Inger Rubin

Evidence for nitric oxide and nitric oxide synthase activity in proximal stumps of transected peripheral nerves

Nitric oxide may be liberated as an inflammatory mediator within injured peripheral nerve trunks. We evaluated the proximal stumps of injured peripheral nerve stumps that later form neuromas or regenerative nerve sprouts, for evidence of local nitric oxide elaboration and activity. Proximal stumps were created in male Sprague-Dawley rats by sectioning of the sciatic nerve and resection of its distal portions and branches. There was striking physiological evidence of nitric oxide activity at the tips of 48-h and 14-day-old proximal nerve stumps. We detected local nitric oxide-mediated hyperemia of both extrinsic plexus and endoneurial microvessels that was reversible, in a dose-dependent stereospecific fashion, by the broad-spectrum nitric oxide synthase inhibitors, Nomega-nitro-L-arginine-methyl ester or Nomega-nitro-L-arginine, but not by 7-nitroindazole, an inhibitor with relative selectivity for neuronal nitric oxide. Immunohistochemical studies provided evidence for the localization of nitric oxide generators at the same sites. In 48-h but not 14-day stumps increased expression of two isoforms of nitric oxide synthase was detected: endothelial nitric oxide and to a much lesser extent neuronal nitric oxide synthase. Both isoforms appeared in axonal endbulb-like profiles that co-localized with neurofilament immunostaining. Western immunoblots identified a band consistent with endothelial nitric oxide synthase expression. In 14-day stumps with early neuroma formation, but not 48-h stumps, there was staining for immunological nitric oxide synthase in some endoneurial and epineurial macrophages. Total nitric oxide synthase biochemical enzymatic activity, measured by labelled arginine to citrulline conversion, was increased in 14-day but not 48-h stumps. Injured peripheral nerves have evidence of early nitric oxide action, nitric oxide synthase expression and nitric oxide activity in proximal nerve stumps. Nitric oxide may have an important impact on the regenerative milieu.

Role of Nitric Oxide Synthase Inhibition in Leukocyte-Endothelium Interaction in the Rat Pial Microvasculature

We investigated the role of nitric oxide (NO) in leukocyte-endothelium interaction, blood-brain barrier (BBB) function and oxygen free-radical production in the rat pial microcirculation. In a closed cranial window preparation (dura removed) over the parietal cortex of pentobarbital-anesthetized Wistar rats, NO synthase (NOS) was inhibited by systemic and/or topical application of Nω-nitro-l-arginine (l-NNA) under physiological conditions and during leukotriene B4 (LTB4) activation. Circulating leukocytes were labeled by intravenous injection of rhodamine 6G. We used a confocal laser scanning microscope (CLSM) and studied leukocyte rolling and sticking in pial veins and arteries before and after NOS inhibition. At the end of the experiments, sodium-fluorescein was injected intravenously to test BBB integrity. Brain cortex oxygen free-radical production was investigated in the cranial window preparation using lucigenin-enhanced chemiluminescence (CL). l-NNA application did not lead to significant changes in leukocyte-endothelium interaction, BBB function, and oxygen free-radical production under physiological conditions [leukocyte-endothelium interaction: control (n = 5), l-NNA systemically (n = 5), l-NNA topically (n = 5): at baseline rollers/100 μm: 0.76 ± 0.55, 0.64 ± 0.94, 0.44 ± 0.55 and stickers/100 μm: 0.90 ± 0.28, 0.76 ± 0.24, 0.84 ± 0.42; at 60 min rollers/100 μm: 1.49 ± 0.66, 1.21 ± 0.99, 0.67 ± 0.66 and stickers/100 μm: 1.04 ± 0.20, 1.19 ± 0.23, 1.21 ± 0.54; oxygen free-radical production (n = 4): CL count before l-NNA application 35 ± 17 cps, after 1 h of topical superfusion of l-NNA 38 ± 14 cps; p < 0.05]. In contrast to the results achieved under physiological conditions, a significant further increase of rolling leukocytes and BBB permeability occurred due to NOS inhibition under LTB4-activated conditions [76 ± 47% significant (p ≤ 0.01, n = 7) further increase of rollers/100 μm due to 60 min l-NNA application following the activation period of 120 min LTB4 superfusion]. Our results support a modulatory role for NO in leukocyte-endothelium interaction and BBB permeability in the pial microcirculation when this interaction is increased.

