Revisiting the roles of progesterone and allopregnanolone in the nervous system: resurgence of the progesterone receptors.

Progesterone is commonly considered as a female reproductive hormone and is well-known for its role in pregnancy. It is less well appreciated that progesterone and its metabolite allopregnanolone are also male hormones, as they are produced in both sexes by the adrenal glands. In addition, they are synthesized within the nervous system. Progesterone and allopregnanolone are associated with adaptation to stress, and increased production of progesterone within the brain may be part of the response of neural cells to injury. Progesterone receptors (PR) are widely distributed throughout the brain, but their study has been mainly limited to the hypothalamus and reproductive functions, and the extra-hypothalamic receptors have been neglected. This lack of information about brain functions of PR is unexpected, as the protective and trophic effects of progesterone are much investigated, and as the therapeutic potential of progesterone as a neuroprotective and promyelinating agent is currently being assessed in clinical trials. The little attention devoted to the brain functions of PR may relate to the widely accepted assumption that non-reproductive actions of progesterone may be mainly mediated by allopregnanolone, which does not bind to PR, but acts as a potent positive modulator of γ-aminobutyric acid type A (GABA(A) receptors. The aim of this review is to critically discuss effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABA(A) receptors, with main focus on the brain.

Metallothionein stroma reaction in tumor adjacent healthy tissue in head and neck squamous cell carcinoma and breast adenocarcinoma.

INTRODUCTION: An accumulation of genetic alterations forming the field of cancerization is an important event for the transformation from normal to cancer cell in multistep carcinogenesis. Histopathologically healthy tumor adjacent tissue might be considered as a cancerization field which is typified by genetic changes required for the development of cancer. Metallothionein (MT) is considered to be a protective and anti-apoptotic protein. The aim of our study was to evaluate the MT expression in head and neck squamous cells carcinoma and breast adenocarcinoma and their histologically healthy adjacent tissue. MATERIALS AND METHODS: We have sampled 29 tissue samples in total derived from head and neck cancers and 29 samples of their clear surgical margins, 33 breast adenocarcinomas and 33 clear surgical margins. Antibody recognizing MT-1 was used for immunohistochemical analysis. RESULTS: MT expression was revealed in 85,7% of head and neck cancers and 94% of breast adenocarcinomas. It was found in all tumor adjacent tissue. MT expression was statistically significantly higher in tumor adjacent tissue than in cancer tissue in cases with the presence of lymph node metastases in both, breast adenocarcinoma and head and neck squamous cell carcinoma. Generally stroma seems to respond to the presence of cancer by the expression of MT, even in tissues which normally do not express MT. CONCLUSIONS: MT might be a normal or protective reaction of healthy adjacent tissue to the presence of tumor.

Modulation of HSP25 expression during anterior horn motor neuron degeneration in the paralysé mouse mutant.

The paralysé spontaneous mutation in mice involves degeneration and death of anterior horn motor neurons. Mutant mice are not viable past postnatal day 16. At present, the mechanisms involved in motor neuron death are unknown. Here, we investigate the expression of the small heat shock protein Hsp25, in the spinal cord of paralysé at two different stages during postnatal development, i.e., day 11 and day 14. Western blot analysis reveals that the level of Hsp25 was strikingly different in paralysé as compared to control littermates. Hsp25 expression level in paralysé at day 11 was much lower than in control mice. At day 14, an opposite pattern was observed. Such pattern seems to be restricted to spinal cord, since level of Hsp25 in other tissues (lung, brain, liver, and heart) was quite similar. Immunofluorescence examination of the lumbar spinal cord sections reveals that in control mice, Hsp25 was expressed at high level in motor neurons located in the ventral horn at both day 11 and day 14. By contrast, in paralysé mice, Hsp25 staining within the motor neurons was barely detectable except as a spot in the nucleolus (day 11). At the end stage of the disease (day 14), not only was Hsp25 staining even less intense in motor neurons, but also a strong Hsp25 staining was observed in reactive astrocytes within the gray matter. Taken together, these data suggest that Hsp25 expression is differently modulated in neuronal and glial cells during neurodegenerative processes leading to motor neuron death.

Annexin V inhibits protein kinase C activity via a mechanism of phospholipid sequestration.

In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCalpha. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCalpha. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.

Inhibition of cytosolic phospholipase A2 by annexin V in differentiated permeabilized HL-60 cells. Evidence of crucial importance of domain I type II Ca2+-binding site in the mechanism of inhibition.

