Ilari Paakkari

Nitric Oxide in the Central Nervous System

The majority of the data on nitric oxide (NO) in the central nervous system (CNS) relies on histochemical and immunohistochemical evidence concerning the distribution of the nitric oxide synthase (NOS), its inhibition by specific antagonists and its co-localization with the receptor enzyme guanylate cyclase (GC) in the same functional region. All three isoforms, endothelial (eNOS), neural (nNOS) and macrophage type inducible (iNOS), are of importance to the normal and pathological function of the CNS. In nNOS gene deleted mice eNOS seems to contribute to the maintenance of neuronal function. NO may contribute to synaptic plasticity as a retrograde mediator that is released by postsynaptic NMDA-receptor activation. Microglia contains membrane-bound inducible iNOS that may be important in host defence function. Glia and pericytes surrounding the blood vessels contain GC that is stimulated by NO released from endothelium and nerve endings. Excessive production of highly reactive NO may be responsible for the neurotoxicity mediated by NMDA receptors that contributes to the symptomatology of strokes and neurodegenerative diseases. Moreover, after initial stimulation by cytokines, large amounts of NO produced by iNOS in the microglia (brain-based macrophages) may cause cellular damage.

Endothelial Dysfunction After Repeated Chlamydia pneumoniae Infection in Apolipoprotein E–Knockout Mice

BackgroundArterial relaxation is largely regulated by endothelial nitric oxide (NO). Its diminished activity has been associated with incipient atherosclerosis. We investigated the endothelium-dependent relaxation of aorta in apolipoprotein E–knockout (apoE-KO) mice exposed to single or repeated Chlamydia pneumoniae inoculation. Methods and ResultsForty-eight apoE-KO mice, 8 weeks old, were inoculated intranasally with C pneumoniae (n=24) or saline (n=24) every 2 weeks over a 6-week period. Twenty mice (10 infected and 10 controls) were killed at 2 weeks and 6 weeks, respectively, after the first inoculation. The smooth muscle tone of aortic rings was measured in vitro at both time points. The norepinephrine-precontracted thoracic aortic rings were successively exposed to methacholine in the absence and presence of NG-nitro-l-arginine methyl ester (L-NAME) and diclofenac. The methacholine-induced relaxation was attenuated in the infected mice at 6 weeks in both the absence and presence of L-NAME (P <0.05 and P <0.01, respectively). When administered together with L-NAME, diclofenac enhanced the relaxation of the L-NAME–pretreated aortas in infected mice at 2 weeks (P <0.05) but not in noninfected mice. The relaxation response from infected mice tended to differ in the same manner at 6 weeks (P <0.1). No intimal thickening was detected at either time point. ConclusionsC pneumoniae impairs arterial endothelial function, and the NO pathway is principally involved. Cyclooxygenase-dependent vasoconstricting products may also account for the infection-induced impaired relaxation. These findings further support the role of C pneumoniae infection in atherosclerosis development.

The long-term effects of oral and transdermal postmenopausal hormone replacement therapy on nitric oxide, endothelin-1, prostacyclin, and thromboxane.

OBJECTIVE Oral postmenopausal hormone replacement therapy (HRT) decreases the risk of cardiovascular disorders, but the mechanisms of this protection are largely unknown. We compared the long-term effects of sequential oral HRT and transdermal HRT on vasodilatory nitric oxide and prostacyclin as well as vasoconstrictive endothelin- and thromboxane A2, all of which may be factors in the protective effect of HRT against cardiovascular disorders. DESIGN Prospective, randomized study. SETTING Department of Obstetrics and Gynecology at a university hospital. PATIENT(S) Fifty-two healthy postmenopausal female nonsmokers (n = 42) or smokers (n = 10) who had climacteric symptoms. INTERVENTION(S) The women received either oral HRT (2 mg of estradiol on days 1-12, 2 mg of estradiol plus 1 mg of norethisterone acetate on days 13-22, and 1 mg of estradiol on days 23-28; n = 21) or transdermal HRT (50 microg/d of estradiol on days 1-28 followed by 250 microg/d of norethisterone acetate on days 14-28; n = 21) for 1 year. Ten female smokers received transdermal HRT for 1 year. MAIN OUTCOME MEASURE(S) Plasma levels of nitrate as an index of nitric oxide production, endothelin-1, and urinary output of the prostacyclin metabolite (2,3-dinor-6-keto-PGF1alpha) and that of the thromboxane A2 metabolite (2,3-dinorthromboxane B2) were measured before and during the combined phases of the 2nd, 6th, and 12th treatment months. RESULT(S) Both regimens increased plasma estradiol levels and alleviated vasomotor symptoms. Neither regimen caused significant changes in nitrate, endothelin-1, prostacyclin, or thromboxane A2 in nonsmoking women. Female smokers had significantly higher levels of endothelin-1, which were significantly reduced by transdermal HRT at 6 months of treatment. CONCLUSION(S) Nitric oxide, endothelin-1, prostacyclin, and thromboxane A2 are not of primary importance in the protective effect of sequential oral HRT against cardiovascular disorders in otherwise healthy nonsmoking postmenopausal women. In this regard, transdermal HRT appears comparable to oral HRT. Postmenopausal female smokers have high levels of endothelin-1 that are reduced by transdermal HRT.

