Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1

The receptor specificity and signal transduction pathway has been identified and characterized for a truncated form of myeloid progenitor inhibitory factor-1 (MPIF-1(24-99)). MPIF-1 binds specifically to sites, in particular CCR1, shared with macrophage inflammatory protein-1alpha (MIP-1alpha) on the surface of human monocytes and dendritic cells, as inferred by its ability to compete for [125I]MIP-1alpha, but not for [125I]MIP-1beta or [125I]monocyte chemotactic protein-1(MCP-1) binding to intact cells. Based on calcium flux, MPIF-1 is an agonist on CCR1-transfected HEK-293 cells, monocytes, and dendritic cells, but not on CCR5-, CCR8-, or CX3CR1-transfected cells. The inhibitory effect of guanosine 5'-O-(3-thio-triphosphate) (GTP-gammaS) or pertussis toxin pretreatment on MPIF-1 binding and calcium mobilization, respectively, indicates the involvement of G proteins in the interaction of MPIF-1 and its receptor(s). The increase in intracellular free calcium concentration following MPIF-1 treatment is mainly due to the influx of calcium from an extracellular pool. However, a portion of the intracellular free calcium concentration is derived from a phospholipase C inhibitor-sensitive intracellular pool. MPIF-1 induces a rapid dose-dependent release of [3H]arachidonic acid from monocytes that is dependent on extracellular calcium and is blocked by phospholipase A2 (PLA2) inhibitors. Furthermore, PLA2 activation is shown to be necessary for filamentous actin formation in monocytes. Thus, the MPIF-1 signal transduction pathway appears to include binding to CCR1; transduction by G proteins; effector function by phospholipase C, protein kinase C, calcium flux, and PLA2; and cytoskeletal remodeling.     

Effects of the antioxidant butylated hydroxyanisole on cytosolic free calcium concentration

The effect of the antioxidant butylated hydroxyanisole on free intracellular calcium was investigated in rat cardiomyocytes, rat pituitary cells, human umbilical vein endothelial cells, granulocytes and BHK-cells. The effects of incubation time and butylated hydroxyanisole (BHA) concentrations were examined with respect to the extracellular calcium concentration. Butylated hydroxyanisole increased the free cytosolic calcium concentration in a dose- and time-dependent manner irrespective of the presence of extracellular calcium. Differences between all cell types were observed in the sequel of the re-uptake of the calcium ions released by BHA. Dependence on the concentration of extracellular calcium on the extent of sequestration was seen in rat pituitary cells and cardiomyocytes only.     

Manipulation of xenogeneic skin and/or renal graft survival in the rat-mouse concordant combination by portal vein pretransplant transfusion

In cell culture, human osteoblasts and the osteosarcoma cell line MG-63 express annexins I, II, IV, V and VI. Small proportions of annexins IV and V are lost from MG-63 cells into the culture medium in a sedimentable form. however, the bulk of these annexins is intracellular. In non-confluent cells 3 days after passaging, annexin IV and annexin V are strongly present throughout the nucleus and are also present in the cytoplasm. On elevation of the intracellular calcium concentration with the lonophore ionomycin, the intranuclear pools of annexin IV in 38 +/- 4% of cells and annexin V in 70 +/- 5% of cells show relocation to the nuclear membrane within 40 s. Extracellular ATP, which causes a transient increase in the cytosolic free calcium concentration by acting at P2-purinoceptors, also causes relocation of the intranuclear pool of annexin IV in 22 +/- 4% of cells and of annexin V in 38 +/- 8% of cells. After stimulation no significant reversal of the relocation is observed. Elevation of intracellular calcium with ionophore and ATP also causes relocation of the cytoplasmic pools of annexins IV and V. The results support a role for annexins at cellular membranes in response to elevation of cytosolic calcium levels.     

Cell signaling by the type IV pili of pathogenic Neisseria

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+]i) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+]i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.     

