Localization of Rho GTPase in sea urchin eggs

OBJECTIVE: Previous studies in our laboratory showed that the platelet anti-aggregating effect exerted by insulin, mediated by a nitric oxide (NO)-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP), is lost in the insulin-resistant of obesity and obese NIDDM. It is not clear 1) whether the alterations observed in obese NIDDM patients are attributable to the obesity-related insulin resistance or to diabetes per se and 2) whether insulin-resistant states present a normal or a blunted response to NO. This study has been conducted to investigate 1) the platelet sensitivity to insulin in lean NIDDM and 2) the platelet sensitivity to an NO donor, glyceryl trinitrate (GTN), in obesity and in both lean and obese NIDDM. RESEARCH DESIGN AND METHODS: We determined 1) ADP-induced platelet aggregation and platelet cGMP content in platelet-rich plasma (PRP) obtained from 11 lean NIDDM patients, after a 3-min incubation with insulin (0, 240, 480, 960, 1,920 pmol/l) and 2) ADP-induced platelet aggregation and platelet cGMP content in PRP obtained from 9 obese subjects, 11 lean and 8 obese NIDDM patients, and 18 control subjects, after a 3-min incubation with 0, 20, 40, and 100 mumol/l GTN. RESULTS: Insulin dose-dependently decreased platelet aggregation in lean NIDDM patients (P = 0.0001): with 1,920 pmol/l of insulin, ADP ED50 was 141.5 +/- 6.4% of basal values (P = 0.0001). Furthermore, insulin increased platelet cGMP (P = 0.0001) from 7.5 +/- 0.2 to 21.1 +/- 3.7 pmol/10(9) platelets. These results were similar to those previously described in healthy subjects. GTN reduced platelet aggregation in all the groups (P = 0.0001) at all the concentrations tested (P = 0.0001), but GTN IC50 values were much higher in insulin-resistant patients: 36.3 +/- 5.0 mumol/l in healthy control subjects, 26.0 +/- 6.0 mumol/l in lean NIDDM patients (NS vs. control subjects), 123.6 +/- 24.0 mumol/l in obese subjects (P = 0.0001 vs. control subjects), and 110.1 +/- 19.2 mumol/l in obese NIDDM patients (P = 0.0001 vs. control subjects). GTN dose-dependently increased platelet cGMP in all the groups (P = 0.0001 in control subjects, lean NIDDM patients, and obese subjects; P = 0.04 in obese NIDDM patients). Values reached by obese subjects and obese NIDDM patients, however, were lower than those reached by control subjects (with 100 mumol/l of GTN, P = 0.001 and P = 0.0001, respectively). In healthy control subjects and in obese subjects, the insulin:glucose ratio, used as an indirect measure of insulin sensitivity, was positively correlated to GTN IC50 (r = 0.530, P = 0.008), further suggesting that the sensitivity to NO is reduced in the presence of insulin resistance. CONCLUSIONS: The insulin anti-aggregating effect is preserved in lean NIDDM; platelet sensitivity to GTN in preserved in lean NIDDM but is reduced in the insulin-resistant states of obesity and obese NIDDM. Resistance to nitrates, therefore, could be considered another feature of the insulin-resistance syndrome.     

Antagonist for atrial natriuretic peptide receptors ameliorates glomerular hyperfiltration in diabetic rats

To evaluate the possible contribution of atrial natriuretic peptide (ANP) to the development of glomerular hyperfiltration, we examined the effect of non-peptide competitive antagonist for biological receptors of ANP, HS-142-1, on glomerular filtration rate (GFR) and renal plasma flow (RPF) in diabetic rats. Increased GFR and RPF in diabetic rats were significantly ameliorated by the injection of HS-142-1, while blood pressure remained unchanged. Urinary cyclic GMP excretion was significantly higher in diabetic rats than in control rats and HS-142-1 decreased urinary cGMP excretion significantly. These results indicate that atrial natriuretic peptide contributes to the development of glomerular hyperfiltration and hyperperfusion in diabetes and HS-142-1 might be useful in the treatment of them.     

