Confirmation of direct angiotensin formation by kallikrein.

This study was undertaken to confirm our previous preliminary observation that hog pancreas kallikrein (EC 3.4.21.35) directly liberated an angiotensin-like substance from human plasma protein Cohn fraction IV-4 at an acidic pH of 4.0-5.0. First, the possibility of proangiotensin or des-Asp1-angiotensin being the pressor substance was ruled out by t.l.c. Secondly, the pressor substance was purified by Sephadex G-25 and Bio-Gel P-2 gel filtration, and finally by high-performance liquid chromatography. The amino acid composition of the isolated pressor substance (residues/mol) was: Asp, 1.03; Val, 1.03; Ile, 1.00; Tyr, 0.69; Phe, 1.04; His, 0.91; Arg, 0.86; Pro, 0.86. This composition was identical with that of angiotensin. Since the reaction mixture was not contaminated with common proteolytic enzymes, such as trypsin, chymotrypsin, renin, cathepsin D and proangiotensin-converting enzyme, and other enzymes activated by kallikrein, it is clear that hog kallikrein directly produces angiotensin in vitro.     

Identification of [hydroxyproline3]-bradykinin released from human plasma and plasma protein Cohn's fraction IV-4 by trypsin

Aside from bradykinin (BK), a novel kinin, [Hydroxyproline3]-bradykinin ( [Hyp3]-BK), was isolated from the reaction mixture of human plasma and plasma protein Cohn's fraction IV-4 with trypsin. The liberated kinins were isolated based on procedures which we previously described for the isolation of [Hyp3]-lysyl-bradykinin ( [Hyp3]-Lys-BK) formed by kallikrein. The ratio of the amounts of two kinins thus formed from human plasma protein Cohn's fraction IV-4 were [Hyp3]-BK 25 +/- 4% and BK 75 +/- 4%, similarly to that of [Hyp3]-Lys-BK and Lys-BK, formed by kallikrein, but it varied by persons. The isolation of [Hyp3]-BK and [Hyp3]-Lys-BK suggests that a novel kininogen containing hydroxyproline in the third position of the bradykinin sequence in human plasma protein, possibly undergone post-translational modifications.     

Characterization of a vasopressor substance generated in human plasma by incubation

Incubation of human plasma at 37 degrees C for several hours leads to the formation of a non-dialysable vasopressor substance termed the active pressor principle. Some of the chemical and physical natures of active pressor principle were investigated in anesthetized and ganglion blocked rats. It was found to have properties characteristic of protein. The substance was crudely purified to about 25-fold in alcoholic trichloroacetic acid solution after placing the plasma in a boiling water bath for 5 minutes ("active fraction"). After treatment of the vasoactive plasma or "active fraction" with Pronase, the pressor activity was almost abolished. The molecular weight of this fraction as determined by gel filtration was about 68,000. With addition of diisopropyl fluorophosphate before incubation of the plasma, no vasopressor substance was generated. After treatment of the rat with captopril, an angiotensin converting enzyme inhibitor, the pressor effect of incubated plasma was not inhibited. These findings suggest that a vasoactive protein, which is clearly different from renin, is generated during simple incubation of plasma, and that a serine protease is involved in the formation of this substance.     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

Chemical identity of tryptensin with angiotensin.

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.     

Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.     

Nature of the pressor substance in rabbit placenta

1. The renin-like substance isolated from the placenta of the rabbit produces a prolonged increase of blood pressure in the nephrectomized rat. If incubated with renin substrate from ox serum, it forms a pressor substance that elevates blood pressure in exactly the same way as does angiotensin. 2. This angiotensin-like principle was concentrated by means of ion-exchange chromatography and compared with Val-5-angiotensins I and II in two paper-chromatography systems and in paper electrophoresis. 3. In all the three methods the unknown principle behaved like a mixture of the two reference compounds. 4. It is concluded that incubation of the renin-like substance of placental origin with substrate from ox serum gave a mixture of Val-5-angiotensins I and II. This is evidence that the enzyme isolated from the placenta is either closely related to, or identical with, renin.     

