Liv Johansen

Role of kallikrein in the hypertensive effect of captopril after sympathetic stimulation of the rat submandibular gland.

After cervical sympathetic nerve stimulation, the rat submandibular gland releases a significant amount of kallikrein into the circulation. To determine whether this glandular kallikrein has kininogenase activity (kinin-generating capabilities) in the peripheral circulation and whether it plays a role in blood pressure regulation, we studied the effect of captopril on blood pressure in 48 hour-nephrectomized rats with and without prior sympathetic stimulation of the submandibular gland. Administration of captopril 10 minutes after gland stimulation resulted in a mean blood pressure decrease (A BP) of 43 ± 8.3 mm Hg (P 0.05). To confirm that the effect of captopril was due to a blockage of kinin destruction generated by glandular kallikrein and not to the inhibition of angiotensin II formation, we determined the effect of captopril after gland stimulation in rats pretreated with either IgG from nonimmunized rabbits (normal-IgG), or IgG from rabbits immunized against kinins (antikinin-IgG) or kallikrein (antikallikrein-IgG). Normal-IgG did not significantly alter the hypotensive effect of captopril (A BP —30 ± 7.7; P < 0.01), while pretreatment with antikinin or antikallikrein almost completely blocked its hypotensive effect (A BP —5.8 ± 1.9 and —6.4 ± 0.4, respectively). In the latter two groups, the decrease in BP was significantly smaller (P < 0.001) than in the groups that were not pretreated or in the group pretreated with normal-IgG. These data suggest that, upon adrenergic stimulation of the submandibular gland, glandular kallikrein released into the vascular compartment has kininogenase activity in the peripheral circulation. The kinins released by glandular kallikrein induced hypotension when their breakdown was prevented by the kininase II inhibitor, captopril. These results suggest that kinins may be responsible in part for the antihypertensive effect of captopril in situations in which glandular kallikrein in blood is increased. (C/rc Res 51: 385-390, 1982)

Uptake and release for glutamine and glutamate in a crude synaptosomal fraction from rat brain.

Abstract[14C]Glutamine uptake in a crude synaptosomal (P2) fraction, (representing the sum of [14C]glutamine accumulated and [14C]glutamate formed by hydrolysis), is distinct from glutamate uptake. Glutamine uptake is Na+-independent and unaffected by the Na+−K+-ATPase inhibitor ouabain, whereas glutamate uptake is Na+-dependent and inhibited by ouabain. The uptake of both glutamine and glutamate is unaffected by the gamma-glutamyltransferase inhibitor, Acivicin. This indicates that glutamine uptake is not mediated by a carrier, as distinct from that of glutamate, and also not linked to gamma-glutamyl-transferase. Na+ affects the distribution of glutamine-derived glutamate by increasing the synaptosomal content and reducing that of the medium. When glutamate release from synaptosomes preloaded with [14C]glutamate is measured by superfusion technique in order to prevent reuptake, Na+ has been found to inhibit release in a non-depolarizing medium (Ringer buffer with no Ca2+) of the [14C]glutamate as well as of endogenous glutamate. The specific activity of the [14C]glutamine-derived glutamate in the incubation medium is much higher than that in the synaptosomes, indicating that there exists a readily releasable pool of newly formed glutamate in addition to another pool. The latter glutamate pool is partially reduced by Na+.

Formation of 2,3-pentanediol from 2,3-pentanedione and acetylethylcarbinol by diacetyl(acetoin)reductase from Aerobacter aerogenes. A possible new pathway.

Diacetyl(acetoin)reductase catalyzes the reversible reduction of acetoin (acetylmethylcarbinol) to 2,3butanediol and the irreversible reduction of diacetyl to acetoin [ 1,2] . The enzyme has been characterized kinetically and found to follow an ordered bi-bi mechanism [3,4] . During work with the three enzymes leading from pyruvate to 2,3-butanediol, fig. 1, we have observed that the first enzyme, the pH 6 acetolactate-forming enzyme, in addition to forming acetolactate from pyruvate, is able to form acetohydroxybutyrate from pyruvate and 2-oxobutyrate [S] . This enzyme requires cocarboxylase and Mn2+ for its activity [6,7] . The second enzyme, acetolactate decarboxylase, decarboxylates acetolactate or acetohydroxybutyrate to yield acetoin or acetylethylcarbinol, respectively [8]. In the present report, we have shown that the substrates for diacetyl(acetoin)reductase, diacetyl, acetoin, and 2,3-butanediol, can be replaced by their respective analogues 2,3-pentanedione, acetylethylcarbinol, and 2,3-pentanediol.