Atrial natriuretic peptide and oxytocin induce natriuresis by release of cGMP

Our hypothesis is that oxytocin (OT) causes natriuresis by activation of renal NO synthase that releases NO followed by cGMP that mediates the natriuresis. To test this hypothesis, an inhibitor of NO synthase, L-nitroarginine methyl ester (NAME), was injected into male rats. Blockade of NO release by NAME had no effect on natriuresis induced by atrial natriuretic peptide (ANP). This natriuresis presumably is caused by cGMP because ANP also activates guanylyl cyclase, which synthesizes cGMP from GTP. The 18-fold increase in sodium (Na+) excretion induced by OT (1 microgram) was accompanied by an increase in urinary cGMP and preceded by 20 min a 20-fold increase in NO3- excretion. NAME almost completely inhibited OT-induced natriuresis and increased NO3- excretion; however, when the dose of OT was increased 10-fold, a dose that markedly increases plasma ANP concentrations, NAME only partly inhibited the natriuresis. We conclude that the natriuretic action of OT is caused by a dual action: generation of NO leading to increased cGMP and at higher doses release of ANP that also releases cGMP. OT-induced natriuresis is caused mainly by decreased tubular Na+ reabsorption mediated by cGMP. In contrast to ANP that releases cGMP in the renal vessels and the tubules, OT acts on its receptors on NOergic cells demonstrated in the macula densa and proximal tubules to release cGMP that closes Na+ channels. Both ANP- and OT-induced kaliuresis also appear to be mediated by cGMP. We conclude that cGMP mediates natriuresis and kaliuresis induced by both ANP and OT.     

Relationship between myoglobin contents and increases in cyclic GMP produced by glyceryl trinitrate and nitric oxide in rabbit aorta, right atrium and papillary muscle

Effects of glyceryl trinitrate (GTN) and nitric oxide (NO) on the cardiac functions and myocardial cyclic GMP (cGMP) contents were examined in comparison with those in the aorta and correlated with myoglobin (an inhibitor of soluble guanylate cyclase) contents using the preparations isolated from the reserpinized rabbit. GTN (10(-10)-10(-4) mol/l) produced a dose-dependent relaxation in the aorta. However, this compound exerted no effect on the rate of the spontaneous beat of the right atrium and the contraction of the papillary muscle. A transient and significant increase in cGMP was observed in the aorta with GTN (3 x 10(-6) mol/l). Although the increase was also observed in the right atrium, it was much smaller. No definite change was observed in papillary muscle. Increases in cGMP produced by NO (3 x 10(-6) mol/l) were larger and significant in all tissues; (AUCcGMP(GTN)/AUCcGMP(NO)) ratio was 30.1 for the aorta, 65.0 for the right atrium and 16.3% for the papillary muscle. Although higher concentrations of NO were necessary in the right atrium and papillary muscle to induce increases in cGMP, no differences were noted in the three tissues as regards the maximum accumulation of this substance. Furthermore, kinetic analysis of NO-induced increases in tissue cGMP indicated no marked difference in the production rate among the three tissues, while the rate of elimination of cGMP was lower in the aorta than in the atrium or the papillary muscle. The increases in cGMP observed in these three tissues were inversely related to the contents of myoglobin in respective tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detecting panic disorder in emergency department chest pain patients: a validated model to improve recognition

Non-insulin-dependent diabetes mellitus (NIDDM) and the renal disease attributable to it have been characterized extensively in the Pima Indians, a group of American Indians who form the Gila River Indian Community in Arizona. Both of these diseases are common in this community, and their onset and duration are known with greater certainty than in other populations because research examinations, which include oral glucose tolerance tests and measures of urinary protein excretion, have been performed frequently on most members of the population for the past 30 years. Studies of glomerular structure and hemodynamic function in diabetic Pima Indians indicate that glomerular hyperfiltration often develops at the onset of NIDDM and remains elevated until after overt nephropathy appears. Structurally, the glomeruli in subjects with microalbuminuria are not clearly distinguishable from those in subjects with normoalbuminuria, but macroalbuminuria is characterized by extensive glomerular sclerosis, mesangial expansion, and widening of epithelial cell foot processes that together lead to a rapid decline in the glomerular filtration rate. The decline in glomerular function in subjects with macroalbuminuria is due both to a loss of ultrafiltration surface area and to reduction in glomerular hydraulic permeability.     