Conversion of substance P to C-terminal fragments in human plasma

Substance P is rapidly converted by enzyme(s) in human plasma to des-[Arg1Pro2]-substance P (fragment 3-11) and to des-[Arg1Pro2Lys3Pro4]-substance P (fragment 5-11). These metabolites were isolated by HPLC and partially sequenced. No evidence was obtained for deamidation of substance P in plasma or for the formation of the N-terminal tetrapeptide [Arg-Pro-Lys-Pro]. The data suggest that substance P is metabolized in human plasma by an enzyme with the specificity of dipeptidyl-aminopeptidase IV. Consistent with this hypothesis, the rate of degradation of substance P measured with an antibody directed against the N-terminal region is 2-3-fold greater than measured with a C-terminally directed antibody. The degrading activity of plasma was purified 522-fold and was eluted from a gel filtration column in the molecular weight zone 150 000-170 000 and from a chromatofocusing column in the pH range 4.5 to 5.5.     

Alteration and restoration of endotoxin activity after complexing with plasma proteins

Rudbach, Jon A. (The University of Michigan, Ann Arbor), and Arthur G. Johnson. Alteration of endotoxin activity after complexing with plasma proteins. J. Bacteriol. 92:892-898. 1966.-A substantial decrease in the ability of endotoxin to be precipitated by homologous antiserum and to cause fever occurred after incubation with human plasma or human plasma, Cohn fraction IV-1. The endotoxin, thus altered, also displayed decreased lethality for rabbits. These alterations in endotoxin activity could be restored when the endotoxin-plasma protein mixture was treated with a proteolytic enzyme, and the endotoxin was precipitated with ethyl alcohol. Inactivation of the antigenic and toxigenic properties of the endotoxin molecule by plasma is discussed as resulting from complexing with plasma proteins rather than from enzymatic degradation.     

Biological activities of angiotensin II-(1-6)-hexapeptide and angiotensin II-(1-7)-heptapeptide in man

Biological activities of angiotensin II-(1-6)-hexapeptide [ANG-(1-6)] and angiotensin II-(1-7)-heptapeptide [ANG-(1-7)] were studied in 5 normal men and 3 patients with Bartter's syndrome. The angiotensins were infused iv in each subject from 0900 h to 0915 h at a rate of 21 nmol(16.8 micrograms)/kg X min and 18 nmol(16.2 micrograms)/kg X min for ANG-(1-6) and ANG-(1-7), respectively. In the normal men a significant rise in blood pressure was observed by the infusions of both peptides. Average increments of blood pressure for ANG-(1-6) were 17/14, 23/18, 22/15 and 17/14 mmHg at 2, 5, 10 and 15 min, respectively, and those for ANG-(1-7) were 19/15, 20/17, 13/13 and 15/13 mmHg at 2, 5, 10 and 15 min, respectively. The duration of pressor actions after the cessation of the infusions (T) was 10 min for ANG-(1-6) and 20 (for systolic) and 30 (for diastolic) min for ANG-(1-7). T for ANG-(1-6) was shorter than and T for ANG-(1-7) was similar to T for Ile5-angiotensin II (Ile5-ANG II) reported previously in 7 normal men 5 of whom were the same as examined in the present study. On the other hand, both peptides did not cause a rise in blood pressure in the 3 patients with Bartter's syndrome. Both angiotensins did not cause an increase in plasma aldosterone but did cause a significant decrease in plasma renin activity both in the normal men and in the patients. From these results and our previous observations of inactivity of angiotensin II-(5-8)-tetrapeptide, a pressor action of angiotensin II-(4-8)-pentapeptide, and pressor, renin-suppressing and steroidogenic actions of angiotensin II-(3-8)-hexapeptide in normal men, it is thought that ANG-(1-6) and ANG-(1-7) are bound to angiotensin II (ANG II) receptor in the peripheral arterioles and show pressor actions (less than 0.024% and less than 0.028% of Ile5-ANG II, respectively) and suppress renin mainly via short loop feedback and that the shortest biologically active ANG II molecules for pressor, renin-suppressing and steroidogenic actions are Tyr-Ile-His, Val-Tyr-Ile-His and Val-Tyr-Ile-His-Pro-Phe, respectively, in man. It is also evident that ANG-(1-6) is more rapidly metabolized than ANG-(1-7) or Ile5-ANG II in man.     

