[Cross-link in fibrin polymerized by factor 13: epsilon-(gamma-glutamyl)lysine].

(epsilon)-((gamma)-Glutamyl)lysine has been isolated from enzymatic hydrolyzates of cross-linked human fibrin. This compound was not detected in "non-cross-linked" fibrin prepared with ethylenediaminetetraacetic acid, which inhibits factor XIII; intermediate amounts were observed when the fibrin was prepared with glycine ethyl ester, which inhibits factor XIII competitively. These and ancillary experiments furnish conclusive evidence that epsilon-(gamma-glutamyl)lysine cross-links form in human fibrin during polymerization catalyzed by factor XIII.

Droplet countercurrent chromatography.

A new form of countercurrent chromatography, named droplet counter-current chromatography, has been developed. This all-liquid separation technique is based on the partitioning of solute between a steady stream of droplets of moving phase and a column of surrounding stationary liquid phase. Milligram quantities of dinitrophenyl (DNP) amino acids were separated with an efficiency comparable to that of gas chromatography.

Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, vasopressin, and deoxycorticosterone.

Several factors interact to maintain precise control of electrolyte transport in the mammalian cortical collecting duct. We have studied the effects of deoxycorticosterone, arginine vasopressin, and bradykinin on net transepithelial sodium and potassium transport in isolated, perfused rat cortical collecting ducts. Chronic administration of deoxycorticosterone to rats increased both sodium absorption and potassium secretion above very low basal levels. Consequently, deoxycorticosterone-treated rats were used for all remaining studies. Arginine vasopressin (10(-10) M in the bath) caused a sustained fourfold increase in net sodium absorption and a sustained threefold increase in net potassium secretion. Bradykinin (10(-9) M in the bath) caused a reversible 40-50% inhibition of net sodium absorption without affecting net potassium transport or the transepithelial potential difference. In the perfusate, up to 10(-6) M bradykinin had no effect. We conclude: As in rabbits, chronic deoxycorticosterone administration to rats increases sodium absorption and potassium secretion in cortical collecting ducts perfused in vitro. Arginine vasopressin causes a reversible increase in net potassium secretion and net sodium absorption. Bradykinin in the peritubular bathing solution reversibly inhibits net sodium absorption, possibly by affecting an electroneutral sodium transport pathway.

Effects of vasopressin and bradykinin on anion transport by the rat cortical collecting duct. Evidence for an electroneutral sodium chloride transport pathway.

Our previous studies in cortical collecting ducts isolated from rat kidneys have shown that vasopressin increases both sodium absorption and potassium secretion, while bradykinin inhibits sodium absorption without affecting potassium transport. To determine which anions are affected by these agents, we perfused cortical collecting ducts from rats treated with deoxycorticosterone and measured net chloride flux, net bicarbonate flux (measured as total CO2), transepithelial voltage, and the rate of fluid absorption. Arginine vasopressin (10(-10) M in the peritubular bath) caused a sustained sixfold increase in net chloride absorption and a two- to threefold increase in the magnitude of the lumen negative transepithelial voltage. Before addition of vasopressin, the tubules secreted bicarbonate. Vasopressin abolished the bicarbonate secretion, resulting in net bicarbonate absorption (presumably due to proton secretion) in many tubules. Bradykinin (10(-9) M added to the peritubular bath) caused a reversible 40% inhibition of net chloride absorption, but did not affect the transepithelial voltage or the bicarbonate flux. We concluded: (a) that arginine vasopressin stimulates absorption of chloride and inhibits bicarbonate secretion (or stimulates proton secretion) in the rat cortical collecting duct; and (b) that bradykinin inhibits net chloride absorption in the rat cortical collecting duct without affecting transepithelial voltage or bicarbonate flux. Combining these results with the previous observations on cation fluxes described above, we conclude that bradykinin inhibits electroneutral NaCl absorption (or stimulates electroneutral NaCl secretion) in the rat cortical collecting duct.

Regulation of postocclusive hyperemia by endogenously synthesized prostaglandins in the dog heart.

Experiments were performed to evaluate the role of prostaglandin synthesis in the regulation of coronary blood flow in dog hearts. The left main coronary artery was cannulated and flow measured both in otherwise intact animals and in canine heart-lung preparations. Prostaglandin E was measured by radioimmunoassay. Reactive hyperemia (flow after occlusion release) was induced by coronary occlusion for 10, 15, and 20 s and was 39 plus or minus 13 (mean plus or minus SEM), 66 plus or minus 21, and 82 plus or minus 24 ml, respectively. Indomethacin, an inhibitor of prostaglandin synthetase, reduced reactive hyperemia at 10, 15, and 20 s to 15 plus or minus 5, 33 plus or minus 11, and 47 plus or minus 17 ml, respectively (P smaller than 0.05). Meclofenamate, a different prostaglandin synthetase inhibitor, gave similar results. In a second group of five dogs, prostaglandin production of the heart was examined in response to 20-s occlusions. There was a significant increase in prostaglandin production from a basal level of 18.6 plus or minus 4.9 mg/min to 35.3 plus or minus 5.8 ng/min after occlusion of the coronary artery for 20 s (P smaller than 0.05). After indomethacin, this increase in prostaglandin production was not observed and reactive hyperemia was significantly reduced. Thus, prostaglandin synthesis appears to be important to modulating canine coronary blood flow in response to brief periods of coronary occlusion.