Prostaglandins maintain renal vasodilation and hyperfiltration during chronic nitric oxide synthase blockade in conscious pregnant rats

Rats demonstrate renal vasodilation and hyperfiltration in pregnancy. Because both NO and cGMP biosynthesis are increased in gravid rats and because acute administration of NO synthase inhibitors abrogates renal vasodilation and hyperfiltration, NO most likely mediates the renal circulatory changes of gestation. In the present study, we tested the effect of chronic inhibition of NO synthase on effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) in chronically instrumented, conscious, gravid rats. Because gestation is a relatively long-term condition, we postulated that chronic withdrawal of NO would result in sustained inhibition of renal vasodilation and hyperfiltration. Contrary to our hypothesis, the renal circulatory changes of pregnancy were maintained during chronic blockade of NO synthase. That is, subcutaneous administration of 10 micrograms/min N omega-nitro-L-arginine methyl ester (NAME) for 48 hours did not significantly reduce GFR in either virgin or pregnant rats; thus, hyperfiltration persisted in the latter despite chronic NO synthase blockade. In contrast, ERPF was reduced and effective renal vascular resistance (ERVR) increased in both groups of rats during NAME administration but in a parallel fashion, such that renal vasodilation persisted in the gravid animals despite chronic inhibition of NO synthase. However, with superimposition of acute prostaglandin synthesis inhibition (meclofenamate, 10 mg/kg IV), renal vasodilation and hyperfiltration were abolished; ie, the combined treatments of chronic NO synthase blockade and acute prostaglandin synthesis inhibition led to the equalization of GFR, ERPF, and ERVR in conscious virgin and pregnant rats. Inhibition of prostaglandin synthesis alone had little affect on the renal circulation, as previously reported. In summary, prostaglandins are recruited to maintain renal vasodilation and hyperfiltration during chronic NO synthase blockade in conscious pregnant rats.     

Identification and characterization of a new gene from human chromosome 21 between markers D21S343 and D21S268 encoding a leucine-rich protein

Organic nitrates undergo enzymatic metabolization in the vasculature to release the active compound nitric oxide (NO). The resulting preferential venodilation has been suggested to be related to the vascular bioactivation process of organic nitrates because sodium nitroprusside, which is bioactivated differently, is not venoselective. We sought to determine whether NO has an influence on vascular bioconversion of organic nitrates because endogenous endothelial production of NO is smaller in veins than in arteries. Rings of porcine coronary arteries were subjected to radioactive glyceryl trinitrate (GTN) after preincubation with defined amounts of NO. The vascular content of GTN and the dinitrates (GDNs) 1,2-GDN and 1,3-GDN then was quantified. NO (3 microM, 30 min) significantly impaired bioactivation of GTN as indicated by a 30-50% reduction in the accumulation of 1,2-GDN and 1,3-GDN, whereas unchanged GTN was increased. Incubation with NO also reduced the stimulated specific activity of soluble guanylate cyclase isolated from human platelets. Its specific activity was reduced from 2.6 +/- 0.2 to 2.1 +/- 0.13 nmol of cGMP/mg/min. Relaxation studies with rings of porcine coronary arteries showed that NO-induced inhibition of vascular GTN metabolism and cGMP accumulation decreased the vasodilator potency of GTN by 10-fold. Further experiments showed that the duration of NO treatment is more important for this effect than the concentration of NO. We suggest that NO can inhibit vascular bioactivation of organic nitrates and might slightly desensitize soluble guanylate cyclase. The preferential venodilation induced by organic nitrates might be the result of the comparably low production of endogenous NO in veins.     