Relative biological activities of Asn1-,Val5-angiotensin II, Ile5-angiotensin II and Sar1-angiotensin II in man

Biological activities of asn1-,val5-angiotensin II (Hypertensin, Ciba, Asn1-,Val5-ANG II), ile5-angiotensin II (human angiotensin II, Ile5-ANG II) and sar1-angiotensin II (Sar1-ANG II) were compared in man. In 7 normal men 5 pmol/kg X min each of Asn1-,Val5-ANG II, Ile5-ANG II and Sar1-ANG II was infused iv from 0900 h to 0930 h at 1-week intervals. Average increments of blood pressure at the end of the infusions were 11/12, 23/20 and 36/30 mmHg, respectively (significant differences among the 3: P less than 0.001), average decrements of plasma renin activity were 0.30, 0.32 and 0.27 ng/ml X H, respectively (no significant difference among the 3), average increments of plasma aldosterone were 1.1, 2.3 and 4.4 ng/100 ml, respectively (significant difference between the former 2: P les than 0.001, between the latter 2: P less than 0.02), and durations of blood pressure rise after the cessation of these infusions (T) were 2-5 (average 5) min, 10-25 (average 20) min and 35-60 (average 40) min, respectively (significant difference between the former 2:less than P 0.01, between the latter 2: P less than 0.001). From these results it is evident that the pressor and steroidogenic actions of Ile5-ANG II are significantly stronger than those of Asn1-,Val5-ANG II and that the duration of pressor action of the former is much longer than that of the latter. Therefore, when the activities of angiotensin II (ANG II) derivatives are compared with those of ANG II in man, Ile5-ANG II--natural human ANG II--should always be used instead of Asn1-,Val5-ANG II. The pressor and steroidogenic actions and T of Sar1-ANG II are significantly stronger or longer than those of Ile5-ANG II. The reason for this is thought to be that Sar1-ANG II is bound tightly to the vascular and adrenal ANG II receptors and is not readily metabolized.     

Subcellular distribution of leukotriene C4 binding units in cultured bovine aortic endothelial cells

The subcellular distribution of specific binding sites for [3H]leukotriene C4 ([3H]LTC4) was analyzed after sedimentation of organelles from disrupted bovine aortic endothelial cells on sucrose density gradients and was shown to be in membrane fractions I (20% sucrose) and IV (35% sucrose). Saturation binding studies of [3H]LTC4 on endothelial cell monolayers at 4 degrees C demonstrated high-affinity binding sites with a dissociation constant (Kd) of 6.8 +/- 2.2 nM (mean +/- SD) and a density of 0.12 +/- 0.02 pmol/10(6) cells. At 4 degrees C, the specific binding of [3H]LTC4 by each of the subcellular fractions reached equilibrium at 30 min and remained stable for an additional 60 min. After 30 min of incubation with [3H]LTC4, the addition of excess unlabeled LTC4 to each subcellular fraction reversed more than 70% of [3H]LTC4 binding in 10 min. The [3H]LTC4 binding activities of subcellular fractions were enhanced approximately twofold to fourfold in the presence of Ca2+, Mg2+, and Mn2+, whereas Na+, K+, and Li+ were without effect. As measured by saturation experiments, the Kd and density of LTC4 binding sites in fraction I were 4.8 +/- 1.6 nM and 16.5 +/- 1.9 pmol/mg of protein, respectively, and in fraction IV were 4.7 +/- 1.5 nM and 81.4 +/- 19 pmol/mg of protein, respectively. Inhibition of [3H]LTC4 binding in membrane-enriched subcellular fractions I and IV by LTC4 occurred with molar inhibition constant (Ki) values of 4.5 +/- 0.1 nM and 4.7 +/- 1.2 nM, respectively, whereas Ki values for LTD4 were 570 +/- 330 nM and 62.5 +/- 32.8 nM, respectively, and for LTE4 were greater than 1000 nM for each fraction; LTB4 and reduced glutathione were even less active. FPL55712, a putative antagonist of the sulfidopeptide LT components of slow reacting substance of anaphylaxis, had Ki values of 1520 +/- 800 nM and 1180 +/- 720 nM for [3H]LTC4 binding sites on membrane-enriched subcellular fractions I and IV, respectively. Thus as defined by Kd, Ki, and specificity, the LTC4 binding units that are distributed to the plasma membrane and the binding units in the subcellular fraction of greater density were similar to each other. Pretreatment of the isolated subcellular membrane fractions with trypsin abolished [3H]LTC4 binding by fraction I, enriched for the plasma membrane marker 5' nucleotidase, and that by fraction IV, enriched for the mitochondrial membrane marker succinate-cytochrome C reductase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of Angiotensin Peptides by Neuronal and Glial Cultures from Rat Brain