Amelanotic malignant melanoma

The purpose of this study was to investigate whether extracellular matrix proteins which influence human keratinocyte behaviour are capable of altering intracellular signalling systems in these cells. The effects of extracellular matrix proteins on two major signal transduction pathways, intracellular calcium and cyclic adenosine monophosphate (cyclic AMP), were investigated. The extracellular matrix proteins examined were the basement membrane preparation matrigel, collagens type I and IV, vitronectin and its active tripeptide component Arg-Gly-Asp (RGD). Acute additions of matrigel, vitronectin and RGD caused rapid transient increases in intracellular calcium and, together with collagen type I, also caused sustained elevations in basal calcium when cells were grown on these substrates. Cyclic AMP production was unaffected by acute exposure to these extracellular matrix proteins. Culture of cells on matrigel, collagen type I or IV, however, significantly reduced basal cyclic AMP accumulation and increased the response of the cells to the receptor-independent agonist forskolin. It is concluded that in vitro some extracellular matrix proteins can initiate both acute and sustained changes in intracellular signalling in human keratinocytes.     

Effects of provitamin A or non-provitamin A carotenoids on liver xenobiotic-metabolizing enzymes in mice

Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 micrograms/ml. The highest Ca2+ rise (104 +/- 47 nM) was observed for 10 micrograms/ml SSP. It was mainly dependent (81 +/- 11%) on extracellular calcium influx, however, SSP mobilized 68 +/- 7% of Ca2+ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca2+ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca2+ concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (RLIS), phase I response, followed by an increase in tight junctional resistance (RTJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC50 of 6 microM). The objective of the study was to determine the degree to which the changes in [Ca2+]i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La3+, and Mn2+ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease in RLIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La3+ also inhibited the ATP-induced phase II response, but Mn2+ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase in RTJ. KCl-induced membrane depolarization stimulated an acute decrease in RLIS and a late increase in RTJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca2+]i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases in RTJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic, ATP-like changes in RTE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease in RLIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase in RTJ.     

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.     

Oxidant stress causes alteration in the attachment of mononuclear cells to collagen

The effect of oxidant stress on the attachment of blood mononuclear cells to collagen was investigated. Study of the kinetics of attachment of mononuclear cells pretreated with 0.2% H2O2 for 10' showed significantly lower attachment to collagen I substrata when compared to untreated controls. Reduced glutathione at 2.5mM concentration partially reversed the H2O2 induced alteration in attachment. Pretreatment with H2O2 caused a reduction in the number of free thiol groups both at the cell surface and intracellular sites. Changes in cell surface free thiol groups could be reversed by reduced glutathione. These results point to the importance of a stable redox status in the cellular environment for normal interaction of mononuclear cells with extracellular matrix components.     

Jejunal myenteric denervation induced by benzalkonium chloride

We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes.     

Growth hormone-releasing factor mobilizes cytosolic free calcium through different mechanisms in two somatotrope subpopulations from porcine pituitary

Porcine somatotropes can be separated by Percoll density gradient centrifugation into low (LD) and high density (HD) subpopulations that differ ultrastructurally and functionally. Here, we report the effects of growth hormone-releasing factor (GRF) on the cytosolic free calcium concentration ([Ca2+]i) of single LD and HD somatotropes. Resting [Ca2+]i in LD somatotropes was 2-fold higher than in HD cells. GRF induced [Ca2+]i increases in a similar percentage of somatotropes from both subsets. However, amplitude and kinetics of the responses were markedly different. In all responsive LD somatotropes, GRF evoked a rapid initial peak followed by a sustained plateau (plateau-type response). Blockade of extracellular Ca2+ entry by 3 mM EDTA, 2 mM CoCl2, or 100 microM verapamil completely abolished the plateau phase without affecting the initial Ca2+ spike. Conversely, only the plateau phase was preserved in thapsigargin (TG)-treated LD cells. The vast majority of GRF-responsive HD somatotropes exhibited a transient [Ca2+]i peak that returned gradually to baseline (transient-type response). This response was completely blocked by removal of extracellular Ca2+, whereas TG treatment had no effect. Taken together, our results indicate that the response of LD somatotropes to GRF depends on mobilization of Ca2+ of both extra- and intracellular origin, whereas that of HD somatotropes seems to be exclusively dependent on extracellular Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC). These findings are the first to demonstrate a differential effect of GRF on Ca2+ mobilization in two somatotrope subpopulations, and suggest the existence of differences in the GRF receptor(s) expressed in each subpopulation and/or in the intracellular signalling pathways activated upon GRF binding.     