The cost of quality assurance

The relative importance of endothelial derived relaxing factor (EDRF)/nitric oxide (NO) in maintaining kidney function in normal condition and in acute renal failure (ARF) were evaluated in inactin anesthetized rats. ARF was induced by unilateral occlusion of the left renal artery (40 min) followed by reperfusion, with the contralateral kidney serving as normal control. This protocol resulted in marked reductions in renal plasma flow (RPF), glomerular filtration rate (GFR) and increases in fractional sodium excretion (FENa) and urinary protein excretion in the post-ischemic kidney in comparison to the contralateral normal kidney. Administration of the nitric oxide (NO) synthase inhibitor NG--monomethyl-L-arginine (0.25 mg/kg per min, L-NMMA) exacerbated the ischemia-induced changes in renal functions as reflected by further reductions in urine flow (V), GFR, marked sodium wasting and renal edema. Pretreatment of the animals with NO precursor L-arginine (2.5 mg/kg per min, L-Arg) abolished the detrimental effects of L-NMMA in ARF. In contrast, D-Arginine (2.5 mg/kg per min, D-Arg) failed to reverse the detrimental effects of L-NMMA. Infusion of L-Arg alone also resulted in improvements in RPF and GFR in the ischemic kidney. The results of the present study suggest that the function of the ischemic kidney is sustained by EDRF/NO and is thus more sensitive to NO synthase inhibition.     

Synthesis of the esterified cerebroside in the epidermis of guinea pigs

BACKGROUND: The level of glomerular filtration rate (GFR) and its determinants in non-insulin-dependent diabetes mellitus (NIDDM) are currently controversial. DESIGN OF THE STUDY: We measured GFR and effective renal plasma flow (ERPF) in 121 consecutive NIDDM without evidence of overt diabetic nephropathy. Age varied from 28 to 70 years, 61.2% were women and known duration of NIDDM was 0-37 years. Hypertension was detected in 36.4% of patients and 47.8% had microalbuminuria. RESULTS: An inverse correlation was found between GFR and age, but not with known duration of NIDDM: It was a weak correlation (r = -0.41) but statistically significant (P < 0.001). The other variables considered were not significant by multiple stepwise regression analysis, but patients with lower GFR tended to have diabetic retinopathy more frequently. GFR was lower in hypertensive compared to normotensive patients (123 +/- 28.4 versus 136 +/- 32.5 ml/min/1.73 m2; P < 0.05), but was not different between patients with normal and elevated albumin excretion rate. ERPF also had an inverse correlation with age (r = -0.45, P < 0.001). CONCLUSION: We conclude that (i) age should be considered as a confounding variable when evaluating GFR in patients with NIDDM, and (ii) the age-dependent decline in GFR may mask hyperfiltration in the early stages of diabetic nephropathy in NIDDM:     

Role of superoxide anions in changes of endothelial vasoactive response during acute hyperglycemia

The effects of acute hyperglycemia on endothelial Ca2+ signaling, formation of endothelium-derived relaxing factor (EDRF) and bioactivity of EDRF were investigated. Hyperglycemia increased 2,5-tert-butyl-1,4-hydrochinone (BHQ)-initiated Ca2+ signaling and EDRF formation in a concentration-dependent manner. The effect of elevated D-glucose on Ca2+/EDRF response could be diminished by co-incubation with the antioxidants vitamin E, probucol, GSH, vitamin C and superoxide dismutase. Convincingly, hyperglycemic conditions yielded an increase in superoxide anion release from endothelial cells and the superoxide anion-generating mixture xanthine oxidase/hypoxanthine mimicked the effect of hyperglycemia on Ca2+/EDRF signaling. Besides an enhanced formation of the vasodilatatory NO compound EDRF, hyperglycemia enhanced NO degradation by endothelial cells and, thus, reduced bioactivity of EDRF. We suggest that vasoactivity during acute hyperglycemia depends on the superoxide anion scavenging properties of the vascular wall. In acute hyperglycemia and early stages of diabetes, radical scavenging capacity may be suitable to protect NO degradation, resulting in an enhanced vasodilation. In contrast, decreased free radical scavenging properties of the vasculature in prolonged hyperglycemia and in later stages of diabetes might promote NO degradation by an overshoot of superoxide anions, resulting in an attenuation of endothelium-dependent vasodilation.     