The degradation pattern and rate of [Ile5]-Angiotensin (Ang) I, II, and III were studied in neuron-enriched and glia-enriched cells in primary cultures from rat brain. Metabolites were separated by HPLC, and their identities were evaluated by comparison of their retention times with those of synthetic Ang peptide fragments and by analysis of their amino acid composition. Major metabolites were identified as des-Asp1-[Ile5]-Ang I, des-Asp1-[Ile5]-Ang II, [Ile5]-Ang II (3-8) hexapeptide, [Ile5]-Ang II (4-8) pentapeptide, and [Ile5]-Ang II (5-8) tetrapeptide. Glia-enriched cells degraded [Ile5]-Ang I and [Ile5]-Ang III significantly faster than neuron-enriched cells, whereas no difference between the two types of cells was found in the degradation rate of [Ile5]-Ang II. Although the half-lives of [Ile5]-Ang I and [Ile5]-Ang III in neuron-enriched cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were not significantly different, neuron-enriched cultures from WKY rats metabolized [Ile5]-Ang II about 2.6 times faster than neuron-enriched cells derived from SHR.     

Insulin-like growth factor characteristics of an acidic non-suppressible insulin-like activity.

The biological activities of an acidic form of non-suppressible insulin-like activity (ILA pI 4.8) have been studied. ILA pI 4.8 was isolated from Cohn fraction IV-1 of human serum by pH 5.5 ion-exchange chromatography on SP-Sephadex. Carrier-bound ILA was eluted at pH 9.7 and then sequentially gel chromatographed in 1% formic acid on Sephadex G-75 and Bio-Gel P-30. The low-Mr (7000) active material was subjected to flat bed isoelectric focusing. Overall recovery was 87 munit of insulin equivalents/100 g of Cohn fraction IV-1, with a specific activity in the range 4-10 munit/mg of protein, representing a purity of 1-6%. This material has been tested in a variety of insulin-like growth factor (IGF)/somatomedin assay systems. It stimulated, in a dose-related manner, [14C]glucose conversion into lipid by isolated rat adipocytes, 35SO4(2-) incorporation into weanling rat costal cartilage and [3H]thymidine incorporation into DNA of cultured human fibroblasts. Like IGF-I and -II, ILA pI 4.8 was able to inhibit degradation of 125I-insulin by crude homogenates of rat liver. In addition, the biological activity of ILA pI 4.8 was completely suppressible by a recently described inhibitor of IGF-I and IGF-II. ILA pI 4.8 was able to compete, in a parallel manner, with 125I-IGF-I and 125I-IGF-II and, at higher doses, with 125I-insulin in a placental radioreceptor assay. No cross-reactivity was seen in a radioimmunoassay for IGF-I and -II C-peptides, but at higher concentrations parallel displacement was observed in a somatomedin C/IGF-I radioimmunoassay using two different antisera. These data indicate that ILA pI 4.8 does possess many of the biological activities previously reported for the IGFs. Since ILA pI 4.8 does occur naturally in serum, it would appear reasonable to tentatively include it as one of the IGF/somatomedin family.     