Cardiotoxicity of new antihistamines and cisapride.

Although the new second-generation nonsedative antihistamines terfenadine and astemizole were launched as highly selective and specific H(1)-receptor antagonists, they were later found to cause prolongation of the QT-interval and severe cardiac arrhythmias. The prolongation of the QT-interval is caused by the blockade of one or more of the cardiac potassium channels, among which the delayed rectifier I(Kr), encoded by the HERG-gene, appears to be the most significant. The potency of the prokinetic drug cisapride to block I(Kr) appears to be similar to that of terfenadine (IC(50) about 50 nmol/l). These drugs cause problems when overdosed, used in combination with inhibitors of their CYP3A4-mediated metabolism, or when given to individuals with altered drug kinetics (the aged) or patients with existing cardiac disease (congenitally long QT). Moreover, interactions with other QT-interval prolonging drugs require special attention. Active hydrophilic metabolites of the second-generation antihistaminic compounds (ebastine-carebastine, loratadine-desloratadine, terfenadine-fexofenadine, astemizole-norastemizole) are new compounds with probably reduced risk for drug interactions and cardiac toxicity.

Effects of a COX-2 preferential agent nimesulide on TNBS-induced acute inflammation in the gut.

In inflammatory bowel disease, increased production of prostaglandins by cyclooxy- genase-2 (COX-2) contributes to bowel dysfunction, inflammatory edema, and hyperemia suggesting that inhibitors of COX-2 may have beneficial effect in gut inflammation. We compared the effects of nimesulide, a preferential COX-2 inhibitor, with those of indomethacin, acetylsalicylic acid (ASA), and dexamethasone in a 24-h model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in the rat. TNBS-induced colitis was associated with enhanced COX-2 expression in the gut and increased circulating concentrations of PGE2 metabolite (PGEM). Treatment with nimesulide (10 mg/kg), indomethacin (10 mg/kg), or dexamethasone (1 mg/kg) reduced plasma PGEM concentrations and edema in the inflamed bowel. In addition, nimesulide and dexamethasone treatments decreased neutrophil infiltration into the inflamed colon mucosa. ASA (10 mg/kg) did not have a significant effect on any of these measures of inflammation. None of the studied drugs reduced the size of inflammatory mucosal lesions in the colon. In TNBS-induced acute inflammation of the colon, nimesulide reduced the formation of inflammatory edema, probably by a mechanism related to inhibition of PGE2 production by COX-2 pathway. In addition, nimesulide inhibited neutrophil infiltration into inflamed mucosa mimicking the action of dexamethasone.

Acute Chlamydia pneumoniae infection causes coronary endothelial dysfunction in pigs.

BACKGROUND Coronary endothelial dysfunction contributes to the pathogenesis of acute coronary syndromes (ACSs). Acute Chlamydia pneumoniae infection has been epidemiologically associated with ACS. In this study, we investigated whether acute C. pneumoniae infection could alter the endothelial vasomotor function of porcine coronary vessels. METHODS AND RESULTS Twenty pigs, 7-9 kg in weight, were inoculated intratracheally with C. pneumoniae (n=12) or saline (n=8), and investigated at 3 days (five infected/four non-infected) and 2 weeks (5+2 infected/four non-infected) after inoculation. The endothelium-dependent reactivity of coronary microcirculation was assessed at both time points by measuring peak coronary flow velocity (CFV) in response to bradykinin, before and after infusions with glutathione, an antioxidant, and L-arginine, a substrate for nitric oxide synthase (NOS). CFV after bradykinin was significantly decreased in infected animals at both time points. At 2 weeks, both glutathione and L-arginine significantly improved CFV after bradykinin. CFV after sodium nitroprusside (SNP) was similar in both groups. At 3 days, the relaxation responses of bradykinin-induced pre-contracted left anterior descending (LAD) coronary rings to bradykinin were significantly less in infected animals. N(G)-nitro-L-arginine-methyl-ester, an NOS inhibitor, had significantly greater inhibitory effect on bradykinin-induced relaxation in infected animals. Plasma nitrate-nitrite and fibrinogen, and NOS activity from LAD coronary samples were significantly increased in infected animals. CONCLUSION Acute C. pneumoniae infection causes endothelial dysfunction of both resistance and epicardial coronary vessels, and favours a pro-coagulant status. These effects could in part account for the epidemiologically suggested association between acute infection and ACS.