Investigations into the role of nitric oxide and the large intracranial arteries in migraine headache

Previous studies suggest that nitric oxide (NO) is involved in headaches induced by i.v. infusion of the vasodilator and NO donor glyceryl trinitrate (GTN) in healthy subjects. Extending these studies to sufferers of migraine without aura, it was found that migraineurs experienced a stronger headache than non-migraineurs. In addition, most migraineurs experienced a delayed migraine attack at variable times (mean 5.5 h) after GTN provocation. This biphasic headache response in migraineurs may be linked to hypersensitivity in the NO-cGMP pathway. Thus, compared to controls, migraineurs were found to be more sensitive to GTN-induced intracranial arterial dilatation, which is known to be mediated via liberation of NO and subsequent synthesis of cGMP Furthermore, histamine infusions in migraineurs induced headache responses and intracranial arterial responses resembling those induced by GTN in migraineurs. Histamine is known to liberate NO from the endothelium via stimulation of the H1 receptor, which is present in the large intracranial arteries in man. Because both immediate histamine-induced headache and intracranial arterial dilatation and delayed histamine-induced migraine are blocked by H1-receptor blockade, a likely common pathway for GTN and histamine-induced headaches/migraines and intracranial arterial responses may be via activation of the NO-cGMP pathway. The delay in the development of these experimental migraines may reflect activation of multiple physiological processes. The intracranial arteries of migraineurs were found supersensitive to the vasodilating effect of GTN (exogenous NO). This relates to clinical findings suggesting dilatation of the large intracranial arteries on the headache side during spontaneous migraine attacks. The function of arterial regulatory mechanisms involving NO in migraine was therefore studied. In peripheral arteries, no endothelial dysfunction of NO was found and cardiovascular and intracranial arterial sympathetic function was normal. A mild parasympathetic dysfunction may be involved and may, via denervation supersensitivity, be responsible for the observed supersensitivity to NO. Another possibility is that NO initiates a perivascular neurogenic inflammation with liberation of vasoactive peptides. NO also mediates a variety of other physiological phenomena. One of these, the pain-modulating effect observed in animals, was evaluated in a human study using GTN infusion and measurements of pain thresholds. No definite effects of GTN were demonstrated. The precise mechanisms involved in NO-triggered migraines and which part of the NO-activated cascade that is involved remain to be determined. The possibilities for pharmacological stimulation and/or inhibition of several steps of the NO-activated cascade increase rapidly and soon may be available for human studies.     

Prorenin and renal function in NIDDM patients with normo- and microalbuminuria

OBJECTIVES: To assess whether prorenin is elevated and perhaps a predictor of deteriorations in albuminuria and/or renal function in NIDDM patients with normo- and microalbuminuria. DESIGN: A cross-sectional and a longitudinal study. SETTING: Outpatient diabetic clinic. SUBJECTS: Twenty-eight NIDDM patients (16 with normoalbuminuria, 12 with microalbuminuric) and 16 healthy subjects, matched for sex, age and BMI. Fifteen patients were reinvestigated after (mean [range]) 3.1 (2.1-4.3) years. MAIN OUTCOME MEASURES: Serum prorenin and renin, urinary albumin excretion rate, and glomerular filtration rate. RESULTS: Serum prorenin was similar in both normoalbuminuric (116x/divided by) 1.9 microU ml-1 (geometric meanx/divided by antilog SD) and microalbuminuric (124x/divided by) 1.7 microU ml-1) as well as in healthy control subjects (90x/divided by) 1.7 microU ml-1). Prorenin did not correlate to either urinary albumin excretion rate or glomerular filtration rate. No difference between baseline and follow-up levels of albuminuria, glomerular filtration rate or prorenin were observed. The annual changes in albuminuria, glomerular filtration rate and prorenin were not correlated, and no correlation was found between baseline prorenin levels and annual changes in albuminuria or glomerular filtration rate. CONCLUSIONS: Serum prorenin levels are not elevated in either normoalbuminuric or microalbuminuric NIDDM patients, and serum prorenin is not a valid predictor of long-term changes in albuminuria in this patient group.     