High magnesium concentration inhibits ligand-stimulated calcium influx and hormone secretion in rat pituitary lactotropes with involvement of intracellular free magnesium

The effects of extracellular magnesium concentration ([Mg2+]ex) on thyrotropin-releasing hormone (TRH)-stimulated intracellular free calcium mobilization and prolactin secretion were investigated concomitantly with measurement of the intracellular free magnesium concentration ([Mg2+]i). TRH-stimulated intracellular free calcium mobilization was significantly inhibited when the medium was replaced by high Mg2+ medium ([Mg2+]ex = 10 mM) in normal Ca2+ medium. The inhibitory effects of high Mg2+ became apparent concomitantly with an increase in [Mg2+]i from 0.7 to 1.3 mM. High Mg2+ significantly inhibited TRH-induced PRL secretion in a dose-dependent manner in normal Ca2+ medium. TRH-stimulated inositol triphosphate (IP3) production was rather augmented by the replacement with high Mg2+ medium. In summary, high Mg2+ inhibits Ca2+ influx stimulated by TRH in the rat pituitary lactotropes, possibly with the involvement of [Mg2+]i increase. These results have general importance in relation to high Mg(2+)-induced suppression of the biological functions of cells.     

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: voltage-gated calcium channels, and/or phospholipase C coupled to a pertussis-sensitive G-protein

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca2+]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca2+]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 micrograms/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca2+]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca2+ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced [Ca2+]i but did not block the effect of IGF-2. 2) IGF-1, IGF-2, and insulin also induced a Ca2+ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhibitors, neomycin, or U-73122 partially blocked the intracellular [Ca2+]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca2+ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF1 receptor antibodies diminished the IGF-1-induced Ca2+ spike and totally abolished the Ca2+ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca2+ influx involves the IGF1 receptor, while part of IGF-1 and all of IGF-2 Ca2+ mobilization do not implicate this receptor.     

High affinity binding of latent matrix metalloproteinase-9 to the alpha2(IV) chain of collagen IV

Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular matrix degradation. Previously, we showed the surface association of pro-MMP-9 in human breast epithelial MCF10A cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity (Kd approximately 22 nM) to MCF10A cells and other cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in zymogen activation and is not followed by ligand internalization, even after complex formation with tissue inhibitor of metalloproteinase-1 (TIMP-1). A 190-kDa cell surface protein was identified by ligand blot analysis and affinity purification with immobilized pro-MMP-9. Microsequencing and immunoblot analysis revealed that the 190-kDa protein is the alpha2(IV) chain of collagen IV. Specific pro-MMP-9 surface binding was competed with purified alpha2(IV) and was significantly reduced after treatment of the cells with active MMP-9 before the binding assay since alpha2(IV) is hydrolyzed by MMP-9. A pro-MMP-9.TIMP-1 complex and MMP-9 bind to alpha2(IV), suggesting that neither the C-terminal nor the N-terminal domain of the enzyme is directly involved in alpha2(IV) binding. The closely related pro-MMP-2 exhibits a weaker affinity for alpha2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the binding of alpha2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with alpha2(IV), the proenzyme does not bind to triple-helical collagen IV. These studies suggest a unique interaction between pro-MMP-9 and alpha2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network.     

