Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line

The role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106-01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose-dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 microM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH- and forskolin-induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106-01 cells can be induced by PTH and forskolin via a calcium-dependent cAMP messenger system.     

Prolactin stimulation of ornithine decarboxylase activity in the mammary gland may involve an activation of protein kinase C

Phorbol myristate acetate (TPA), a protein kinase C activator, stimulates ornithine decarboxylase (ODC) activity in mammary gland explants derived from 12-14 day pregnant mice. The calcium ionophore A23187 similarly stimulates ODC activity. Maximally stimulatory concentrations of TPA and A-23187 produce additive responses. The prolactin (PRL) stimulation of ODC activity is nonadditive to that caused by TPA, A23187 or TPA plus A23187. These observations are compatible with the thesis that the stimulation of ODC activity by PRL may occur via an activation of protein kinase C.     

Prolactin stimulates the L-type calcium channel-mediated transepithelial calcium transport in the duodenum of male rats

Elevated plasma levels of prolactin (PRL) have been reported in several physiological and pathological conditions, such as lactation, prolactinoma, and dopaminergic antipsychotic drug uses. Although PRL is a calcium-regulating hormone that stimulates intestinal calcium absorption in lactating rats, whether PRL is capable of stimulating calcium absorption in male rats has been elusive. Herein, the transepithelial calcium transport and electrical characteristics were determined in ex vivo duodenal tissues of male rats by Ussing chamber technique. We found that PRL receptors were abundantly present in the basolateral membrane of the duodenal epithelial cells. PRL (200–800 ng/mL) markedly increased the active duodenal calcium transport in a dose-dependent fashion without effect on the transepithelial resistance. The PRL-enhanced active duodenal calcium transport was completely abolished by L-type calcium channel blocker (nifedipine) as well as inhibitors of the major basolateral calcium transporters, namely plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger. Several intracellular mediators, such as JAK2, MEK, PI3K and Src kinase, were involved in the PRL-enhanced transcellular calcium transport. Moreover, PRL also stimulated the paracellular calcium transport in the duodenum of male rats in a PI3K-dependent manner. In conclusion, PRL appeared to be a calcium-regulating hormone in male rats by enhancing the L-type calcium channel-mediated transcellular and the paracellular passive duodenal calcium transport. This phenomenon could help restrict or alleviate negative calcium balance and osteoporosis that often accompany hyperprolactinemia in male patients.     

Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptides

Prolactin (PRL)-stimulated ornithine decarboxylase (ODC) activity and subsequent proliferation are inhibited by the cyclopeptides cyclosporine (CsA) and didemnin B (DB) in Nb 2 node lymphoma cells. Similar concentrations of these agents also inhibit 125I-PRL binding, suggesting that their inhibitory effects on these PRL-dependent physiologic responses are mediated at least in part at the level of PRL receptor interactions. The phorbol ester TPA stimulated ODC activity and [3H]thymidine incorporation to 54% and 31% that of a near-optimal mitogenic concentration of PRL (10 ng/ml), suggesting that mitogenesis in these cells is coupled to some degree to the activation of protein kinase C (PKC). The calcium ionophore A23187 increased ODC activity only slightly and actually decreased [3H]thymidine incorporation to a value below the "cells only" controls. The addition of TPA plus A23187 did not further enhance the effects of TPA to elevate ODC activity and [3H]thymidine incorporation. However, A23187 significantly elevated PRL-stimulated ODC activity with a subsequent inhibition of [3H]thymidine incorporation, suggesting a block of entry into S phase. Both cyclopeptides decreased the elevation of ODC activity in G1 phase of cell cycle in response to PRL, suggestive of a site of action for these agents in early G1, a conclusion compatible with their ability to inhibit PRL binding to these cells. Addition of CsA or DB 2 hr after PRL had no effect on PRL-stimulated ODC activity detectable at 6 hr, but addition of either as late as 6 hr still affected the extent of mitogenesis. This is in line with the requirement for PRL to be present in the culture medium for a minimum of 3 to 6 hr to invoke a maximal effect on mitogenesis. Addition of either cyclopeptide after the cells were in S phase had no effect on the extent of [3H]thymidine incorporation. An inhibitor of the cyclooxygenase pathway (indomethacin) enhanced both PRL-stimulated ODC activity and proliferation, whereas inhibition of the lipoxygenase pathway by NDGA attenuated only proliferation, suggesting that in Nb 2 cells, products of the lipoxygenase pathway may contribute to the mechanism of PRL-stimulated mitogenesis. Because Nb 2 lymphoma cells were derived from estrogenized rats, estrogen was tested as a mitogen. By itself it was not mitogenic, but in conjunction with PRL, estradiol-17 beta elevated the ODC response and inhibited proliferation. Inhibitors of PKC known to have minimal effects on RNA synthesis, quercetin and gossypol, totally inhibited both the elevations of ODC activity and [3H]thymidine incorporation in response to PRL in Nb 2 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulatory function of protein kinase C in the activation of ornithine decarboxylase and in cAMP production in rat osteoblasts