The kallikrein-kinin system in Bartter's syndrome and its response to prostaglandin synthetase inhibition.

The kallikrein-kinin system was characterized in seven patients with Bartter's syndrome on constant metabolic regimens before, during, and after treatment with prostaglandin synthetase inhibitors. Patients with Bartter's syndrome had high values for plasma bradykinin, plasma renin activity (PRA), urinary kallikrein, urinary immunoreactive prostaglandin E excretion, and urinary aldosterone; urinary kinins were subnormal and plasma prekallikrein was normal. Treatment with indomethacin or ibuprofen which decreased urinary immunoreactive prostaglandin E excretion by 67%, decreased mean PRA (patients recumbent) from 17.3+/-5.3 (S.E.M.) ng/ml per h to 3.3+/-1.1 ng/ml per h, mean plasma bradykinin (patients recumbent) from 15.4+/-4.4 ng/ml to 3.9+/-0.9 ng/ml, mean urinary kallikrein excretion from 24.8+/-3.2 tosyl-arginine-methyl ester units (TU)/day to 12.4+/-2.0 TU/day, but increased mean urinary kinin excretion from 3.8+/-1.3 mug/day to 8.5+/-2.5 mug/day. Plasma prekallikrein remained unchanged at 1.4 TU/ml. Thus, with prostaglandin synthetase inhibition, values for urinary kallikrein and kinin and plasma bradykinin returned to normal pari passu with changes in PRA, in aldosterone, and in prostaglandin E. The results suggest that, in Bartter's syndrome, prostaglandins mediate the low urinary kinins and the high plasma bradykinin, and that urinary kallikrein, which is aldosterone dependent, does not control kinin excretion. The high plasma bradykinin may be a cause of the pressor hyporesponsiveness to angiotensin II which characterizes the syndrome.

Preparation of dipeptidyl aminopeptidase IV for polypeptide sequencing.

Dipeptidyl aminopeptidase IV is a dipeptidylpeptide hydrolase (EC 3.4.14) which hydrolyzes bond at the carboxyl group of proline releasing X-Pro dipeptides from the amino-terminus of polypeptides. The enzyme was purified 440-fold in 37% yield from swine kidney by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration and affinity chromatography with dipeptide-substituted Sepharose 4B. The enzyme released X-Pro from all X-Pro-beta-naphthylamides and polypeptides tested. The released dipeptides were not further degraded, and were readily identified in digests. The enzyme is suitable for use in the dipeptidyl aminopeptidase method for sequence analysis of polypeptides.

Activation of ganglionic tyrosine hydroxylase by peptides of the secretin-glucagon family: structure-function studies.

The hydroxylation of tyrosine to dopa is the rate-limiting reaction in catecholamine biosynthesis. It has been previously reported that secretin, vasoactive intestinal peptide and peptide histidine isoleucine amide, all members of the secretin-glucagon family of peptides, increase dopa synthesis in superior cervical ganglia in vitro. We report here that two other members of this peptide family, rat growth hormone-releasing factor and helodermin H38, a component of Gila monster venom, also increase the rate of dopa synthesis, while glucagon-like peptides I and II and a number of other peptides tested produce no effect. Since analogs of cAMP also increase dopa synthesis, it is of particular interest that all of the peptides that increase catechol synthesis also raise the levels of this cyclic nucleotide in the superior cervical ganglion. Helodermin H38 stimulated the rate of dopa synthesis and the level of cAMP with similar potencies (EC50S of approximately 10 nM) and with maximal effects of two- and two-fold, respectively. By either measure, rat growth hormone-releasing factor produced a two-fold increase at 10 microM and a three- to four-fold increase at 30 microM. Analogs of peptides of the secretin-glucagon family with a deletion or modification of the N-terminal histidine were much less effective in these assays at the concentrations tested than were their parent compounds, demonstrating an important role for this amino acid in conferring activity on these peptides. In addition to increasing dopa synthesis in intact tissue, incubation of ganglia with rat growth hormone-releasing factor, secretin, vasoactive intestinal peptide or peptide histidine isoleucine amide also increased the activity of tyrosine hydroxylase measured subsequently in ganglion homogenates. Thus, the peptidergic stimulation of dopa synthesis observed in the intact superior cervical ganglion appears to be due, at least in part, to the activation of tyrosine hydroxylase. Together with previous studies, these findings support the hypothesis that certain members of the secretin-glucagon family increase catecholamine synthesis in sympathetic neurons by a cAMP-dependent activation of tyrosine hydroxylase.