Influence of extracellular matrix proteins on the development of cultured human dendritic cells

The development of dendritic cells (DC) is still only partly understood. Recently established culture systems using CD34+ cells or monocytes as precursor cells for the generation of DC indicate the necessity of pro-inflammatory cytokines for their development. In vivo the contact to other cells or to the proteins of the extracellular matrix might also be essential for their development. In our experiments we used granulocyte-macrophage colony-stimulating factor- and IL-4-treated human monocytes as precursor cells to investigate the interaction of DC at different maturation stages with the matrix proteins fibronectin, collagen type I and collagen type IV. We demonstrate a strong beta1-integrin-mediated adherence of immature DC to fibronectin that is lost completely during maturation. The binding to collagen type I was less strong but induced a maturation of the precursor cells. After 3 days of culture on this protein, the cells showed all features of fully matured DC such as expression of CD83 and an excellent allostimulatory capacity. The reason for this effect was shown to be the induction of TNF-alpha production by the DC themselves. In contrast to the adhesion to fibronectin, the maturation and the cytokine production of DC induced by collagen type I could not be inhibited by blocking of beta1-integrins. These results indicate that proteins of the extracellular matrix play an important role in the development and function of human DC.     

Saturable P-glycoprotein kinetics assayed by fluorescence studies of drug efflux from suspended human KB8-5 cells

This article describes a new and rapid method to determine the pumping rate of P-glycoprotein (P-gp) in intact cells. Multidrug resistant (MDR) human epidermoid carcinoma KB8-5 cells (containing P-gp) were loaded with daunorubicin (DNR) in the absence or in the presence of verapamil, sufficient to inhibit DNR pumping by P-gp. In either case, the cells were resuspended in medium devoid of DNR and the subsequent increase of the DNR fluorescence intensity was measured as a function of time. For cells loaded with the same amount of drug, the free cytosolic drug concentration (Ci(t)) was a unique function of the DNR medium concentration (Co(t)). The cellular drug content in the presence of verapamil decreased nonlinearly with decreasing extracellular drug concentration, indicating that the intracellular drug apparent distribution volume increased with decreasing cellular drug content. At each fluorescence intensity, we calculated the P-gp mediated (verapamil-inhibitable) DNR transport rate from the rate of increase of the DNR fluorescence intensity in the absence of verapamil minus the rate of increase of the DNR fluorescence intensity in the presence of verapamil. When plotted against the intracellular free drug concentration (as calculated from the total cellular drug content and a separately determined relation between the total cellular drug content and the intracellular free drug concentration: the apparent distribution volume), this P-gp mediated DNR transport rate showed saturation of P-gp at higher DNR concentrations. The results imply that P-gp mediated DNR transport is saturable (the value of Km is in the order of 1 microM).     

A simulation model including ovulation rate, potential embryonic viability, and uterine capacity to explain litter size in mice: II. Responses to alternative criteria of selection

We have used BCECF- or Fura-2-loaded rat pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). Ca2+-mobilizing agonists CCK-8 and ACh induced a transient acidification totally dependent on release of Ca2+ from internal stores. Employment of different physiological tools including ionomycin and thapsigargin to increase the cytosolic Ca2+ concentration and capacitative calcium influx also induced cellular acidification. Application of 1mM LaCl3 reduced the CCK-8-evoked acidification. These data indicate that the mobilization of intracellular Ca2+ stores by CCK-8 decreases cellular pH by Ca2+/H+ exchanger.     

Analogues of the thrombin receptor tethered-ligand enhance mesangial cell proliferation

Thrombin stimulates cytosolic calcium mobilization and tritiated thymidine incorporation in rat glomerular mesangial cells. This effect may be mediated by a thrombin receptor similar to the receptor found in human platelets. In order to test this possibility, a series of analogues of the thrombin receptor peptide, SFLL-RNPNDKYEPF, was evaluated for their effects on mesangial cells. Analogues of the thrombin receptor peptide containing five, six, seven and 14 amino acids were as efficacious as thrombin with respect to calcium mobilization and thymidine incorporation, although they were significantly less potent. The dissimilarity in potency between thrombin and the thrombin receptor peptides is consistent with the kinetics of the proposed mechanism of action of the enzyme, since the cleavage by thrombin of its receptor results in a tethered ligand which is at a relatively high concentration compared to the free peptides in solution. Those thrombin receptor peptide analogues which showed decreased activity in platelets were tested in mesangial cells. Removal of serine at position one, N-acetylation, or replacement of the phenylalanine at position two with alanine resulted in analogues which were inactive in stimulating mesangial cell proliferation or calcium mobilization. In addition, those analogues which had no stimulatory effects in mesangial cells were not antagonists of SFLLRN-mediated calcium mobilization and thymidine incorporation in mesangial cells.     