Proteolytic enzymes; further studies on protein,polypeptide, and other inhibitors of serum proteinase,leucoproteinase, trypsin,and papain

1. Serum proteinase precursor was found in plasma protein fractions I and III of Cohn. Inhibitors of serum proteinase, leucoproteinase, trypsin, and papain were found in fractions IV-1 and IV-4, and to a lesser extent in fractions V and I. 2. Pancreatic, soy bean, lima bean, and egg white inhibitors inhibited trypsin stoichiometrically. Pancreatic inhibitor had comparable inhibitory activity against serum proteinase; soy bean inhibitor had somewhat less, lima bean inhibitor even less, and egg white inhibitor very little. None of these inhibitors appreciably inhibited leucoproteinase or papain. 3. Serum and fractions IV - 1 and IV - 4 had marked inhibitory activity against trypsin and leucoproteinase, and somewhat less against serum proteinase and papain. The inhibitory activity of the plasma proteins against trypsin and leucoproteinase was due almost entirely to fractions IV - 1 and IV - 4; against serum proteinase and papain fraction V was slightly more important. The "reconstituted plasma proteins" accounted for 8 to 25 per cent of the proteinase-inhibitory activity of whole serum or plasma. 4. The proteinase-inhibitory activity of serum, plasma protein fractions, and soy bean inhibitor was heat labile, while that of pancreatic, lima bean, and egg white inhibitors was relatively heat stable. 5. Reducing and oxidizing agents, in very high concentration, inhibited serum proteinase, as well as trypsin and leucoproteinase. These proteinases were not influenced by mercurial sulfhydryl inhibitors, indicating that free sulfhydryl groups do not play an important part in their activity.     

Change of hepatitis B virions (Dane particles) phosphorylation pattern by human hepatoma cell particulate fraction

Protein kinase activity has been found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B surface antigen carriers [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302]. Dane particles were purified from the pooled, HBeAg-positive plasma. When this preparation was incubated with [gamma 32P]ATP in the presence of 10mM MnCl2 and 0.5% NP-40 for 15 seconds at 30 degrees C, several phosphorylated polypeptides of 20,000, 42,000, 48,000, 50,000 and 56,000 daltons were detected in sodium dodecyl sulfate-polyacrylamide gels. When the Dane particles were incubated with [gamma 32P]ATP, 10 mM MnCl2, and 0.5% NP-40 in the presence of human hepatoma cell (J-5) particulate fraction at 30 degrees C, 15 seconds, the 42,000, 48,000 and 50,000 daltons phosphorylated polypeptides were not found. When human peripheral blood lymphocytes particulate fraction was incubated with Dane particles under the same conditions, no change of Dane particle phosphorylated polypeptides was detected. Previous publications [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302; Gerlich, W.H. et al. (1982) J. Virol. 42, 761-766] showed that when hepatitis B core particles purified from hepatoma tissues contained protein kinase activity, only phosphorylated polypeptide was 20,000 daltons. Our data suggested that when Dane particles were put in an environment of hepatoma cells (or tissues), the protein kinase could only phosphorylate selected polypeptides in these particles.     

