Digitalis-like activity in human plasma: relation to blood pressure and sodium balance

PURPOSE: On the assumption that renal tubular cells are more important as the target cells for a natriuretic factor than blood cells, we used a well-characterized cultured renal tubular cell line, Madin-Darby canine kidney (MDCK), cells to monitor the circulating digitalis-like factor in human plasma and examine its role in the regulation of blood pressure and sodium balance. SUBJECTS AND METHODS: We investigated the effects of plasma on binding of radioactive ouabain to monolayered MDCK cells in order to determine the level of a circulating digitalis-like factor. First, we measured specific 3H-ouabain binding to MDCK cells in the presence of plasma from 71 outpatients (34 normotensive subjects and 37 hypertensive patients) after incubation for 4 hours. Second, we measured specific 3H-ouabain binding after incubation of cells with plasma from 16 hospitalized subjects (eight normotensive subjects and eight hypertensive patients) receiving low and high sodium diets. RESULTS: In Study 1, ouabain binding was lower by 30% with plasma from hypertensive patients than with plasma from normotensive subjects (p less than 0.01). There was a significant negative correlation between individual subject's systolic or mean blood pressure and ouabain binding (r = -0.34, p less than 0.01 or r = -0.29, p less than 0.01). In Study 2, ouabain binding was also significantly reduced by 25% in the presence of plasma from hypertensive subjects as compared with plasma from normotensive subjects irrespective of sodium intake (p less than 0.01). A significant negative correlation was also found for all subjects between either systolic, diastolic, or mean blood pressure and ouabain binding (r = -0.58, p less than 0.01, r = -0.51, p less than 0.01, or r = -0.55, p less than 0.01, respectively). With the changes from low to high sodium intake, there was a corresponding decrease in ouabain binding (p less than 0.01) and an increase in sodium excretion (p less than 0.01). A significant negative correlation was observed between these two parameters (r = -0.47, p less than 0.05). CONCLUSIONS: These findings suggest that a circulating digitalis-like factor, which may act on renal tubular cells as the ouabain-displacing compound, is increased in patients with essential hypertension and also demonstrate that plasma levels may be influenced by changes in dietary sodium intake.     

T-cell colony formation in healthy homosexual men without anti-human immunodeficiency virus antibody

In order to study the abnormal differentiation and proliferation of precursor T cells in homosexual men, a study was made of T-cell colony formation in 15 healthy homosexual men without anti-human immunodeficiency virus (HIV) antibody. The colony forming method used was the one-step monolayer technique using phytohemagglutinin as an inducer. Our results revealed that colony formation was extremely reduced and 3 cases showed no colony growth. However, with the addition of recombinant interleukin 2 to the culture system, the number of colonies increased up to the same level as in heterosexual controls. In addition, the analysis of CD3+, 4+, and 8+ cells of colony component cells revealed a decrease in CD3+ and 4+ cells. However, with the addition of recombinant interleukin 2, the numbers of CD3+ cells distinctly increased. The above results suggest that a disorder in the differentiation and proliferation of precursor T cells in homosexual men might be one of the causes of immunodeficiency.     

Identification of protein Salpha gene mutations including four novel mutations in eight unrelated patients with protein S deficiency

Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Salpha (PSalpha) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A-->G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A-->G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient's family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A-->G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A-->G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A-->G mutation itself might influence phenotypic expression of PS status in different individuals.     

Familial Creutzfeldt-Jakob Disease with a codon 200 mutation presenting as thalamic syndrome: diagnosis by single photon emission computed tomography using (99m)Tc-ethyl cysteinate dimer

The clinical features of familial Creutzfeldt-Jakob disease with a codon 200 point mutation [fCJD (E200K)] are similar to those of sporadic CJD (sCJD). MRI diffusion-weighted imaging (MRI-DWI) has been reported to be useful for the early diagnosis of CJD. We describe a Japanese fCJD (E200K) case in which thalamic symptoms were the initial manifestations. On admission, electroencephalography (ECG) showed no periodic synchronous discharge (PSD), and MRI showed no abnormalities. However, single photon emission computed tomography (SPECT) using (99m)Tc-ethyl cysteinate dimer ((99m)Tc-ECD) revealed hypoperfusion in the right thalamus. We conclude that the thalamic form of CJD tends to show no high-intensity area (HIA) by MRI-DWI, and that SPECT may be more useful for visualizing the affected area responsible for the thalamic symptoms at an early stage.     

Role of anatomic architecture in sustained atrial reentry and double potentials.

To determine the role of anatomic architecture in atrial flutter, electrophysiologic findings were correlated with anatomic features in a modified model of atrial flutter with ligation of the crista terminalis. Crista ligation in the middle right atrium prolonged intraatrial conduction time in a rate-dependent manner in 12 dogs, particularly in the low right atrium. With burst atrial pacing, unidirectional block occurred either in the low right atrium or in the interatrial septal region near the superior vena cava, leading to initiation of atrial flutter. Atrial activation mapping revealed a slow conduction area in the low right atrium where conduction had been delayed by crista ligation. On the intact tissues between the venae cavae, double potentials were recorded, a finding indicative of functional block in the center of the reentrant circuit. The interdeflection time of double potentials changed with the activation sequence of atrial flutter. This change could be explained by assuming that the functional center of the reentrant circuit leaned on the right atrial free wall side. Anatomic study demonstrated that areas of slow conduction, unidirectional block, and functional block in the center of the reentrant circuit were closely related to the location of the intact crista terminalis. In conclusion, the intact portion of the crista terminalis played an important role in the genesis of atrial flutter after blockage of longitudinal conduction through the crista.     

