Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphatase

BACKGROUND: Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. OBJECTIVES: We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. METHODS: Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. RESULTS: We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. CONCLUSIONS: Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.     

Effect of oral and inhaled cetirizine in allergen induced bronchoconstriction

Cetirizine is a potent, selective H₁ histamine receptor antagonist. The effect of oral and inhaled eetirizine was assessed on the early bronchoconstrictor response to inhaled allergen in 10 mild atopie asthmatic patients in a double-blind, randomized, plaeebo controlled trial. All were sensitive to Dermatophagoides pleronyssinus and this was used as the provoking allergen. The geometric mean PD20 FEV] values obtained at allergen challenge were measured as cumulative breath units (c.b.u.) and following oral cetirizine, inhaled cetirizine and placebo were 124–5, 75–7 and 76–7 c.b.u. respectively. These did not differ significantly. We conclude that neither oral nor inhaled cetirizine significantly attenuates the early response to inhaled allergen in atopie asthmatic subjects. However, the method of repeated allergen challenge is likely to be relatively insensitive. Clinical and Experimental Allergy, [o]. 23, pp. 528–531.     

CERVICAL MYELOPATHY COMPLICATING MULTIPLE JOINT REPLACEMENT IN RHEUMATOID DISEASE

The incidence of cervical myelopathy and subluxation was investigatedin 48 patients with rheumatoid disease who had three or moremajor lower limb joint replacements. Eight (17%) developed cervicalmyelopathy requiring cervical fusion. This was the subsequentcause of death in two. Four further patients demonstrated clinicalfeatures of myelopathy. Cervical subluxation was present in29 of 44 (66%) in whom adequate radiographs were available.The development of cervical symptoms and signs could not havebeen predicted from the sex, age of onset, duration of diseaseor steriod therapy. Radiographic changes in the cervical spinewere independent of major lower limb joint destruction and wereoften not present when planning a programme of joint replacement.Fifty-one control patients were studied. Cervical myelopathyoccurred in 2 (4%) and subluxation in 24 (47%). The developmentof rheumatoid changes in the cervical spine was unrelated toinvolvement of the hip or knee joints in the control group. There was a significant (p<0.05) increase in the incidenceof cervical myelopathy in patients with multiple lower limbjoint replacements compared with the control population. KEY WORDS: Rheumatoid arthritis, Cervical myelopathy, Cervical subluxation, Arthroplasty     

Effect of minimizing preoperative fasting on perioperative blood glucose homeostasis in children

Two hundred healthy, unpremedicated children, ages 1–10 years, scheduled for elective outpatient surgery were studied in order to examine the effect of minimizing preoperative fasting on perioperative blood glucose concentrations in paediatric patients. None of the patients ingested solids after midnight. On the day of surgery, the children were assigned to one of two groups. Group A children (n= 113) were not allowed any liquids for at least 6 h prior to surgery (NPO). Children in Group B (n= 87) ingested 10 ml·kg^?1 of apple juice 2–4 h prior to the induction of anaesthesia. All patients received lactated Ringer's solution intraoperatively, unless BG at induction was < 50 mg·dl^?1 (2.8 m·mol·l^?1) in which case dextrose 2.5% in lactated Ringer's solution was administered. None of the patients who received apple juice was hypoglycaemic during induction of anaesthesia. However, two children in the NPO group had blood glucose values ± 50 mg·dl^?1 (2.8 m·mol·l^?1) at the time of induction of anaesthesia. Thirteen (11%) patients in Group A and 6 (7%) patients in Group B showed either no change or a further decrease in their postoperative BG concentration as compared with their induction values. Two of 43 patients in Group A and 2 of 41 patients in Group B had gastric fluid volumes > 0.4 ml/kg. All patients in both groups had gastric pH < 2.5. This study shows that gastric fluid volume and pH following a 2–4 h fast are not different from the values measured in children who were subjected to a traditional fasting period of 6 h or longer. Moreover, apple juice consumed 2–4 h prior to surgery neither buffers gastric pH nor does it modify intraoperative glucose homeostasis in children.     

Detection of ambulatory ischaemia is not of practical clinical value in the routine management of patients with stable angina: A long-term follow-up study

It has been reported that medically treated patients with stableangina and positive exercise test for ischaemia have an adverse1–2 year outlook if they are shown also to have transient,and predominantly silent, ischaemic episodes detected by ambulatoryST segment monitoring during their daily activities: it hasbeen suggested that this investigation could be used to identifypatients more likely to benefit from early investigation andtreatment. We assessed the long-term (up to 65 months) prognosticsignificance of transient iscliaemic episodes during daily activitiesin 172 patients routinely attending cardiac outpatients withmedically treated stable angina who had undergone exercise testingand 48 h of ambulatory ST segment monitoring between February1988 and August 1989 for this purpose. A positive exercise testfor ischaemia was not a prerequisite for inclusion. One hundred and four patients (60.5%) had a positive exercisetest for iscliaemia and 72 (42%) had transient ischaemia duringdaily activities (63 had both tests positive). Over a median50-month follow-up period 54 patients suffered at least onecardiac event (primary event: cardiac death n=7; non-fatal myocardialinfarction n=11; unstable angina n=18; elective CABGIPTCA n=18).Two further patients suffered non-cardiac death. Cardiac events,either objective (cardiac death or non-fatal myocardial infarction)or subjective (unstable angina or revascularisation) were nomore likely to occur in those with transient ischaemia duringdaily life when compared with those without, at follow-up timesup to 65 months. The detection of transient ischaemia during daily life is oflimited practical clinical value in the management of ‘lowrisk’ medically treated patients with stable angina, anddoes not appear to help identify subgroups at increased riskof an adverse outcome at follow-up to more than 5 years.     

Defining ''full-length'' recombinant factor VIII: a comparative structural analysis

Coagulation factor VIII (FVIII) is an important glycoprotein co-factor involved in haemostasis, functioning to accelerate activation of factor X by activated factor IX. Insertion of expression vectors containing the full-length cDNA sequence of human FVIII into mammalian cell lines results in the production of recombinant factor VIII (rFVIII), typically referred to as 'full-length' rFVIII (FLrFVIII). Both FLrFVIII and plasma-derived FVIII exist primarily as heterodimeric proteins, consisting of a heterogenous light and heavy chain. The objectives of this study were to compare the structural heterogeneity of high-purity FVIII preparations and further define the term 'full length' as it refers to rFVIII protein structure. Five commercially available FVIII concentrates were characterized based on SDS-PAGE, N-terminal sequencing, and peptide and domain mapping coupled to mass spectrometry. The major heavy chain species identified in FLrFVIII included various B-domain-truncated forms of FVIII, with the predominant species terminating at Arg(1313). This study demonstrates that the use of full-sequence FVIII cDNA for the production of rFVIII does not result in a homogeneous FLrFVIII protein product. Rather, commercially available FLrFVIII represents a heterogenous mixture of various B-domain-truncated forms of the molecule, with no evidence of a contiguous, intact B-domain.