Comparison of spontaneously released endothelium-derived relaxing factor in cerebral and extracerebral arteries in rabbits

To investigate regional differences in spontaneously released endothelium-derived relaxing factor (EDRF), a bioassay of spontaneously released EDRF was performed on rabbit basilar, ear, common carotid and thoracic arteries using an isometric tension measurement technique and a measurement of cyclic guanosine monophosphate (cGMP) content in the vascular smooth muscle. The amount of spontaneously released EDRF was higher in the basilar artery than in any other arteries examined (p < 0.01). The levels of cGMP were 57.3 +/- 4.4 (n = 7) in basilar, 26.5 +/- 4.3 (n = 6) in ear, 24.5 +/- 2.3 (n = 11) in common carotid, and 30.3 +/- 3.8 pmol/g tissue (n = 8) in thoracic artery with endothelium, while endothelium-denuded arteries showed 24.2 +/- 6.6 (n = 5), 17.5 +/- 5.1 (n = 6), 20.1 +/- 2.9 (n = 7) and 14.4 +/- 2.3 pmol/g tissue (n = 8) in the same order. Haemoglobin (10(-5) M, incubated with the artery for 5 min, significantly reduced the level of cGMP in all vessels with endothelium: 35.3 +/- 4.4 (basilar), 16.0 +/- 2.1 (ear), 14.0 +/- 1.9 (common carotid) and 8.7 +/- 1.2 pmol/g tissue (thoracic artery). Since endothelium-dependent relaxation is associated with a rise in the cGMP content of the smooth muscle cell, the data of cGMP measurement in addition to the bioassay of spontaneously released EDRF in tension measurement suggests that the spontaneous release of EDRF is much greater in the basilar artery than in extracerebral arteries. It is concluded that the intensity of the spontaneously released EDRF is relatively higher in the intracerebral artery than in the extracerebral artery.     

Isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by B1 repetitive sequence-representational difference analysis

1. We examined whether zaprinast, a putative cGMP-specific phosphodiesterase inhibitor, affects neural control of renal function in pentobarbital-anaesthetized dogs. 2. Renal nerve stimulation (1 Hz, 1 ms duration) reduced urine flow rate, urinary Na+ excretion (UNaV) and fractional excretion of Na+ (FENa) with little change in either renal blood flow (RBF) or glomerular filtration rate (GFR). 3. Intrarenal arterial infusion of zaprinast (10 and 100 micrograms/kg per min) increased basal urine flow rate, UNaV and FENa but not RBF or GFR. Zaprinast infusion (100 micrograms/kg per min) also increased renal venous plasma cGMP concentration and urinary cGMP excretion. 4. Renal nerve stimulation-induced reductions in UNaV and FENa were attenuated during zaprinast infusion, whereas the reduction in urine flow rate was resistant to zaprinast. 5. Renal nerve stimulation increased the renal venous plasma noradrenaline concentration and renal noradrenaline efflux, which remained unaffected during infusion of zaprinast (100 micrograms/kg per min). 6. The results of the present study suggest that zaprinast induces natriuresis and counteracts adrenergically induced antinatriuresis by acting on renal tubular sites in the dog kidney in vivo.     

Homodimers of the 60 kDa phosphatidylinositol-anchored transforming growth factor-beta 2 binding proteins in FBHEC and MG-63 cells

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His+ revertants to a maximum of 4 times over background at a GTN dose of 5 mumol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 mumol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C-->T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Glomerular filtration and tubular reabsorption of albumin in preproteinuric and proteinuric diabetic rats