Nitric oxide system is involved in glomerular hyperfiltration in Japanese normo- and micro-albuminuric patients with type 2 diabetes

Glomerular hyperfiltration plays a pathogenic role in the early stages of diabetic nephropathy. Experimental studies in laboratory animals suggest that nitric oxide (NO) might be involved in the pathogenesis of glomerular hyperfiltration. We performed a cross-sectional study to determine the relationship between diabetic glomerular hyperfiltration and the NO system. Normoalbuminuric (n=41), microalbuminuric (n=25), and macroalbuminuric (n=16) patients with type 2 diabetes were recruited in this study and compared with age-matched 84 non-diabetic control subjects. Creatinine clearance and urinary NO(2)(-)/NO(3)(-) excretion (urinary NOx) were measured, and the expression of endothelial cell nitric oxide synthase (ecNOS) was evaluated in human renal tissues. Glomerular hyperfiltration was present in 19 (37.5%) and nine (36.6%) of normoalbuminuric and microalbuminuric type 2 diabetic patients, respectively. The urinary NOx was significantly higher in normoalbuminuric patients compared with normal subjects. Creatinine clearance correlated significantly with urinary NOx in normoalbuminuric and microalbuminuric patients. Immunohistochemical staining intensities for ecNOS were significantly increased in glomerular endothelial cells of microalbuminuric type 2 diabetic patients as compared with the control subjects. These results suggest that NO may contribute to the pathogenesis of glomerular hyperfiltration in Japanese type 2 diabetic patients.     

Inhibition of EGF Signaling Protects the Diabetic Retina from Insulin-Induced Vascular Leakage

Diabetes mellitus is a disease with considerable morbidity and mortality worldwide. Breakdown of the blood–retinal barrier and leakage from the retinal vasculature leads to diabetic macular edema, an important cause of vision loss in patients with diabetes. Although epidemiologic studies and randomized clinical trials suggest that glycemic control plays a major role in the development of vascular complications of diabetes, insulin therapies for control of glucose metabolism cannot prevent long-term retinal complications. The phenomenon of temporary paradoxical worsening of diabetic macular edema after insulin treatment has been observed in a number of studies. In prospective studies on non–insulin-dependent (type 2) diabetes mellitus patients, a change in treatment from oral drugs to insulin was often associated with a significant increased risk of retinopathy progression and visual impairment. Although insulin therapies are critical for regulation of the metabolic disease, their role in the retina is controversial. In this study with diabetic mice, insulin treatment resulted in increased vascular leakage apparently mediated by betacellulin and signaling via the epidermal growth factor (EGF) receptor. In addition, treatment with EGF receptor inhibitors reduced retinal vascular leakage in diabetic mice on insulin. These findings provide unique insight into the role of insulin signaling in mediating retinal effects in diabetes and open new avenues for therapeutics to treat the retinal complications of diabetes mellitus.Diabetic maculopathy, an important cause of vision loss in patients with type 2 diabetes, is characterized by hyperpermeability of retinal blood vessels and subsequent formation of macular edema and hard exudates. Although the increase in retinal vascular permeability occurs both diffusely and in focal regions, the basic physiological defect that causes retinal vascular leakage is unknown. The blood–retinal barrier (BRB) isolates the retina from the bloodstream, establishing a favorable environmental milieu with the regulation of ionic balance, nutrient availability, and blockage of potentially toxic molecules that allows for optimal retinal function. The BRB consists of an inner BRB, formed by endothelial cells lining the retinal blood vessels and the outer BRB formed by the retinal pigment epithelium (RPE), a layer of epithelial cells between the retina and the non-neuronal choroid.^1,2 Disruption of the BRB is an important feature of diabetic retinopathy.Based on data from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), a prospective population-based cohort study of patients with type 1 and 2 diabetes mellitus, the prevalence of clinically significant macular edema is 5.9% for type 1 and 7.5% for type 2 diabetes.³ Although epidemiologic studies and randomized clinical trials suggest that glycemic control plays a major role in the development of vascular complications of diabetes,⁴ insulin therapies for control of glucose metabolism may not prevent long-term complications.^5,6 Even though both laser photocoagulation and anti-VEGF therapies have shown significant promise in the treatment of proliferating vessels in proliferative diabetic retinopathy, diabetic macular edema (DME) appears to be more resistant to these treatment approaches, suggesting that other factors might contribute to this complication. We have recently reported the potential role of betacellulin (Btc) in inducing retinal vascular permeability in diabetes.⁷ Clinical trials and other studies have determined that initiation of acute intensive insulin therapy in patients with long-standing poor glycemic control results in a transient worsening of diabetic retinopathy.^8–13 A change in treatment from oral drugs to insulin in patients with non–insulin-dependent (type 2) diabetes mellitus was associated with a significantly increased risk of retinopathy progression and visual impairment.^14–16 In addition, it has been reported that patients who undergo total pancreatectomy for cancer develop severe diabetes because of the complete absence of insulin but rarely if ever develop proliferative diabetic retinopathy,¹⁷ even when they survive for more than one or two decades. These reports led us to model and evaluate the pathophysiological effects of insulin on the retinal vasculature and the potential crosstalk between insulin and Btc in the regulation of retinal vascular permeability.