Annexin V belongs to a family of proteins that interact with phospholipids in a Ca2+-dependent manner. This protein has been demonstrated to have anti-phospholipase A2 activity. However, this effect has never yet been reported with the 85-kDa cytosolic PLA2 (cPLA2). We studied, in a model of differentiated and streptolysin O-permeabilized HL-60 cells, the effect of annexin V on cPLA2 activity after stimulation by calcium, GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), formyl-Met-Leu-Phe, or phorbol 12-myristate 13-acetate. Both recombinant and human placental purified annexin V inhibit cPLA2 activity whatever the stimulus used. The decrease of arachidonic acid release is of 40 and 50%, respectively, at [Ca2+] of 3 and 10 microM. The mechanism of inhibition was also analyzed. cPLA2 requires calcium and protein kinase C (PKC) or mitogen-activated protein kinase phosphorylation for its activation. As annexin V was shown to be an endogenous inhibitor of PKC, PKC-stimulated cPLA2 activity was analyzed. Using GF109203x, a specific PKC inhibitor, we demonstrated that this pathway is of minor importance in our model. cPLA2 inhibition by annexin V is not linked to PKC inhibition. To test the hypothesis of phospholipid depletion, mutants of annexin V were constructed using mutagenesis directed to Ca2+ site. We demonstrate that the Ca2+ site located in domain I is necessary for the inhibitory effect of annexin V on cPLA2 activity. The site in domain IV is also involved but with less efficiency. In contrast, mutations in site II and III do not modify this effect. Moreover, annexin V mutated on all sites does not inhibit cPLA2. Thus, we propose a predominant role of module (I/IV) in the biological action of annexin V, which, in physiological conditions, may control cPLA2 activity by depletion of the phospholipid substrate.

[Environment and health--studies using biomarkers]. / Srodowisko a zdrowie--badania przy uzyciu biomarkerów.

Present day society is questioning more and more the effect of the evolution of modern agriculture on the quality of life. Two new tools, biomarkers and satellite images, may be used to provide a better understanding of the links which exist between the quality of the environment, the food chain and human health. This multi-disciplinary approach has for some years now joined together diverse specialists from INSERM, the CNRS and the University. Experiments are undertaken in real conditions on several sites of large scale farming land in the north of the Paris Basin. The aim is to study the quantitative and qualitative transfers of pollutants travelling in soils and waters towards agricultural food products, and then to man. The first results of laboratory analysis, concerning the use certain genes of thermic shock proteins HSP 70 and metallothionein MT2A as biomarkers of disequilibrium, are very conclusive. Furthermore, the connection of these genes with a fluorescent (reporter) gene and found in farmed vegetation, could enable the use of satellite images, and this possibility seems to be very promising. The ultimate goal is to improve knowledge of the direct effects of modern agricultural practices on human health and to participate in the debate about the place of modern agriculture in the development of modern society.

Annexins and protein kinases C.

Annexins and protein kinases C belong to two distinct families of ubiquitous cytoplasmic proteins involved in signal transduction. All annexins share the property of binding calcium and phospholipids in the presence of calcium. Protein kinases C belong to three distinct groups of kinases: cPKCs (conventional PKCs) depend on calcium, diacylglycerol and negatively charged phospholipids for their activity, nPKCs (novel PKCs) depend on diacylglycerol and negatively charged phospholipids and aPKCs (atypical PKCs) only require negatively charged phospholipids. Almost all annexins are both in vitro and in vivo substrates for PKCs except annexin V. All annexins have a putative binding site for PKCs but only annexin V would possess a potential pseudo-substrate site. We propose that annexin V modulates the activity of some cPKCs on their substrates which may be the other annexins.

Inhibitory effect of annexin V on protein kinase C activity in mesangial cell lysates.

Annexin V belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of the protein kinase C (PKC) activity. This study examines the effect of annexin V on the in vitro PKC activity in cultured mesangial cells using histone H1, the peptide [Ser25]PKC-(19-31), or endogenous proteins as substrates. The SDS/PAGE pattern of 32P-labeled mesangial proteins showed that the calcium-independent PKC [(n+a)PKC] phosphorylated several proteins from 70 kDa to 40 kDa and 22 kDa to 15 kDa. Three additional proteins from 34 kDa to 29 kDa, including annexin I and its proteolytic forms, were detected after activation of calcium-dependent PKC (cPKC). Increasing concentrations of annexin V did not alter the phosphorylation of (n+a)PKC substrates. By contrast, specific phosphorylation of proteins and annexin I by cPKC, was reduced in a dose-dependent manner. Addition of high concentration of calcium and phosphatidylserine did not reverse the inhibitory effect of annexin V. Annexin V also inhibited the phosphorylation of histone H1 or peptide [Ser25]PKC-(19-31) by cPKC. Moreover, removal of annexin V from cytosols increased the annexin I phosphorylation by these isoforms. From these results, we propose that annexin V may regulate the signal-transduction pathway involving the activation of cPKC, as they act in vitro as an inhibitor of these kinases.