Microalbuminuria (26-250 mg/d) is considered to be an indicator of incipient diabetic nephropathy in humans in insulin-dependent diabetes (IDD). However, before microalbuminuria is observed, glomerular alterations, such as glycosylation of the glomerular basement membrane and glomerular hyperfiltration, in IDD may result in increased filtration of albumin before any observed increase in albumin excretion. Glomerular and tubular albumin kinetics were examined in streptozotocin (65 mg/kg body wt, i.v.) diabetic, Munich-Wistar rats at 7-10 (untreated) and 50-70 d (poorly controlled with small doses of insulin) after the onset of diabetes and compared with nondiabetic controls. Additional rats in each condition received acute lysine treatment to prevent tubular protein reabsorption. Urinary albumin excretion and nonvascular albumin distribution volumes were measured in the renal cortex and compared with morphometric measurements of interstitial space and the proximal tubule to assess intracellular uptake of albumin in the proximal tubule. Urinary albumin excretion under anesthesia was not different in 7-10-d IDD versus controls (19 +/- 3 vs. 20 +/- 3 micrograms/min) but increased in the 50-70-d IDD (118 +/- 13 micrograms/min, P < 0.05). Lysine treatment resulted in increased albumin excretion compared with respective nontreatment in 7-10-d IDD (67 +/- 10 micrograms/min, P < 0.05) but not in controls (30 +/- 6 micrograms/min) or in 50-70-d IDD (126 +/- 11 micrograms/min). Glomerular filtration rate was increased both in 7-10-d IDD (2.7 +/- 0.1 ml/min, P < 0.05) and in 50-70-d IDD (2.6 +/- 0.1 ml/min, P < 0.05) compared with control (2.2 +/- 0.1 ml/min). Calculated urinary space albumin concentrations increased early in IDD with 2.5 +/- 0.4 mg% in 7-10-d IDD and 4.9 +/- 0.6 mg% in 50-70-d IDD compared with control (1.4 +/- 0.3 mg%). The increase in filtration of albumin is in excess of that attributable to hyperfiltration before increased albumin excretion early in diabetes. In 50-70-d IDD, absolute tubular reabsorption of albumin is decreased, correlating to the decrease in brush border height of the proximal tubule.     

Urinary glycosaminoglycan excretion in NIDDM subjects: its relationship to albuminura

Nephropathy is a serious microvascular complication of diabetes mellitus which is preceded by a period of microalbuminura. Increased loss of proteoglycan (PG) from glomerular basement (GBM) has been postulated to alter glomerular charge selectivity which contributes to urinary loss of albumin. In this study we measured the excretion of urinary glycosaminoglycans (GAG), the degradation products of PG, in 82 non-insulin-dependent (NIDDM) (Type 2) diabetic and 34 non-diabetic subjects. We found that diabetic subjects had a significantly higher GAG urinary excretion rate compared to non-diabetic subjects (12.54 +/- 5.67 vs 8.80 +/- 3.99 micrograms glucuronic acid min-1, p = 0.0001). Categorizing for albuminuric status shows that the diabetic normo-, micro- and macroalbuminuric groups have a higher GAG excretion rate than non-diabetic subjects. Heparan sulphate (HS) GAG urinary excretion was measured in 25 samples from diabetic subjects and 18 non-diabetic subjects. Diabetic subjects excreted more HS GAG than controls both as a rate or as a percentage of total GAG (3.70 +/- 1.94 vs 2.38 +/- 1.48 micrograms glucosamine min-1, p = 0.02; 31.6% +/- 12.5 vs 23.1% +/- 10.4, p = 0.02). Categorizing for albuminuric status shows that micro- and macro-albuminuric groups have a significantly higher HS GAG excretion rate than non-diabetic subjects. We conclude that, as in IDDM, excretion of GAG and HS GAG is higher in NIDDM and may precede the development of microalbuminuria.     