Effect of orthostatic changes on urokinase excretion.

The effects of orthostatic changes on the excretion of urokinase were studied in healthy normal volunteers. Urokinase excretion rose (average +69%) significantly (p less than .005) while urine volume fell (average-59%) significantly (p less than .001) after the subjects had been standing. There was no difference between men and women nor was there an apparent diurnal variation in urokinase excretion of recumbent subjects. A relationship between urokinase excretion and sympathetic nervous system activity is suggested.

Effects of ranatensin, a polypeptide from frog skin, on isolated smooth muscle.

1. The actions of bretylium tosylate on neuromuscular transmission in the rat phrenic nerve diaphragm preparation have been investigated by electro-2. On the guinea-pig ileum, threshold doses elicit repeated maximal spike contractions which are blocked by atropine. In the presence of atropine, higher concentrations of ranatensin elicit small contraction spikes superimposed on a relatively weak sustained contraction. These latter two actions are not blocked by increasing the concentration of atropine.3. Other smooth muscle preparations respond as follows: rat uterus, rhythmic contractions; rat duodenum, relaxation; rabbit aortic strip, contraction. Atropine has no effect on the above responses. The rat aortic strip does not respond to ranatensin. Ranatensin is four times as active as bradykinin on the rat uterus.4. Ranatensin can be readily distinguished from other known peptides such as angiotensin, bradykinin and the eledoisin-like peptides, by bioassay.

The action of ranatensin, a new polypeptide from amphibian skin, on the blood pressure of experimental animals.

1. The blood pressure response to ranatensin, an undecapeptide from the skin of the frog, Rana pipiens, has been studied in various experimental animals.2. Ranatensin raised blood pressure in the dog and rabbit. The response was not altered by atropine, phentolamine, propranolol or hexamethonium, suggesting a direct peripheral vasoconstrictor action. In both animals ranatensin was about one-tenth as potent as angiotensin. Tachyphylaxis to ranatensin did occur, but there was no cross-tachyphylaxis with angiotensin, bradykinin, or noradrenaline.3. The peptide lowered blood pressure in the monkey, being as potent as eledoisin. The response was not altered by atropine, phentolamine, propranolol, tripelennamine, tetraethylammonium, bretylium, or methysergide. This again suggests a direct peripheral action on vascular smooth muscle. There was no tachyphylaxis to the depressor action, nor was there cross-tachyphylaxis with angiotensin, eledoisin, bradykinin, or noradrenaline.4. Ranatensin did not alter the blood pressure in cats and had a variable action in the guinea-pig with a rapid onset of tachyphylaxis.5. Ranatensin has a variable effect on the blood pressure in the rat that is related to the basal level of blood pressure. When the blood pressure is high, the response to the peptide is hypotension. Ranatensin raises blood pressure in the rat when the basal blood pressure is low. The pressor response to ranatensin appears to be due, in part, to the release of noradrenaline from peripheral sympathetic nerve endings.6. The composite action of ranatensin on blood pressure of various experimental animals is unlike that of any other peptide. Its hypertensive action in the dog or rabbit, together with a potent hypotensive action in the monkey, readily distinguishes it from all other vasoactive peptides.

Survey of plant inhibitors of polymorphonuclear leukocyte elastase, pancreatic elastase, cathepsin G, cathepsin B, Hageman factor fragments, and other serine proteinases.

Various flower bulbs and vegetable and legume seeds were tested for inhibitors of polymorphonuclear leukocyte elastase, pancreatic elastase, cathepsin G, cathepsin B, trypsin, alpha-chymotrypsin, Hageman factor fragments, plasma kallikrein, and plasmin. Calla bulbs contained a 33,000 dalton polymorphonuclear leukocyte elastase inhibitor and a 4,000 dalton cathepsin G inhibitor. Seeds of some members in the Cruciferae family, such as radish and broccoli, were found to contain one or more 2,500-4,000 dalton inhibitors which inhibited cathepsin G, trypsin, Hageman factor fragments, and plasmin, but not plasma kallikrein. These seeds also contained a 1,000 dalton cathepsin B inhibitor. The above inhibitors were probably polypeptides which inhibited proteinases by making an enzyme-inhibitor complex, with the exception of the cathepsin B inhibitor. These newly found inhibitors with their characteristic profiles of inhibition should be useful in biochemical and pathophysiological studies on granulocyte proteinases and enzymes of the coagulation and fibrinolytic pathways.

