Cyclic 3′:5′-Nucleotide Phosphodiesterase in Human Myometrium at the End of Pregnancy: Partial Purification and Characterization of the Different Soluble Isoenzymes

Abstract

Most of the cyclic nucleotide phosphodiesterase (PDE) activity of pregnant human myometrium was found in the soluble fraction. Chromatography of this fraction on DEAE-cellulose resolved two peaks of PDE activities. Peak I hydrolyzed both cAMP and cGMP and was activated by the Ca²⁺-calmodulin complex. Peak II, insensitive to this complex, hydrolyzed specifically cAMP. Sucrose gradient centrifugation of the soluble fraction resolved three peaks (A, B, C) of cAMP PDE activities, and only the first two peaks (A, B) were active towards cGMP. A subsequent sucrose gradient centrifugation of peak I, previously determined by DEAE-cellulose, allowed us to restore two peaks A and B identical to those directly obtained from the soluble fraction: peak A hydrolyzes both substrates, while peak B is specific to cGMP hydrolysis. For peak II, a single large cAMP PDE activity peak is generated. Considering Ca²⁺ and cAMP as intracellular messengers in the control of uterine motility, the characterization of the different forms of PDE in pregnant human myometrium will be of importance in developing improved tocolytic therapy.

Characterization by DEAE-Cellulose Chromatography of a Single Molecular Form of Cyclic Nucleotide Phosphodiesterase in the Myometrium of Nonpregnant Women

“…The kinetic analysis performed in the crude homogenate of nonpregnant women's myometria shows that, whatever the phase of the menstrual cycle, PDE presents two affinities for cAMP and only one for cGMP, whereas in myometria of pregnant women near term, two affinities towards both sub strates were described [5]. Biphasic kinetic patterns for cAMP PDE and generally linear kinetics for cGMP PDE have been described previously in the spayed rhesus monkey [3].…”

“…The separation of the different soluble forms was realized with a currently used chromatographic method (DEAE-cellulose) enabling their comparison with the isoenzymes described in other tissues [4,7], In myometria of non pregnant women, whatever the phase of the menstrual cycle (follicular or luteal), a single peak of soluble calcium-calmodulin-sensi tive PDE activity, without substrate specific ity, is identified. This peak, previously ob served in myometria of pregnant women [5], seems to possess properties identical to those isolated from uteri of nonpregnant rats [4] or from vascular smooth muscles [7,16]. How ever, the intensity of activation by the cal cium-calmodulin complex, similar in myom etria of nonpregnant and pregnant women, is really lower than in those tissues.…”

“…Nev ertheless, the proteolytic environment in the uterus is certainly different between the non pregnant and pregnant states and a proteoly sis phenomenon can exist in vivo. The set ting of hydrolytic enzymes which may be involved in the regulation of uterine growth during the pregnancy and in the postpartum involution has been described for the human uterus [22], In the longitudinal myometrial layer, the existence of a form of PDE which presents a great affinity for cAMP [5] and is largely represented near term must be taken into account. At this time, an important deg radation of cAMP by PDE is in good agree ment with the fact that the myometrium gets ready to develop an important contractile activity during the parturition process.…”

“…In myometrium of various species [3,4] including man [5], PDE has been found pre dominantly in the soluble fraction. Contrast ing with several other tissues, little informa tion is available for the existence of soluble myometrial isoenzymes.…”

“…The data in uteri of 20-day-old Sprague-Dawley rats are conflict ing: only a single form which catalyzes the hydrolysis of both substrates, cAMP and cGMP, was identified [6], whereas the exis tence of an additional form highly specific for cAMP was also established [7]. Using the same classical reliable procedure [8,9]; i.e., DEAE-cellulose chromatography, we have previously shown that two peaks of PDE activity were resolved in the longitudinal layer of human myometrium near term [5].…”