Dot immunobinding and immunoblotting of picogram and nanogram quantities of small peptides on activated nitrocellulose

Nitrocellulose was activated with divinyl sulfone, a spacer of ethylenediamine, and glutaraldehyde. The aldehyde groups on the activated nitrocellulose, Nit-CHO, were stable through one month at 4 degrees C. Peptides were attached to the membrane by reaction of the amino group with the free carbonyl, forming peptide bonds. The decapeptide angiotensin I (AI), the octapeptide angiotensin II (AII), angiotensin analogues, Met- and Leu-enkephalin (Met-E and Leu-E) were tested on the membranes with specific rabbit antibodies (sRaAb) against the peptides, and visualized by horseradish peroxidase conjugated anti-rabbit antibody (HRP-anti-RaAb). With this technique AII could be detected with a sensitivity of 20 pg/cm2 and AI by 500 pg/cm2. Substitution of Ala7 for Pro7 in AI and AII caused a marked reduced binding of anti-AI and antid-AII antisera, respectively, and it completely abolished crossreactivity of anti-AI with Ala7-AII as well as anti-AII with Ala7-AI. Peptides from the gp41 and gp36 antigens corresponding to the sequence aa596-618 of the human immunodeficiency viruses type 1 and 2, HIV-1 and HIV-2, were tested on Nit-CHO with two human sera from infected patients. The serological reactions were specific for both the HIV-1 and HIV-2 peptide, respectively. This indicated that the technique could be exploited for serological testing of humans. Separation of peptides by high performance thin layer chromatography (HPTLC) and identification by immunoblotting was demonstrated with angiotensin analogues. After separation by HPTLC on silica aluminium plates the peptides were electrotransfered by semidry electroblotting on Nit-CHO, followed by specific antibody overlays and developed as for the dot immunobinding technique. This combined method enabled us to differentiate between closely related peptide analogues and it improved the sensitivity of peptide detection 100-1000 fold as compared to visualization by quenched fluorescence on chromatography plates.

NO- and non-NO-, non-prostanoid-dependent vasodilatation in rat sciatic nerve during maturation and developing experimental diabetic neuropathy

This study examined NO‐ and non‐NO‐, non‐prostanoid‐dependent pathways of agonist‐induced vasodilatation in streptozotocin (STZ)‐induced diabetic rats and their age‐matched controls at 1–2, 8–10 and 18–20 weeks after induction of diabetes. Using laser Doppler flowmetry, vasodilatory responses to acetylcholine (ACh; 0.1 mM) and morpholino‐sydnonimine (SIN‐1) were determined in the presence of Ringer solution, during inhibition of NO synthase (NOS) and cyclo‐oxygenase (COX) with Nω‐nitro‐L‐arginine (L‐NNA; 1 mM) + indomethacin (10−5 M), and during inhibition of K+ channels, NOS and COX with tetraethylammonium (TEA; 10 mM) + L‐NNA + indomethacin. Basal NOS activity and nerve conduction velocity were also determined. In age‐matched controls, SIN‐1‐induced vasodilatation in the presence of TEA + L‐NNA + indomethacin, basal NOS activity and the initial vasodilatory response to ACh during NOS and COX inhibition all decreased with maturation. In STZ‐induced diabetics, SIN‐1‐induced vasodilatation in the presence of TEA + L‐NNA + indomethacin was impaired immediately after induction of diabetes, but not at 18–20 weeks. NOS activity in STZ‐induced diabetics displayed a transient 2‐fold increase at 8–10 weeks, decreasing to age‐matched control levels at 18–20 weeks. At 18–20 weeks of STZ‐induced diabetes, ACh‐induced vasodilatation during NOS and COX inhibition was prolonged due to increased K+ channel activity and experimental diabetic sensory neuropathy (EDN) had developed. Thus, in sciatic nerve microcirculation of STZ‐induced diabetic rats: (1) diabetic impairment of vasodilatation in response to exogenous NO was transient; (2) non‐NO‐, non‐prostanoid‐dependent vasodilatation and K+ channel activity were augmented in STZ‐induced diabetes; and (3) alterations in NO bioactivity were not related to the development of EDN.