In vivo and in vitro phosphorylation of annexin II in T cells: potential regulation by annexin V.

In order to understand how signal transduction occurs during T cell activation, it is necessary to identify the key regulatory molecules whose function is influenced by phosphorylation. Annexins II (A-II) and V (A-V) belong to a large family of Ca(2+)-dependent phospholipid-binding proteins. Among many putative functions, annexins may be involved in signal transduction during cellular proliferation and differentiation. In the present study we show that A-II is phosphorylated in vivo in the Jurkat human T cell line. Indeed, A-II is phosphorylated after stimulation by phorbol myristate acetate and on serine residues after T cell antigen receptor (TcR) stimulation. In cytosol from Jurkat cells, A-II is phosphorylated only by Ca2+/phospholipid-stimulated kinases such as Ca(2+)-dependent protein kinases C (cPKCs). A-V inhibits the phosphorylation of A-II and other substrates of cPKCs and has no effect on kinases activated only by phospholipids. In conclusion, A-II is phosphorylated both in vitro and in vivo in Jurkat cells, and may play a role as a substrate during signal transduction in lymphocytes via the TcR through the PKC pathway. On the other hand, A-V could act as a potent modulator of cPKCs in Jurkat cells.

Protein kinase C-dependent phosphorylation of annexins I and II in mesangial cells.

In this study we describe the phosphorylation of annexins from cultured rat mesangial cells by protein kinase C (PKC) both in vitro and in vivo. Annexins I and II were detected either by Western-blot analysis or by immunoprecipitation using specific antibodies. In the presence of [gamma-32P]ATP, cytosolic annexin I and annexin II were phosphorylated in vitro only when Ca2+ and phospholipids were added, but not in the presence of phospholipids alone. Annexin I was shown to be a better substrate than annexin II. In experiments in vivo performed on 32P-labelled mesangial cells, the addition of two well-known activators of PKC, namely angiotensin II (AII) and phorbol myristate acetate (PMA), increased preferentially the phosphorylation of annexin I. Annexin II was phosphorylated to a much lesser extent after AII treatment. Phosphoamino acid analysis of annexins, either by two-dimensional chromatography or by using a specific antiphosphotyrosine antibody, revealed only phosphoserine in these experiments in vivo. The addition of AII to mesangial cells increased serine phosphorylation of annexin I and annexin II, whereas PMA only increased serine phosphorylation of annexin I. V8-protease phosphopeptide mapping of annexin I that was phosphorylated both in vitro and in vivo by PKC from mesangial cells shows similar phosphopeptides.

Immunological identification of protein kinase C-alpha and protein kinase C-delta in cultured rat mesangial cells: differential sensitivity of the two isoforms towards the protein kinase inhibitor H7.

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC alpha by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC delta using similar techniques. The inhibition of these two PKC isoforms by the protein kinase inhibitor H7 [1-(iso-quinolinyl sulphonyl)-2-methyl piperazine] was examined using both histone H1 and endogenous proteins as substrates. Phosphorylations catalyzed by the calcium-dependent PKC isoform alpha were almost 90% inhibited when histone H1 was used, and only 55% when endogenous proteins were the substrate. In contrast, the phosphorylation of endogenous proteins catalysed by the calcium-insensitive, phospholipid-dependent PKC delta was not significantly affected by the inhibitor.

Heterogeneity of protein kinase C in cultured rat mesangial cells.

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC alpha isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000-39,000, the 24,000 and the triplet of 17,000-20,000-22,000 molecular weight. The addition of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 molecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes.

Production of tumor necrosis factor by rat mesangial cells in response to bacterial lipopolysaccharide.