Prevalence of a physician-assigned diagnosis of prostatitis: the Olmsted County Study of Urinary Symptoms and Health Status Among Men

OBJECTIVE: To investigate the effect of alloxan-induced diabetes mellitus (DM, a major risk factor for erectile dysfunction and associated with impaired endothelial function) on the formation of nitric oxide (NO), prostacyclin (PGI2), cGMP and cAMP in the corpus cavernosum of rabbits. MATERIALS AND METHODS: Rabbits were rendered diabetic (hyperglycaemic, nonketotic) with alloxan; after 3 and 6 months, the penises were excised and the corpus cavernosum processed for the study of PGI2, NO, cAMP and cGMP formation, using a range of stimulators and radioimmunoassays. RESULTS: PGI2 formation in response to acetylcholine and phorbol ester, but not arachidonate, and cGMP formation in response to A23187 (NO-release dependent), was significantly diminished in diabetic rabbit corpus cavernosum compared with controls at both 3 and 6 months after the induction of DM. cAMP formation in response to forskolin and prostaglandin E1 was reduced after 6 but not 3 months, although nitroprusside-stimulated cGMP (activates guanylyl cyclase directly) was unaffected in cavernosal tissue from diabetic rabbits. CONCLUSIONS: These results show that the formation of NO and PGI2, and adenylyl cyclase activity but not guanylyl cyclase, are impaired in the corpus cavernosum of diabetic rabbits. As NO and PGI2 are produced by the endothelium, these studies consolidate the view that endothelial dysfunction is a major contributor to erectile dysfunction in diabetes mellitus.     

Does luminal release of EDRF contribute to downstream microvascular tone?

The contribution of luminally released endothelium-derived relaxing factor (EDRF) transported with the flowing blood to the control of smooth muscle tone of downstream arterioles was studied. The dilatory response of arterioles in the spinotrapezius muscle of the rat to superfusion of acetylcholine (ACH) was investigated by intravital microscopy before and after blockade of blood flow by micropipette occlusion upstream of the site of observation. Vessels were studied without treatment, after topical application of indomethacin (INDO, 3 10(-5) M) to inhibit prostanoid production and after local treatment with NG-nitro-L-arginine (LNNA, 2.9 10(-3) M) to suppress local production of EDRF. In untreated vessels and after INDO, responses to ACH were not significantly different in the presence or absence of blood flow. After LNNA, the responses to ACH were reduced to about 50%, but were again similar with or without flow. These results indicate that luminally transported EDRF does not play a significant role in controlling arteriolar tone in small skeletal muscle arterioles.     

Glomerular mesangial cells: electrophysiology and regulation of contraction

Mesangial cells are smooth muscle-like pericytes that abut and surround the filtration capillaries within the glomerulus. Studies of the fine ultrastructure of the glomerulus show that the mesangial cell and the capillary basement membrane form a biomechanical unit capable of regulating filtration surface area as well as intraglomerular blood volume. Structural and functional studies suggest that mesangial cells regulate filtration rate in both a static and dynamic fashion. Mesangial excitability enables a homeostatic intraglomerular stretch reflex that integrates an increase in filtration pressure with a reduction in capillary surface area. In addition, mesangial tone is regulated by diverse vasoactive hormones. Agonists, such as angiotensin II, contract mesangial cells through a signal transduction pathway that releases intracellular stores of Ca2+, which subsequently activate nonselective cation channels and Cl- channels to depolarize the plasma membrane. The change in membrane potential activates voltage-gated Ca2+ channels, allowing Ca2+ cell entry and further activation of depolarizing conductances. Contraction and entry of cell Ca2+ are inhibited only when Ca2+-activated K+ channels (BK(Ca)) are activated and the membrane is hyperpolarized toward the K+ equilibrium potential. The mesangial BK(Ca) is a weak regulator of contraction in unstimulated cells; however, the gain of the feedback is increased by atrial natriuretic peptide, nitric oxide, and the second messenger cGMP, which activates protein kinase G and decreases both the voltage and Ca2+ activation thresholds of BK(Ca) independent of sensitivity. This enables BK(Ca) to more effectively counter membrane depolarization and voltage-gated Ca2+ influx. After hyperpolarizing the membrane, BK(Ca) rapidly inactivates because of dephosphorylation by protein phosphatase 2A. Regulation of ion channels has been linked casually to hyperfiltration during early stages of diabetes mellitus. Determining the signaling pathways controlling the electrophysiology of glomerular mesangial cells is important for understanding how glomerular filtration rate is regulated in health and disease.