Rapid purification of tonin, esterase b, antigen ψ and kallikrein from rat submandibular gland by fast protein liquid chromatography

Tonin, esterase B, antigen psi and kallikrein from the rat submandibular gland were purified by fast protein liquid chromatography with Mono P or Mono Q columns. The purity of the separated proteins was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and by isoelectrofocusing in flat-bed polyacrylamide gel. Tonin and esterase B were purified by DE-52 cellulose anion-exchange chromatography and chromatofocusing on Mono P in two and three steps, respectively. Antigen psi and kallikrein were purified by a two-step procedure using DE-52 cellulose and Mono Q anion-exchange chromatography. The high resolution power of Mono Q revealed the different isoenzymes of kallikrein.

Excess antibody immunoassays for rat glandular kallikreins. Measurement of kallikrein from different organs in the presence of cross-reacting antigens.

An immunoradiometric assay has previously been developed for measurement of rat glandular kallikrein. In the present paper, further studies on the specificity and sensitivity of the method are described. Problems of interference of immunologically cross-reacting antigens were overcome by proper preabsorption of the antibody. A method was thus established in which enzymatic activity of the immunoreactive kallikrein could be measured even in the presence of enzymes sharing immunological determinants and substrate specificity with kallikrein. Two variants of the immunoradiometric assay have been evaluated. A simplified version with simultaneous addition of all reagents gave results equal to those obtained in the original assay. A further modification with delayed addition of the solid-phase antibody, gave considerable improvement in assay sensitivity.

Excess antibody immunoassay for rat glandular kallikrein measurement of kallikrein complexed with inhibitors and in plasma

We have recently developed an immunoradiometric assay (IRMA) for specific measurement of immunoreactive kallikrein which allows a simultaneous determination of the enzymatic activity of kallikrein. This paper describes the application of this method for measurements of glandular kallikrein complexed with inhibitors. Interference by low molecular weight inhibitors such as benzamidine and Trasylol was easily overcome by increasing the amount of immobilized anti-kallikrein antibody added in the assay, and by prolonging the incubation time of the antigen-binding step. The recovery of kallikrein in complex with plasma inhibitors was complete only when the anti-kallikrein antibody was immunoadsorbed onto a solid-phase sheep anti-rabbit immunoglobulin. The dose-response curve of glandular kallikrein in plasma paralleled that of purified kallikrein in both the immunoradiometric and the immunoenzymometric assays. The concentration of immunoreactive glandular kallikrein in normal rat plasma was 12.8 +/- 4.3 nU/ml. The enzymatic activity of this immunoreactive kallikrein was 86% inhibited.

Excess antibody immunoassay for the measurement of tonin in rat tissues and plasma

Tonin, a proteolytic enzyme isolated from the rat submandibular gland, can generate angiotensin II directly from angiotensinogen. To date a method for the measurement of tonin in plasma has not been available and the present paper describes a sensitive and specific excess antibody immunoassay for determination of tonin in tissue homogenates and plasma. Interference from immunologically cross-reacting proteins was evaluated and the assay was found to be specific for tonin. Tonin measured in various tissue homogenates was directly proportional to the amount of sample added, giving a linear dose-response curve. The slope of this curve was determined by the recovery of tonin, which was better than 55% for urine and all tissues tested. The highest concentration of tonin was seen in the submandibular and sublingual gland (69 and 0.7 microgram/mg protein, respectively). The parotid gland, the exorbital lacrimal gland, liver, kidney, pancreas, and lung contained only negligible amounts (less than 4 ng/mg protein). Tonin in plasma was bound to one major inhibitor with a molecular weight of about 650,000-750,000. A partial splitting of the tonin-inhibitor complex was obtained by preincubating plasma with guanidine, allowing tonin to be measured with a recovery of 38 +/- 13% (n = 16) and with a linear dose-response curve. The concentration of immunoreactive tonin in normal arterial plasma from adult male rats was 0.90 +/- 0.53 ng/ml (n = 16). The concentration decreased after removal of the submandibular glands and increased after sympathetic stimulation.

Uptake and Release of Amino Acids from Synaptosomes

Synaptosomes are isolated, sealed synaptic nerve endings with intact membrane structure and therefore well suited for uptake and release studies of neurotransmitters (1).