Tumor necrosis factor (TNF) is a cytokine which is produced by mononuclear phagocytes upon activation by bacterial lipopolysaccharide (LPS) and various other stimuli. In immune-mediated glomerulonephritis, infiltration of glomeruli by monocytes-macrophages is associated with production of TNF. The purpose of the present experiments was to determine whether mesangial cells could also contribute to glomerular TNF synthesis. TNF activity has been determined in the culture medium of rat mesangial cells using a L-929 fibroblast lytic assay. This activity was detectable only when the cells were exposed to LPS (0.1 to 10 micrograms/ml) and for periods longer than one hour. The cytotoxic factor was identified as TNF since: (1) the lytic activity was completely inhibited by an anti-mouse TNF polyclonal antibody and was associated with suppression of lipoprotein lipase activity in adipocytes; (2) its molecular weight (110,000 daltons) corresponded to that observed for murine TNF under non-denaturing conditions; and (3) mRNA encoding TNF was expressed by mesangial cells two hours after addition of LPS. To assess the mechanisms whereby TNF production was regulated, the role of prostaglandin E2 (PGE2) was determined. LPS caused a dose-dependent increase of PGE2 synthesis by mesangial cells. Treatment by indomethacin promoted a suppression of PGE2 production together with an increase of TNF synthesis, indicating that PGE2 acted in a negative feedback manner to regulate the production of TNF. Addition of PGE2 (0.1 to 300 nM) or 8-bromo cyclic AMP (0.1 to 100 microM) induced similar dose-dependent reductions of TNF synthesis. Thus the inhibitory effect of PGE2 probably required in part cyclic AMP accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of polymorphonuclear leukocytes on the metabolism of arachidonate in human platelets.

The effect of the association of purified polymorphonuclear leukocytes (PMNL) with platelets on arachidonic acid (AA) metabolism was studied in the presence of various concentrations of this fatty acid. Both thromboxane B2 (TXB2) and 12-hydroxy-eicosatetraenoic acid (12-HETE) were measured. In the presence of tracer doses of AA, addition of increasing amounts of PMNL to platelets inhibited in a concentration-dependent manner their 12-HETE and TXB2 production. This inhibition was not due to diversion of AA metabolism towards other pathways since, apart a negligible amount of 12,20-diHETE, no other product could be detected. Inhibition of platelet-TXB2 synthesis by PMNL persisted at increasing concentrations of AA below 16 microM. Above this concentration, TXB2 production by platelets incubated alone diminished progressively. Addition of PMNL blunted in part this inhibitory effect and even resulted, above 16 microM AA, in an increased production of TXB2. In contrast with what was observed for TXB2 formation, the inhibition of 12-HETE synthesis persisted when PMNL and platelets were coincubated in the presence of high doses of AA (163 microM). At this concentration, 15-HETE generation became apparent for each cell type incubated separately and was markedly enhanced in the coincubation studies. The present investigation demonstrates that the presence of PMNL modifies the metabolism of arachidonate by human platelets. Moreover, this cell-cell interaction markedly depends on the concentration of substrate. PMNL in excess may attenuate synthesis by platelets of their toxic products.

Human glomeruli release fatty acids which stimulate thromboxane synthesis in platelets.

The cell-free medium of isolated human glomeruli exhibited a procoagulant activity and stimulated thromboxane (TXB2) synthesis in human platelets in a dose-dependent manner. The amount of TXB2 measured was 16-fold higher than what could have been predicted (TXB2 synthesized by the platelets under control conditions plus TXB2 present in the glomerular supernatant). The lipid extract of the glomerular supernatant and its purified fraction including the fatty acids was still able to stimulate--although at a lesser degree--TXB2 synthesis in platelets. Stimulation was abolished after treatment of this fraction by charcoal or albumin. Gas chromatography/mass spectrometry analysis demonstrated the presence in the purified glomerular fraction of several long-chain saturated or monoenoic fatty acids at a total concentration of 80 microM with the following order of abundance: stearic, palmitic, myristic and oleic acids. Addition to human platelets of these same exogenous synthetic acids resulted in a dose-dependent stimulation of TX synthesis. It was maximum with three or four fatty acids tested in combination, but still present with myristic acid used separately. Arachidonic acid was absent in the glomerular supernatant. Thus the stimulation observed could not be related to a greater availability of substrate. Fatty acids did not act on platelets through a non-specific detergent effect since addition of high doses of detergents inhibited TXB2 formation in platelets. The combination of fatty acids from glomerular origin identified in the present study represents a novel factor involved in the control of intracapillary hemostasis, but different from the procoagulant activity common to many tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells.

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.

Bioconversion of leukotriene C4 by rat glomeruli and papilla.