Rat Sertoli cell calcium response to basement membrane and follicle-stimulating hormone

Sertoli cells cultured on basement membrane substrates differentiate morphologically into polarized cells and exhibit an enhanced responsiveness to FSH. The signal transduction mechanisms by which the extracellular matrix induces changes in the morphology and function of Sertoli cells are not known. Since calcium has been implicated in mediating changes in cytoskeletal assembly and organization, we investigated to see if basement membrane can modulate cytosolic free calcium concentrations during the process of adhesion and spreading of Sertoli cells. A direct quantification of the intracellular free cytosolic calcium concentration [Ca2+]i in freshly isolated immature rat Sertoli cells plated on laminin was performed by digital imaging microscopy using the fluorescent probe Fura-2 AM. [Ca2+]i levels rose by 1.5-2-fold within 1 h after plating on laminin, suggesting that calcium may be involved in adhesion and spreading of the cells on basement membrane. Furthermore, the possibility that matrix influences [Ca2+]i levels upon stimulation with FSH was examined by adding FSH directly to the cells spreading on laminin. A dramatic decrease in [Ca2+]i was observed compared to the level in untreated cells. Similarly, a significant decrease in [Ca2+]i in response to FSH was observed in cells already spread on laminin or Matrigel. Addition of dibutyryl cAMP did not significantly alter the basal calcium levels. Long-term exposure of Sertoli cells cultured on either laminin or Matrigel to FSH was studied by incubating the cells with 45CaCl2 in the presence or absence of FSH for 24 h. FSH induced a decrease or no change in 45Ca concentration in cells cultured on basement membrane. Addition of dibutyryl cAMP, instead of FSH, did not alter the basal 45Ca concentrations. In cells cultured on the peptides derived from laminin (RGD and SIKVAV), FSH increased the uptake of 45Ca significantly, whereas on YIGSR, also a laminin-derived peptide, it did not have any effect. Thus, basement membrane induces an early increase in [Ca2+]i in cultured Sertoli cells during spreading, and FSH appears to significantly decrease [Ca2+]i levels.     

On the need for evidence-based medicine

We tested the hypothesis that the fludrocortisone in doses sufficient to elevate blood pressure (BP) in normal subjects would increase platelet cytosolic calcium. Eight normal volunteers were given 0.8 mg fludrocortisone daily for 7 days (short protocol). Eight other normal volunteers ingested the drug for 6 weeks (long protocol). In the short protocol, fludrocortisone increased platelet cytosolic calcium and body weight by day 3, while BP was increased by day 7. In the long protocol, platelet cytosolic calcium was increased after 1 week, returned to basal values by 3 weeks and remained at that level for the rest of the study. Stimulation of the subjects' platelets ex vivo with thrombin and vasopressin led to a significant increase in intracellular free calcium concentration; however, fludrocortisone treatment did not alter the calcium response to either agonist. Fludrocortisone decreased serum potassium, plasma renin activity, plasma noradrenaline concentration and serum ionised calcium. These changes, as well as the BP increase, reverted to basal values when the drug was discontinued. We next incubated human platelets with fludrocortisone (1.4 nmol/l) and found a significant increase in cytosolic calcium by 30 min. The data suggest that a blood pressure-raising dose of mineralocorticoid leads to a transient (days to weeks) increase in platelet cytosolic calcium. Platelet cytosolic calcium and blood pressure are dissociated in that cytosolic calcium increases before the BP increase and later decreases to lower values, while the BP increase is sustained. Mineralocorticoid also has a direct effect on platelet cytosolic calcium in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)