Kazimierz Kowalski

“Big” human placental lactogen: Disulfide-linked peptide chains

Abstract “Big” human placental lactogen has been purified by affinity chromatography. On SDS 1 -polyacrylamide disc gel electrophoresis, “big” hPL had a molecular weight of about 45,000. Following reduction with mercaptoethanol, a single band with a molecular weight of 23,000 was noted. These observations suggest that “big” hPL consists of two peptide chains linked by a disulfide bond.

Thyrotropin- and prostaglandin E2-responsive adenyl cyclase in thyroid plasma membranes*

Abstract We studied TSH and PGE 2 effects on adenyl cyclase in bovine thyroid plasma membranes. With [α- 32 P]ATP as substrate, there was no PGE 2 effect on adenyl cyclase in plasma membranes highly responsive to TSH and NaF (7- to 12-fold and 12- to 15-fold increase over control, respectively). Since enhanced plasma membrane Na + -K + -activated ATPase activity (40-fold greater than original homogenate) might have resulted in significant ATP hydrolysis despite the presence of an ATP-regenerating system, we repeated these studies with 32 P-labeled 5′-adenylyl-imidodiphosphate ([ 32 P]AMP-PNP), an ATP analogue which is an ATPase-resistant substrate for adenyl cyclase. With AMP-PNP as substrate, adenyl cyclase in all plasma membranes tested showed a dose-related response (133% to 226% increase) to both TSH (5 to 250 mU/ml) and PGE 2 (10 −6 to 10 −4 M), which response was inhibited, in each instance, by the prostaglandin antagonist 7-oxa-13-prostynoic acid (PY 1 , 10 −5 M). Since guanyl nucleotides, e.g. GTP, have been shown to enhance basal and hormone-stimulated adenyl cyclase in liver plasma membrane, we examined the effect of 0.1 mM GTP on thyroid plasma membrane adenyl cyclase using [α- 32 P]ATP as substrate. In the presence of GTP, both basal and TSH-stimulated adenyl cyclase were enhanced two fold; in addition, a stimulatory PGE 2 effect (two-fold increase) was reproducibly demonstrated. In contrast, the effect of fluoride ion was inhibited by GTP. PY 1 inhibited the stimulatory effects of both TSH and PGE 2 under these conditions. These findings suggest great similarity between TSH and PGE 2 adenyl cyclase receptor(s) in thyroid and are consistent with the premise that prostaglandins play an important role in TSH stimulation of thyroid function.

Effects of thyrotropin, prostaglandin E1 and a prostaglandin antagonist on iodide trapping in isolated thyroid cells.

Abstract We examined effects of two adenyl cyclase activators, thyrotropin [TSH] and prostaglandin E 1 [PGE 1 ], as well as those of a prostaglandin antagonist, 7-oxa-13-prostynoic acid [PY 1 ], on iodide trapping in isolated bovine thyroid cells [ITC]. When maximally effective concentrations of TSH and PGE 1 were combined, no additive effect on I − trapping was seen. PY 1 abolished PGE 1 effects on I − trapping and markedly inhibited TSH effects thereon, but was without effect on the increase induced by dibutyryl cyclic AMP [DBcAMP] or NH 4 + ion. PY 1 also inhibits TSH and PGE 1 activation of ITC adenyl cyclase, whereas NH 4 + ion is without effect on ITC adenyl cyclase. Thus, these data suggest that: 1) TSH effects on iodide trapping are mediated via adenyl cyclase activation, and 2) there may be a single TSH-related prostaglandin receptor in thyroid, activation of which is requisite of TSH atimulation of adenyl cyclasse and hormonogenegis in the gland.

Effects of a prostaglandin antagonist, polyphloretin phosphate, on basal and stimulated thyroid function

Abstract Polyphloretin phosphate [PPP], a phosphorylated polyanionic phloridzin derivative, antagonizes prostaglandin E2 [PGE2] effects on several physiologic systems. We examined the effects of PPP on thyrotropin- [TSH] and PGE2-stimulated thyroid function. PPP, 1 to 25 μg/ml [⋍10−8 to 10−7 M], did not affect basal adenyl cyclase activity in isolated bovine thyroid cells [ITC] but markedly inhibited both TSH [1 to 100 mU/ml] and PGE2 [10−7 to 10−5 M] stimulation thereof. PPP, 10 to 100 μg/ml, stimulated [I31I]-iodide trapping in ITC [27% to 80% increase over control, approximating response to 10 to 100 mU/ml TSH]. PPP effects on iodide trapping were additive with those of TSH or dibutyryl cyclic AMP [DBcAMP], whereas competitive interaction was seen when PPP and PGE2 were combined. PPP, 25 to 100 μg/ml, stimulated ITC phagocytosis of latex beads [maximum response = 100% to 130% increase over control, approximating response to 200 mU/ml TSH], whereas the stimulatory effects of PGE2 but not those of TSH or DBcAMP were consistently antagonized by PPP, 5 μg/ml. PPP was resolved into high and low molecular weight components by Sephadex G-25 column chromatography. The low molecular weight components were responsible for PPP antagonism of TSH- and PGE2-induced adenyl cyclase activation, whereas the stimulatory effects on iodide trapping and bead phagocytosis were due to high molecular weight fractions.

Stimulatory effect of ammonium ion on iodide trapping in isolated thyroid cells

Abstract We studied the effects of ammonium ion on iodide trapping in isolated bovine thyroid cells. A variety of ammonium salts [HCO 3 = , HPO 4 = , SO 4 = , Cl − , CH 3 COO − ] at mEq concentration augmented iodide trapping [cell/medium (C/M) ratio 143% to 174% of control] during 4.5 hr incubations. The stimulatory effect was not related to alteration of medium pH, was NH 4 + ion concentration-dependent, and was not reproduced by other basic ions, eg. tetraethylammonium, tetramethylammonium or pyridine at equivalent or higher concentrations. Other monovalent cation additions were either inhibitory [K + ], without effect [Cs + ], or only weekly stimulatory [Rb + ]. When ineffective concentrations of one stimulator [TSH or NH 4 + ] were added to effective concentrations of the other, a reduction in stimulation of iodide trapping was observed. Combinations of maximally effective concentrations of NH 4 + ion and of TSH had no additive effect on C/M ratio. These findings suggest that NH 4 + ion exerts a specific stimulatory effect on iodide transport in isolated thyroid cells and may do so in a manner similar to TSH.