Since leukotriene C4 (LTC4) may be locally synthesized by bone marrow-derived cells infiltrating the kidney in inflammatory renal diseases we examined the in vitro metabolism of exogenously added [3H] LTC4 by rat glomeruli and papilla using radiometric HPLC. Homogenized as well as intact glomeruli converted [3H] LTC4 mainly into [3H] LTE4 (83%) and, at a smaller extent, into [3H] LTD4 (4%). Intact [3H] LTC4 represented 13% of the sum of radioactive leukotrienes. Addition of L-cysteine resulted in accumulation of LTD4. In contrast, there was nearly no conversion of [3H] LTC4 (87% intact) in the presence of homogenized papilla. The metabolism of [3H] LTC4 by the glomeruli was time- and temperature-dependent. The 10,000 g supernatant and pellet of homogenized glomeruli both retained the ability to metabolize [3H] LTC4. The papillary 10,000 g supernatant was inactive, as found for the total homogenate, whereas the papillary 10,000 g pellet separated from its supernatant could transform [3H] LTC4 into its metabolites, LTD4 and LTE4. Addition of increasing amounts of papillary 10,000 g supernatant to homogenized glomeruli progressively protected [3H] LTC4 from its bioconversion. These results demonstrate that both glomeruli and papilla possess the gamma-glutamyl transpeptidase and dipeptidase necessary to process LTC4. However, the enzyme activity of the papilla is unmasked only when the inhibitor present in the 10,000 g supernatant is separated from the enzyme present in the pellet.

Histamine H2 receptors in rat renal glomeruli.

The aim of this study was to demonstrate histamine-H2 receptors in glomeruli isolated from rat renal cortex and to correlate binding to stimulation by histamine of glomerular cyclic AMP concentration. Binding studies were performed at 10-12 degrees C using [3H]cimetidine as a tracer. Specificity of binding relies on the following: inhibition of [3H]cimetidine binding by the unlabelled drug, other H2-antagonists and agonists in contrast with the very weak inhibitory effects of H1 agonists and antagonists; reversibility of steady-state binding after addition of unlabelled drug; half inhibition of the glomerular cyclic AMP response to histamine at concentrations of cimetidine close to the KD value derived from the binding studies (3 microM); calculated KD value in agreement with the therapeutical concentration of cimetidine and the physiological concentration of histamine. [3H]Cimetidine binding concentration of cimetidine and the physiological concentration of histamine. [3H]Cimetidine binding strikingly increased in the presence of copper chloride (20-300 microM) due to an increase both in number of sites and affinity. However this greater binding did not influence either the inhibitory effect of cimetidine on histamine-induced glomerular cyclic AMP concentration or the stimulatory effect of histamine itself. [3H]Cimetidine binding was temperature-dependent since it progressively diminished from 0 to 37 degrees. This was not due to [3H]cimetidine degradation as shown by thin layer chromatography but rather to a change in drug-receptor interaction at higher temperatures. Glumerular concentration of cyclic AMP increased progressively in the presence of histamine (0.1-1000 microM). This stimulatory effect was markedly inhibited by H2 antagonists. These data demonstrate the presence in rat glomeruli of H2 receptors linked to adenylate cyclase.

In vitro prostaglandin synthesis by various rat renal preparations.

Prostaglandin synthesis by eight different structures from the rat kidney (while cortex, cortical tubules, glomeruli, outer medulla, papilla, glomerular cultured epithelial and mesangial cells, cultured interstitial medullary cells) was measured in vitro after incubation with [14C] arachidonic acid using high-performance liquid chromatography followed by RIA with four specific anti-prostaglandin antibodies (prostaglandin E2, prostaglandin F2 alpha, 6 keto-prostaglandin F1 alpha, thromboxane B2). Prostaglandin production by the whole cortex and cortical tubules was very low. The order of abundance for isolated glomeruli was thromboxane B2 great than prostaglandin E2 greater than prostaglandin F2 alpha greater than 6 keto-prostaglandin F1 alpha. Mesangial cells synthesized prostaglandin E2 at a markedly high rate, in decreasing order: prostaglandin F2 alpha, thromboxane B2 and 6 keto-prostaglandin F1 alpha. The same order of abundance was observed for epithelial cells. The papilla synthesized essentially prostaglandin E2 and prostaglandin F2 alpha, whereas the main product for the outer medullar was 6 keto-prostaglandin F1 alpha. Cultured interstitial cells synthesized mainly prostaglandin E2 and to a lesser extent prostaglandin F2 alpha. Unidentified peaks eluting between 6 keto-prostaglandin F1 alpha and thromboxane B2 were also observed chiefly with glomeruli but they were absent with the medullary preparations. They disappeared after incubation with indomethacin or aspirin and represented for glomeruli the greatest percentage of conversion of [14C] arachidonic acid. These results show that the prostanoid profile varies markedly with the different regions and cells of the rat kidney.