (70 mmol/L Ba2+). Peak average currents were significantly enhanced in heart failure (38.2+/-9.3 fA) versus nonfailing control hearts (13.2+/-4.5 fA, P=0.02) because of an elevation of channel availability (55.9+/-6.7% versus 26.4+/-5.3%, P=0.001) and open probability within active sweeps (7.36+/-1.51% versus 3.18+/-1.33%, P=0.04). These differences closely resembled the effects of a cAMP-dependent stimulation with 8-Br-cAMP (n= 11). Kinetic analysis of the slow gating shows that channels from failing hearts remain available for a longer time, suggesting a defect in the dephosphorylation. Indeed, the phosphatase inhibitor okadaic acid was unable to stimulate channel activity in myocytes from failing hearts (n=5). Expression of calcium channel subunits was measured by Northern blot analysis. Expression of alpha1c- and beta-subunits was unaltered. Whole-cell current measurements did not reveal an increase of current density in heart failure. CONCLUSIONS: Individual L-type calcium channels are fundamentally affected in severe human heart failure. This is probably important for the impairment of cardiac excitation-contraction coupling.     

Chronic L-arginine treatment increases cardiac cyclic guanosine 5'-monophosphate in rats with aortic stenosis: effects on left ventricular mass and beta-adrenergic contractile reserve

OBJECTIVES: We tested the hypothesis that nitric oxide (NO) cyclic guanosine 5'-monophosphate (GMP) signaling is deficient in pressure overload hypertrophy due to ascending aortic stenosis, and that long-term L-arginine treatment will increase cardiac cyclic GMP production and modify left ventricular (LV) pressure overload hypertrophy and beta-adrenergic contractile response. BACKGROUND: Nitric oxide cyclic GMP signaling is postulated to depress vascular growth, but its effects on cardiac hypertrophic growth are controversial. METHODS: Forty control rats and 40 rats with aortic stenosis left ventricular hypertrophy ([LVH] group) were randomized to receive either L-arginine (0.40 g/kg/day) or no drug for 6 weeks. RESULTS: The dose of L-arginine did not alter systemic blood pressure. Animals with LVH had similar LV constitutive nitric oxide synthase (cNOS) mRNA and protein levels, and LV cyclic GMP levels as compared with age-matched controls. In rats with LVH L-arginine treatment led to a 35% increase in cNOS protein levels (p = 0.09 vs untreated animals with LVH) and a 1.7-fold increase in LV cyclic GMP levels (p < 0.05 vs untreated animals with LVH). However, L-arginine treatment did not suppress LVH in the animals with aortic stenosis. In contrast, in vivo LV systolic pressure was depressed in L-arginine treated versus untreated rats with LVH (163 +/- 16 vs 198 +/- 10 mm Hg, p < 0.05). In addition, the contractile response to isoproterenol was blunted in both isolated intact hearts and isolated myocytes from L-arginine treated rats with LVH compared with untreated rats with LVH. This effect was mediated by a blunted increase in peak systolic intracellular calcium in response to beta-adrenergic stimulation. CONCLUSIONS: Left ventricular hypertrophy due to chronic mechanical systolic pressure overload is not characterized by a deficiency of LV cNOS and cyclic GMP levels. In rats with aortic stenosis, L-arginine treatment increased cardiac levels of cyclic GMP, but it did not modify cardiac mass in rats with aortic stenosis. However, long-term stimulation of NO-cyclic GMP signaling depressed in vivo LV systolic function in LVH rats and markedly blunted the contractile response to beta-adrenergic stimulation.     

Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1. The functional role of the nitric oxide (NO)/guanosine 3':5'-cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts. 2. Rat isolated hearts were perfused at constant pressure with Krebs-Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min(-1) for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored. 3. At baseline, heart rate was 287+/-12 beats min(-1), coronary flow was 12.8+/-0.6 ml min(-1), left ventricular developed pressure (LVDevP) was 105+/-4 mmHg and left ventricular end-diastolic pressure 4.6+/-0.2 mmHg in vehicle-treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05). 4. In normoxic perfused hearts, 1 microM N(G)-nitro-L-arginine (L-NOARG) significantly reduced coronary flow from 13.5+/-0.2 to 12.1+/-0.1 ml min(-1) (10%) and LVDevP from 97+/-1 to 92+/-1 mmHg (5%; P<0.05, n=5). 5. Ischaemic contracture was 46+/-2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 microM) but reduced to approximately 30 mmHg (approximately 25%) at higher concentrations (100 or 1000 microM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L-NOARG reduced contracture at 1 microM to 26+/-3 mmHg (23%), but increased it to 63+/-4 mmHg (59%) at 1000 microM (n=6). Dobutamine (10 microM) exacerbated ischaemic contracture (81+/-3 mmHg; n = 7) and the cyclic GMP analogue Sp-8-(4-p-chlorophenylthio)-3',5'-monophosphorothioate (Sp-8-pCPT-cGMPS; 10 microM) blocked this effect (63+/-11 mmHg; P<0.05 vs dobutamine alone, n=5). 6. At the end of reperfusion, LVDevP was 58+/-5 mmHg, i.e. 55% of pre-ischaemic value in control hearts, significantly increased to approximately 80% by high concentrations of nitroprusside (100 or 1000 microM) or L-NOARG at 1 microM, while a high concentration of L-NOARG (1000 microM) reduced LVDevP to approximately 35% (P<0.05 vs control; n=6). 7. Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 microM had no sustained effect, but increased cyclic GMP approximately 6 fold at 1000 microM; L-NOARG (1 or 1000 microM) was without effect (n=6). Nitroprusside (1 or 1000 microM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6). 8. In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.     

Urinary cyclic GMP, endothelin, and prostaglandin E2 in normal pregnancy and preeclampsia

Cyclic GMP, endothelin and prostaglandin E2 (PGE2) all have systemic vasoactive properties (with cyclic GMP acting as a second messenger of nitric oxide). Intrarenally they act as natriuretics and urinary levels reflect intrarenal production. Cyclic GMP and PGE2 also act as important inhibitors of platelet activation and thrombosis. The purpose of this study was to determine if urinary levels of cyclic GMP, endothelin, and PGE2 differ in preeclamptic as compared to normal pregnancies. Parameters were compared in 13 normotensive, nonpreeclamptic pregnancies, and 32 preeclamptic pregnancies. Preeclamptic women had significantly lower levels of urinary cyclic GMP (0.67 +/- 0.12 vs. 2.1 +/- 0.5 nmol/g creatinine), endothelin (0.88 +/- 0.09 vs. 3.75 +/- 1.4 ng/g creatinine), and PGE2 (26 +/- 4 vs. 9 ng/g creatinine) as compared to normals (p < 0.05). Intrarenal production of cyclic GMP, endothelin, and PGE2 are all disturbed in preeclampsia and may have implications in the sodium retention, hypertension, and intrarenal thrombosis and vasospasm of preeclamptic pregnancy.     

Nursing care delivery models in ambulatory oncology

The action of sodium orthovanadate (Na3VO4) on spontaneous mechanical activity of the longitudinal muscle was investigated in isolated segments of rabbit distal ileum. Vanadate (0.3-1000 microM) concentration-dependently enhanced the amplitude of phasic contractions (pendular movements) and caused the muscle tone to slightly increase at the highest concentrations. Both these effects were mimicked by the Ca2+ channel activator BAY K 8644 (10-1000 nM). Vanadate- and BAY K 8644-induced potentiation of mechanical activity was antagonized by the Ca2+ entry blocker nifedipine (3 nM). In Ca(2+)-free, K(+)-depolarized preparations, vanadate (100 microM) failed to contract the musculature, but potentiated the contractile response to applied calcium (CaCl2: 30-300 microM). The action of vanadate was similar to that of BAY K 8644 (3 nM) and was antagonized by nifedipine (0.1 nM). These results suggest that extracellular calcium is required for vanadate-induced smooth muscle excitation which, at least in part, appears to arise from facilitation of calcium influx through voltage-dependent Ca2+ channels.     

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells

The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.     

Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils

1. The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GMP pathways. In addition, inhibition of neutrophil chemotaxis by NO releasing compounds and increases in neutrophil nitrate/nitrite and cyclic GMP levels were examined. The ultimate aim of this work was to resolve the paradox that NO both activates and inhibits human neutrophils. 2. A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis was supported by the finding that the NO synthase (NOS) inhibitor L-NMMA (500 microM) inhibited chemotaxis; EC50 for fMLP 28.76 +/- 5.62 and 41.13 +/- 4.77 pmol/10(6) cells with and without L-NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 microM) inhibited chemotaxis; EC50 for fMLP 19.71 +/- 4.23 and 31.68 +/- 8.50 pmol/10(6) cells with and without carboxy-PTIO, respectively. 3. A role for cyclic GMP as a mediator of chemotaxis was supported by the finding that the guanylyl cyclase inhibitor LY 83583 (100 microM) completely inhibited chemotaxis and suppressed the maximal response; EC50 for fMLP 32.53 +/- 11.18 and 85.21 +/- 15.14 pmol/10(6) cells with and without LY 83583, respectively. The same pattern of inhibition was observed with the G-kinase inhibitor KT 5823 (10 microM); EC50 for fMLP 32.16 +/- 11.35 and > 135 pmol/10(6) cells with and without KT 5823, respectively. 4. The phosphatase inhibitor, 2,3-diphosphoglyceric acid (DPG) (100 microM) which inhibits phospholipase D, attenuated fMLP-induced chemotaxis; EC50 for fMLP 19.15 +/- 4.36 and 61.52 +/- 16.2 pmol/10(6) cells with and without DPG, respectively. 5. Although the NOS inhibitors L-NMMA and L-canavanine (500 microM) failed to inhibit fMLP-induced SAG, carboxy-PTIO caused significant inhibition (EC50 for fMLP 36.15 +/- 7.43 and 86.31 +/- 14.06 nM and reduced the maximal response from 22.14 +/- 1.5 to 9.8 +/- 1.6 nmol O2-/10(6) cells/10 min with and without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6. A role for cyclic GMP as a mediator of SAG was supported by the effects of G-kinase inhibitors KT 5823 (10 microM) and Rp-8-pCPT-cGMPS (100 microM) which inhibited SAG giving EC50 for fMLP of 36.26 +/- 8.77 and 200.01 +/- 43.26 nM with and without KT 5823, and 28.35 +/- 10.8 and 49.25 +/- 16.79 nM with and without Rp-8-pCTP-cGMPS. 7. The phosphatase inhibitor DPG (500 microM) inhibited SAG; EC50 for fMLP 33.93 +/- 4.23 and 61.12 +/- 14.43 nM with and without DPG, respectively. 8. The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC50 = 14.72 +/- 1.6 microM) > GEA 5024 (IC50 = 18.44 +/- 0.43 microM) > SIN-1 (IC50 > 1000 microM). This order of potency correlated with their ability to increase cyclic GMP levels rather than the release of NO, where SIN-1 was most effective (SIN-1 (EC50 = 37.62 +/- 0.9 microM) > GEA 3162 (EC50 = 39.7 +/- 0.53 microM) > GEA 5024 (EC50 = 89.86 +/- 1.62 microM)). 9. In conclusion, chemotaxis and SAG induced by fMLP can be attenuated by inhibitors of phospholipase D, NO and cyclic GMP, suggesting a role for these agents in neutrophil activation. However, the increases in cyclic GMP and NO induced by fMLP, which are associated with neutrophil activation, are very small. In contrast much larger increases in NO and cyclic GMP, as observed with NO releasing compounds, inhibit chemotaxis.     

Effect of cyclic GMP reduction on regional myocardial mechanics and metabolism in experimental left ventricular hypertrophy

We tested the hypotheses that decreased myocardial cyclic GMP levels produced by intracoronary injection of methylene blue would increase local myocardial work and O2 consumption while decreasing intracellular cyclic GMP and that the relation between work, O2 consumption, and cyclic GMP may be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. In 8 control and 8 LVH open-chest anesthetized dogs, 1 mg/kg/min methylene blue was infused into the left anterior descending coronary artery (LAD); the circumflex region (CFX) served as control area. Regional work was calculated as the integrated product of force (miniature transducer) and segment shortening (sonomicrometry). Regional myocardial O2 consumption was calculated from flow measurements (radioactive microspheres), and regional O2 saturations (microspectrophotometry). A radioimmunoassay was used to determine intracellular level of cyclic GMP in the myocardium. Global hemodynamics and blood gases were unchanged by methylene blue in both control and LVH animals. Intracoronary methylene blue increased regional work from 762 +/- 129 to 1,451 +/- 307 g center dot mm/min in controls and from 912 +/- 173 to 1581 +/- 253 g center dot mm/min in the LVH groups. No significant changes in CFX regional work were observed. Regional blood flow, O2 extraction, and O2 consumption remained unchanged after injection of methylene blue in both control and LVH animals. The basal levels of cyclic GMP in the LVH group were fivefold higher than that in controls. In both groups, cyclic GMP levels were significantly decreased by methylene blue and to a greater extent in the LVH animals (from 6.16 +/- 1.2 to 3.34 +/- 0.44 pmol/g) than in the control animals (from 1.32 +/- 0.20 to 1.09 +/- 0.19 pmol/g). Therefore, intracoronary methylene blue increased regional myocardial work equally in control and LVH hearts without affecting regional metabolism (i.e., increased efficiency). For the same increased mechanical function, the hypertrophic myocardium exhibited a greater reduction in cyclic GMP pool size.     

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.