Studies on the native forms of renin in the rat kidney

This paper describes Sephadex G-100 chromatography of rat kidney extract containing various enzyme inhibitors. The high molecular weight renin (molecular weight above 50 000) constitutes about 50% of the total renin activity. Omission of the enzyme inhibitors yield solely low molecular weight renin. Upon rechromatography high molecular weight renin eluted in two peaks at lower molecular weight with a concomitant reduction of renin activity. Renin activity in the fractions from Sephadex G-100 chromatography was increased 70% by dialysis at acid as well as neutral pH through the whole molecular weight range. Cold storage of extract with low molecular weight increased renin activity about 25%. The results suggest that the fully active enzyme is not represented by the lower molecular weight forms of renin and direct connection between activation of renin and reduction of renin molecular size was not indicated.

Elevated Des-Angi-Angiotensinogen Levels by Nephrectomy and Adrenalectomy

This study investigates the time course of plasma levels of angiotensinogen (Aogen) and of the Aogen metabolite des-AngI-angiotensiongen (des-AngI-Aogen) in nephrectomized rats with and without adrenals for 24 h. After nephrectomy the plasma Aogen levels increased 5-fold over the following 24 h. The increase is significantly lower after sham nephrectomy (3.7-fold, P < 0.05) and if the kidneys are withdrawn without decapsulization (2.4-fold, P < 0.05). A small and transient increase arise after nephrectomy plus adrenalectomy (1.6-fold after 8 h, P < 0.005). After adrenalectomy alone Aogen levels continuously shrink to 38% of control values after 24 h. Plasma des-AngI-Aogen levels increase 2.1- to 3.7-fold 24 h after the different nephrectomy procedures. In connection with recent findings these data support the notion that the increase in Aogen plasma levels after bilateral nephrectomy is triggered by renin, released during surgery. High plasma levels of des-AngI-Aogen after nephrectomy indicate that AngI is generated by tissue renin, e.g., in the adrenals. This suggests that after nephrectomy the plasma des-AngI-Aogen levels should be a valuable proof for the evaluation of the amount of generated angiotensin.

An angiotensin I-protein complex isolated by SDS-polyacrylamide gel electrophoresis from rat serum incubated with angiotensin I

Angiotensin I (AI) protein complex with Mr 25,000 and an angiotensin I immunoreactive protein with Mr 60,000 were isolated from rat serum. The proteins were separated from AI by sodiumdodecylsulphate polyacrylamide gel electrophoresis under denaturing and reducing conditions using SDS and 2-mercaptoethanol (SDS-PAGE). The Mr 60,000 protein was isolated from non-incubated serum. Special attention was given to the Mr 25,000 AI-protein complex, called AI-25,000, which was generated in rat serum incubated with both synthetic and native AI for 1-5 h at 24 degrees C and neutral pH. The AI-25,000 was determined in two radioimmunoassays using two anti-AI antisera and 125I-AI. The experiments showed that AI reacts with a serum protein and forms a stable complex with a high molecular mass.

Enzyme Inhibitors are not Helpful in Preserving Big Renin in the Rat Kidney

The effect of four different homogenization methods, proteolytic enzymes and enzyme inhibitors on rat renal renin was studied. Rat kidney homogenate was treated with trypsin or pepsin. Neither of these enzymes had any effect on the molecular weight pattern. Enzyme inhibitors, 10 mM N-ethylmaleimide, 5.4 mM EDTA, 1.0 mM toluenesulfonyl fluoride, 2.3 mM o-phenanthroline, 2.3 mM p-hydroxymercuribenzoate, which was added to the homogenization medium, all or some of them, as well as 1/20 to the buffer solution used throughout the experiments for dialysis and column chromatography, did not alter the molecular weight pattern. These results suggest that renins, with molecular weights from above 60 000 Dalton to 37 000 Dalton were preformed in the rat kidney and extracted in a native form.