Biomedical subjects
Publications and source records attributed to V Pliska.
The analysis of pA2 values for 1,2-substituted oxytocin analogues suggests a significant resonance effect of p-substituted groups in 2-tyrosine when the hormone binds to its uterus receptor, whereas the N-terminal amino group exerts less clearly characterized effects (participation of its lipophilicity and molecular volume can be assumed.
The 2-p-bromoacetylaminophenylalanine analogue of deamino-oxytoxin displayed some features of an irreversible inhibitor of oxytocin on rat uterus (long persistence of inhibitory effect, slow wash-out from the tissue). An isosteric analogue with a propionylamino group at the same position was, under similar experimental conditions, also an antagonist of oxytocin, but the features of an irreversible inhibitor were lacking. pA2 values of the two substances are between 6.5 and 6.9. The "irreversibility" of the former compound is concentration dependent and it is concluded that it cannot be entirely caused by a covalent binding of the inhibitor to the uterus receptor for oxytoxin. Like many other similar inhibitors, the substances display only an inefficient in vivo inhibition of the vasopressor effect of lysine vasopressin in rats.
[Mpa1,Tyr(Et)2]-LVP (1-deamino-2-O-ethyltyrosine-8-lysine-vasopressin), [Mpa1,Tyr(n-Pr)2]-LVP, [Tyr(n-Bu)2]-LVP, [Mpa1,Tyr(n-Bu)2]-LVP, and [Mpa1,Tyr(n-hexyl)2]-LVP were synthesized in solution by the p-nitrophenyl ester method. The previously prepared [Tyr(Et)2]-LVP was resynthesized. All compounds possessed weak agonistic properties in both antidiuretic (0.5-2.0 IU/mumol) and pressor (0.5-3.0 IU/mumol) assays. In the rat none of the analogues inhibited the antidiuretic action of LVP when the two substances were given together in a single injection. However, when administered in low subthreshold doses, most of the deamino compounds suppressed the antidiuresis induced by a continuous infusion of LVP. Complete inhibition was obtained with [Mpa1,Tyr(Et)2]-LVP. The antagonistic potency seemed to decrease with increasing size of the alkyl substituent and [Mpa1,Tyr(n-hexyl)2]-LVP showed no antagonism. The molar inhibitor-LVP ratio for maximal inhibition was well below 100. Neither of the two amino analogues showed a clear-cut antagonism in the antidiuretic assay. Furthermore, none of the reported compounds was antagonistic to LVP in the rat pressor assay.
Both [2-o-iodotyrosine]-oxytocin and [2-o-methyltyrosine]-oxytocin display only weak vasopressor and antidiuretic effects on rats. They inhibit the in vitro uterotonic action of oxytocin; this inhibition is not fully competitive. It is concluded that they are not suitable as markers for studies of uterine receptor for oxytocin.
The question as to whether calcium can be considered to be a mediator of oxytocin-induced myometrial contraction has been investigated. Assuming that the contraction is linearly proportional to the myoplasmic calcium concentration, several possible molecular mechanisms leading to its increase (calcium release from the cell membrane, acceleration of calcium transport from extracellular space by a 'gate' mechanism, release from intracellular organelles, blockade of calcium pumps) were modelled on an analog computer. The oxytocin intervention in the calcium distribution was mimicked by a discontinuous change of the appropriate rate constants. The computed transient simulating the myoplasmic calcium concentration was then compared with an experimental time profile of uterine tension. The result of screening the models shows that oxytocin must act predominantly via release of calcium bound to the cell membrane. A quantitative comparison, however, requires that the kinetics of oxytocin distribution in myometrium also be considered in the model. The problem treated in this paper demonstrates the possibilities and limitations of a screening procedure based upon direct comparison of time profiles of experimental processes with several computed model alternatives.
Analogues of oxytocin and deaminooxytocin with 4-glutamine replaced by 4-glutamic acid methyl ester readily lose their uterotonic activity when incubated with rat serum, presumably by hydrolysis to the much less active 4-glutamic acid derivatives. On the other hand, inactivation of the deaminooxytocin analogue in the rat uterus, as demonstrated by the "oil-bath" technique, is only slightly more rapid than that of deaminooxytocin and distinctly slower than that of oxytocin. Its in situ/in vitro ratio of uterotonic activity is less than 0.1 whereas that for deaminooxytocin is about 3 and also the peristence of the uterotonic effect in situ is slightly less than that of deaminooxytocin. The results with these "rapidly inactivated" analogues can be used as proof of some predictions of the three-compartment model for tissue distribution of neurohypophysial hormones and its influence upon the time course of a biological response published earlier. The potential use of analogues of neurohypophysial hormones as probes for inactivation mechanisms and the results thus far obtained are discussed.
The urine and plasma levels of vasopressin-like immunological activity and of antidiuretic activity were examined following injection of Na-glycyl-glycyl-glycyl-[8-lysine]vasopressin (triglycylvassopressin, TGLVP) in 3 cats. The plasma levels of immunoreactive material were initially high, and fell rapidly. The levels of antidiuretic activity showed considerable variation; the overall pattern was strikingly different from that demonstrated by radioimmunoassay, and all 3 animals showed a rise in plasma antidiuretic activity in the early part of the experiment. Following injection of lysine vasopressin (LVP) the pattern of disappearance of both biological and immunological activity was similar. The total amount of immunoreactive material found in the urine was greater than the amount of antidiuretically active material. These results clearly demonstrate that the antidiuretic activity of TGLVP is mainly due to its conversion to LVP in vivo.
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Our experiments with the neurophysin-related proteins from bovine NSG have demonstrated that these species differ in several important respects from the materials conventionally prepared. (1) In structural terms, the NSG proteins are essentially identical with the conventional neurophysins in amino acid composition and closely similar in immunoreactivity; however, the presence of carbodydrate and lipid moieties in the NSG material, no matter how they are attached, constitutes a structural difference apparently sufficient to cause considerable changes in properties. (2) The affinities for the neurohypophyseal hormones of the NSG proteins are very much higher than those of the "conventional" neurophysins and, moreover, the binding properties of the NSG material are much more stable with time under the conditions of the binding experiments. (3) The low binding capacities of the NSG materials, even when they are purified to apparent molecular homogeneity, indicate a functional heterogeneity perhaps related to supramolecular structure. (4) The conversion of the NSG proteins by acid or alkali treatment to materials resembling the "conventional" neurophysins in their binding properties suggests that the latter may be isolation artifacts. Although we cannot as yet consistently explain the properties of our neurophysins from NSG, we offer the hypothesis that the low binding capacity, as also the Hill coefficient greater than 1 (cf. Reference 25) are indicative of molecular aggregation, perhaps mediated or facilitated by the nonprotein components. It is conceivable that such aggregation, proceeding in the more "natural" environment of the NSG in a more precisely organized manner, might constitute the truly "native," fully functional state of the neurophysins. In this context it is of interest to record our preliminary observations, which suggest the presence of a protein of 10,000 mol. wt. in the NSG membrane fraction (SDS gel) as well as electron-microscopic indications30 of a highly organized ("crystalline") structure within these membranes. Although, therefore, the materials we have described may not merit the description of "native" neurophysins, we believe that they are certainly closer to the native state than the proteins conventionally isolated; and we would suggest that any discussion of the biological role of the neurophysins based on the properties of the conventional preparations may be at best speculative, and at worst misleading.
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