The sites of synthesis and action of prostaglandins (PGs) along the renal tubule are examined. We focused our attention on experiments performed on well-defined nephron segments, using direct quantitative measurements of prostaglandin synthesis by radio- or enzyme-immunoassay. On the other hand, we selected, among the described effects of PGs, those obtained on precisely defined tubular segments. Among PGs, PGE2 synthesis is largely predominant all along the tubule. Its main sites of synthesis are the medullary collecting tubule and, to a lesser extent, the cortical collecting tubule and the thin limb of Henle's loop. Synthesis of PGE2 is amplified approximately tenfold in the presence of an excess exogenous substrate, arachidonic acid, compared with values measured without addition of substrate. Other eicosanoids have roughly the same distribution along the tubule as PGE2. Their rate of synthesis is, however, much less than that of PGE2, approximately 20-fold lower for PGF2 alpha and 6-keto-PGF1 alpha, and 100-fold lower for thromboxane B2 (TxB2). This contrasts with glomerular PG synthesis, where the difference between the production of PGE2 and other eicosanoids is much less marked. Most studies agree that antidiuretic hormone (ADH) and kinins augment PGE2 synthesis, whereas corticosteroids decrease it, at least in the collecting tubule. Direct effects of PGE2 have been described mainly in the medullary thick ascending limb and collecting tubule. They generally consist of a decrease in transepithelial potential difference and reabsorptive rates of water and solutes, in particular sodium and chloride. However, whatever the solute or tubular segment concerned, some studies failed to find such effects. The bulk of evidence suggests that ADH and PGs interact in kidney tubular cells. It is generally accepted that PGs antagonize the hydrosmotic effects of ADH in the collecting tubule. The mechanisms underlying these complex interactions are still under discussion: they probably involve several types of receptors and pathways for ADH action, which intervene in the modulation of both PG synthesis and cyclic nucleotides, and several types of PG receptors, either stimulatory or inhibitory to adenylate cyclase.

The capacity of synthesis of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-PGF1 alpha, and thromboxane (TxB2) along the rabbit nephron was determined. A sensitive enzyme immunoassay was applied to isolated nephron segments, from the glomerulus to the terminal collecting tubule. The three prostaglandins and thromboxane (PG) were measured on the same samples after incubation in the presence of arachidonic acid. In the glomerulus, PGE2 synthesis (29.4 +/- 3.3 pg X glomerulus-1 X 30 min-1) represented 60% of the sum of the four PGs. PGF2 alpha and 6-keto-PGF1 alpha represented 22 and 17%, respectively, and TxB2 1.4%. The contribution of each PG to tubular synthesis was different, since at least 90% of PG synthesis consisted of PGE2. In the medullary collecting tubule (MCT), by far the major tubular site of PG synthesis, it was 809.6 +/- 140.8 pg X mm-1 X 30 min-1 for PGE2, 17.7 +/- 7.2 for PGF2 alpha, 8.3 +/- 1.9 for 6-keto-PGF1 alpha and 0.24 +/- 0.08 for TxB2. These relative proportions were roughly respected all along the tubule. Values were much lower in the convoluted and straight portions of the proximal tubule (proximal convoluted tubule: PGE2 8.2 +/- 2.0, PGF2 alpha 0.4 +/- 0.06, 6-keto-PGF1 alpha 0.26 +/- 0.04, TxB2 0.017) and the medullary and cortical thick ascending limb of the loop. They increased regularly along the distal structures of the tubule (light portion of the cortical collecting tubule (CCT1): PGE2 228.3 +/- 20.4, PGF2 alpha 4.34 +/- 0.6, 6-keto-PGF1 alpha 1.8 +/- 0.3, TxB2 0.22 +/- 0.07).(ABSTRACT TRUNCATED AT 250 WORDS)

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The pure tetrameric form of Acetylcholinesterase (EC-3.1.1.7) from the electric eel electrophorus electricus has been covalently coupled to oxytocin. This conjugate has been used as tracer in a heterologous competitive immunoassay. Microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody were used to separate bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was measured by a colorimetric assay. The minimum detectable concentration was 0.075 pg/well (ie 1.5 pg/ml) and precision was less than 8% at concentration above 0.15 pg/well. An extraction step improved sensitivity up to 10 times with good recoveries. To assess the validity of this assay, basal levels of oxytocin were measured during the oestrous cycle of a cow.

To improve immunoassays of small haptens, we developed two different approaches for their measurement in a non-competitive format. We first devised two-site immunometric assays for small peptides (8-11 amino acids) by selecting two sets of antibodies specifically directed against C- and N-terminal moieties of the peptides. In each case, assay sensitivity improved substantially over that of the corresponding competitive assays. More interestingly, all of these new immunometric assays were much more specific than the competitive assays. In a second approach, we developed a new procedure, solid-phase-immobilized epitope immunoassay (SPIE-IA), in which a single monoclonal antibody uses the same epitope for capture and tracer binding and the hapten is covalently cross-linked to solid-phase proteins. To date, SPIE-IA have been successfully applied to the determination of haptens bearing primary amino groups, including substance P, thyroxine, leukotriene C4, endothelin, and angiotensin II. In each case, assay sensitivity was significantly improved.