Sar1Ile7]angiotensin III, a new selective antagonist of the pressor effect of angiotensin III in conscious rats

Two analogues of angiotensin III were compared as antagonists of the pressor response to angiotensin II (ANG II) and angiotensin III (ANG III) in conscious, unrestrained rats. Dose-mean arterial pressure (MAP) response curves were obtained for ANG II and ANG III in the absence or presence of [Ile7]ANG III (1.3 x 10(-7) mol/kg) or [Sar1 Ile7]ANG III (1.2 x 10(-7) mol/kg). In the presence of [Ile7]ANG III, the dose-MAP response curves for ANG II and ANG III were significantly displaced to the right. [Ile7]ANG III behaved as a partial agonist on ANG II but not ANG III receptors. In the presence of [Sar1 Ile7]ANG III, the dose-MAP response curve for ANG III but not ANG II was significantly displaced to the right. This suggests that [Sar1 Ile7]ANG III is a selective antagonist of ANG III in the vasculature. [Ile7]ANG III, on the other hand, antagonizes both ANG II and ANG III receptors. Our results support the hypothesis of the existence of a sub-class of angiotensin receptors activated by ANG III in the vascular smooth muscle.     

The effect of nivazol and cortivazol, novel synthetic steroids containing a phenylpyrazolo A-ring substitution, on blood pressure in sheep

This study investigated the effect of 5 day infusions of two structurally novel synthetic steroids, nivazol and cortivazol on blood pressure and in vivo indices of "glucocorticoid" and "mineralocorticoid" activity. Cortivazol at 24 mg/day raised mean arterial pressure (MAP) by 16 mmHg (P less than 0.001). This was associated with increased cardiac rate, and increased fasting plasma [glucose], polyuria and polydipsia a trilogy characteristic of glucocorticoid effect. Cortivazol had no consistent action on plasma [Na] or [K], but caused an initial transient urinary Na retention and raised urinary excretion of Na and K on days 3 and 4 of treatment. Nivazol at 24 mg/day raised MAP 10 mmHg (P less than 0.001), but cardiac rate was unchanged. This infusion was also associated with the glucocorticoid effects of increased fasting plasma [glucose] and increased urine volume. Plasma [K] fell from a control of 4.4 +/- 0.1 to 4.0 +/- 0.1 mmol/l (P less than 0.01) after 5 days of infusion. There was no significant effect of nivazol on urinary Na or K excretion. This study demonstrates that replacement of the 3-keto group, by a bulky phenylpyrazolo group fused to the A ring at position 2 and 3, does not diminish either pressor or glucocorticoid activity of steroids containing the typical 4-pregnene-3,20-dione nucleus and confirms that the 3 keto group is not essential for optimal glucocorticoid activity. It is the first demonstration of the pressor effect of these novel steroids.     

Subcellular localization of Gi alpha in human neutrophils

Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.     

Adrenal and vascular renin-like activity in chronic 'two-kidney, one-clip' hypertensive rats

In order to study the vascular and adrenal renin angiotensin system in the chronic phase (4 months after clipping) of 'two-kidney, one-clip' hypertension in rats, systolic blood pressure, plasma renin activity, and tissue renin-like activity in both aorta and adrenal have been measured. Renin activity in adrenal gland was studied in both the zona glomerulosa (GLO) and the remainder of the gland. Results showed an increase in vascular renin activity in chronic hypertensive rats. Moreover it was found that GLO of hypertensive rats presented a significant increase in renin-like activity compared with controls (349.43 +/- 43.86 versus 167 +/- 34.25 ng AI/g/20 h, p less than 0.01) and the fasciculata-reticular-medullar (FRM) portion also showed greater renin activity (345.16 +/- 64.36 versus 57.90 +/- 4.83 ng AI/g/20 h, p less than 0.01). The higher levels of vascular and FRM renin-like activity in chronic renal hypertension are probably a consequence of plasma renin increase. This hypothesis is supported by the fact that bilateral nephrectomy in normal rats induces a significant decrease in plasma renin activity and both aortic and FRM renin-like activity. On the other hand the GLO renin-like activity could depend on both plasma renin and local synthesis since bilateral nephrectomy induces an increase in the renin-like activity in this tissue. These data support the idea that aortic and FRM renin are, at least in part, due to plasma renin uptake and GLO renin is an autonomic system.