Nitric Oxide-based Possibilities for Pharmacotherapy

The goal of nitric oxide (NO) based pharmacotherapy is to reach proper homeostasis of NO metabolism in the target tissue where endogenous production of NO is either too weak or excessively increased. In addition to the classic NO-based therapy of cardiovascular conditions with nitrates, a variety of new therapeutic possibilities have emerged including sexual disorders, gastrointestinal system, immunology, tumour growth regulation and respiratory disorders. NO levels of target tissues can be affected directly by NO donors, or indirectly by increasing the level of L-arginine, a substrate of nitric oxide synthase (NOS). While increased production of NO by induceable NO (iNOS) by, for example, cytokines does not at present seem therapeutically meaningful, increased NO production by constitutive NOS (cNOS) may be involved in the beneficial effects of ACE inhibitors or oestrogens. NO production may be pharmacologically decreased by inhibition of expression of iNOS by glucocorticoids while both cNOS and iNOS derived NO production is inhibited by administration of false substrates, for example L-NAME. Additionally, the respiratory system and related vessels can be reached directly and more selectively by inhalation of pure NO gas. Possible problems in administering NO and perhaps some NO-donors include the toxic nature of the compound itself whereby vital enzyme systems may be inhibited and tissue damaging radicals formed. Future prospects of NO-based pharmacotherapy may feature selective ligands to different NOS isoforms and tissue selective donors that release NO in a controlled fashion.

Randomized comparison of oral and transdermal hormone replacement on carotid and uterine artery resistance to blood flow

Objective To compare the long-term effects of oral and transdermal hormone replacement therapy (HRT) on carotid and uterine vascular impedance. Methods Sixty-three postmenopausal women were randomized to 1 years treatment with oral or transdermal sequential combined HRT. Carotid and uterine artery pulsatility indices (PIs) were assessed by color Doppler at baseline, and after 2, 6, and 12 months of treatment. Fiftyeight women completed the trial, 27 in the oral and 31 in the transdermal group. In a subgroup of 30 women, we also performed Doppler measurements in the estrogen-progestin combined phase. The study had 90% power to detect a difference between treatment groups of 0.05 in the carotid artery and of 0.25 in uterine artery PI at the 5% significance level. Results The carotid PI decreased significantly (P < .001) and similarly during both regimens. This drop was already clearly detectable during the second month, from 0.97 (0.95, 1.01) (mean and 95% confidence intervals [CI]) to 0.94 (0.91, 0.97) in the oral and from 0.98 (0.94, 1.00) to 0.92 (0.89, 0.95) in the transdermal group, but it continued up to 12 months (0.85 [0.82, 0.88], 13% of baseline values in the oral group and 0.84 [0.81, 0.87], 14% in the transdermal group). In the uterine arteries, the drop in PI was steeper and greater and reached its maximum at 6 months (39% and 40%, respectively). Drops in carotid and uterine PI correlated positively with baseline PI values, but were not affected by patient age, time from menopause, previous HRT and smoking. Addition of norethisterone acetate did not counteract drops in carotid and uterine PI in either group. Conclusion Oral and transdermal sequential HRT are similarly effective at 1 year in reducing impedance to flow in carotid and uterine circulation. This long-term vascular effect might explain how HRT protects women from cardio-vascular disease.

A comparison of diclofenac with and without single-dose intravenous steroid to prevent postoperative pain after third molar removal

The efficacy of 40 mg of methylprednisolone given intravenously before operation in combination with orally administered rapid-release and sustained-release diclofenac preparations in preventing postoperative pain after third molar removal was studied. The administration of methylprednisolone and diclofenac resulted in greater pain relief than did administration of diclofenac alone.

Bioactive peptide derived from in vitro proteolysis of bovine β-lactoglobulin and its effect on smooth muscle

Milk proteins have largely been considered as providing essential amino acids, but oligopeptides derived from milk proteins have been shown to possess biological functions. In vitro , opioid activity was first reported in bovine β-casein hydrolysate by Brantl et al . (1979). Precursors of biologically active peptides have been demonstrated in vivo after digestion of milk (Scanff et al . 1992). Peptides that inhibit platelet aggregation (Fiat et al . 1989), stimulate the immune system (Migliore-Samour et al . 1989), inhibit angiotensin I converting enzyme (Maruyama & Suzuki, 1982) and are involved in intestinal Ca solubilization and absorption (Sato et al . 1986) have also been isolated from bovine casein hydrolysates. Bioactive peptides from whey proteins and their physiological effects have received less attention than those from casein. Peptides with opiate-like activity include the lactorphins, residues 50–53 in bovine and human α-lactalbumin and 102–105 in bovine β-lactoglobulin (β-lg) (Chiba & Yoshikawa, 1986; Yoshikawa et al . 1986; Antila et al . 1991). Yamauchi (1992) has reported that a peptide derived from β-lg induced contraction of guinea pig ileum longitudinal muscle in the absence of electric stimulation and agonist. The fragment, containing residues 146–149 of β-lg (His–Ile–Arg–Leu), was called β-lactotensin. The purpose of this study was to determine whether β-lactotensin was released from bovine β-lg by in vitro proteolysis using different proteolytic enzymes. The pharmacological activity of this tetrapeptide was characterized in guinea pig ileum in vitro using a synthetic fragment.