Amino acid sequence of bumblebee MCD peptide: a new mast cell degranulating peptide from the venom of the bumblebee Megabombus pennsylvanicus.

A new 28 amino acid peptide, we recently isolated from the venom of the bumblebee Megabombus pennsylvanicus. has been characterized. The peptide, Met-Cys-Ile-Cys-Lys-Asn-Gly-Lys-Pro-Leu-Pro-Gly-Phe-Ile-Gly-Lys-Ile-Cys- Arg-Lys-Ile-Cys-Met-Met-Gln-Gln-Thr-His(NH2), has been named bumblebee mast cell degranulating (MCD) peptide due to its ability to degranulate rat peritoneal mast cells, and its resemblance to the bee venom MCD peptide. Bumblebee MCD peptide, unlike bombolitins, the other mast cell degranulating heptadecapeptides of bumblebee venom, is not lytic and releases histamine at a dose as low as 0.05 micrograms/ml (1.6 X 10(-8) M).

Biochemical and histochemical characterization of ranatensin immunoreactive peptides in rat brain: lack of coexistence with bombesin/GRP.

An antibody to des-pyroglutamyl ranatensin (RT 2-11) has been prepared and has been used to histochemically and biochemically identify ranatensin-like immunoreactivity (irRT) in the rat brain. The most most prominent stained cell group was situated in the dorsal tegmental pons. Areas of immunoreactive fibers were found in the ventral hippocampus, septal area and the hypothalamus. A similar distribution was found by radioimmunoassay data. The distribution of irRT was compared to that of bombesin-like immunoreactivity (irBN). The two peptides have different though partly overlapping distributions. Gel filtration of brain extracts show that the molecular size of irRT is similar to that of synthetic RT. However, HPLC characterization of irRT indicated that the immunoreactive material is different from synthetic RT and also different from irBN and irGRP. These results indicate the presence of two separate peptide systems, one RT-like, the other BN-like, in the mammalian CNS.

Human plasma prekallikrein and high molecular weight kininogen decrease during parturition.

Prekallikrein and high molecular weight kininogen were measured in plasma taken from nine women during parturition. Prekallikrein decreased significantly (p less than 0.01) from 1.49 +/- 0.15 S-2302 U/ml (mean +/- SEM) in early labor to 1.26 +/- 0.13 S-2302 U/ml in the immediate postpartum period. Immunoreactive high molecular weight kininogen also significantly decreased from 76 +/- 5 micrograms/ml to 68 +/- 5 micrograms/ml one day postpartum (p less than 0.01). Both proteins rose to normal levels within two days. The data suggest that the kallikrein-kinin system is utilized during parturition.

Sensitive radiochemical esterolytic assays for urokinase.

Two radiochemical esterolytic assays for urokinase are described. One assay is based on the urokinase-dependent hydrolysis of Nalpha-acetyl-glycyl-L-lysine [3H]methyl ester and the other on the urokinase-dependent activation of plasminogen and assay of generated plasmin with Nalpha-tosyl-L-arginine [3H]methyl ester. The assays are performed in tubes placed in liquid scintillation counting vials. At the end of the experiment generated [3H]methanol is extracted into the liquid scintillation cocktail and counted. Unhydrolyzed substrate largely remains in the aqueous phase and contributes only a small fraction of the counts. This facile separation of 3H-labeled alcohol from the ester substrate allows the simple and highly sensitive assay for urokinase. The assays give results in good agreement with the classical fibrin plate assay.

Inhibition of [3H]nitrendipine binding by phospholipase A2.

Phospholipase A2 from several sources inhibited [3H]nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC50 values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A2 was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A2 enzymatic activity, shifted the bee venom phospholipase A2 dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A2 (10 ng/ml) for 15 min caused a 2-fold increase in the Kd without changing the Bmax compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the Kd but significantly decreased the Bmax to 71% the value for untreated membranes. [3H]Nitrendipine, preincubated with bee venom phospholipase A2, was recovered and found to be fully active, indicating that the phospholipase A2 did not modify the ligand. It is concluded that phospholipase A2 acts on the membrane at or near the [3H]nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site.

Naloxone attenuates the hypotension induced by Hageman factor.

The hypotension induced in the pentobarbital anesthesized rat by the i.v. administration of an active Hageman factor fragment (Hff) is significantly attenuated by naloxone. This effect is specific because the opiate antagonist does not modify the hypotension elicited by rat urinary kallikrein, bradykinin or nitroglycerin. These results suggest that the contact activation of endogenous Hageman factor could result in the generation of vasoactive opioid peptides derived from circulating large molecular weight precursors.