The effect of activation of protein kinase C on stimulation of ornithine decarboxylase (ODC) activity and cAMP production was studied in fetal rat osteoblasts. Both phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, and 4 alpha-phorbol, ineffective in activating protein kinase C, failed to stimulate ODC activity and cAMP production. We tested the effect of protein kinase C on stimulation of ODC activity by parathyroid hormone (PTH) and forskolin. In contrast to PTH-stimulated ODC activity, which was not affected by PMA, forskolin-stimulated (1 and 10 microM) ODC activity was dose dependently reduced. PMA (400 nM) reduced both 1 and 10 microM forskolin-stimulated ODC activity to the same level, approximately 3 nmol CO2/mg protein, which suggests a controlling role of protein kinase C in forskolin-stimulated ODC activity. The study of the effect of protein kinase C on PTH- and forskolin-stimulated cAMP production also revealed differences between PTH and forskolin. When PMA was added simultaneously with PTH (4 and 20 nM) or forskolin (1 and 10 microM) the PTH-stimulated cAMP production was dose-dependently potentiated by PMA, whereas forskolin-stimulated cAMP production was not affected. However, both PTH- and forskolin-stimulated cAMP production was dose-dependently augmented when PMA was added 3 min prior to PTH or forskolin. With increasing preincubation periods (up to 24 h) with PMA instead of a potentiation an inhibition was observed. This inhibition is not due to PTH receptor desensitization, although, on basis of the present results desensitization can not completely be excluded. In all cases 4 alpha-phorbol was without effect. The present results show that protein kinase C modulates stimulation of ODC activity and cAMP production in fetal rat osteoblasts. The modulation of both ODC activity and cAMP production appears to be dependent on the nature of the stimulator. The present data suggest a role for protein kinase C in limiting the cAMP-mediated stimulation of ODC activity in these cells. Furthermore, it is suggested that protein kinase C can interfere at more than one site in the cAMP-generating system.     

Effects of hyperprolactinemia on ornithine decarboxylase activity and polyamine levels in seminal vesicles of genetically prolactin-deficient adult dwarf mice

Prolactin (PRL) has been shown to exert many different actions in various biological systems. Polyamines are known to influence the growth and function of the seminal vesicles (SV). Furthermore, ornithine decarboxylase (ODC) is considered a key enzyme in the biosynthesis of polyamines and is regulated by PRL in certain target tissues. Adult Ames dwarf mice (df/df), genetically deficient in PRL, were used for this study. The experimental groups were as follows: Group 1, pituitary-grafted; Group 2, sham-operated; Group 3, castrated + testosterone propionate (TP)-treated (25 micrograms/mouse, 3 times/wk, s.c.) + grafted; and Group 4, castrated + TP as above. The animals were killed 40 days later, and polyamines and ODC activity in SV and liver were determined. Serum PRL, FSH, and testosterone (T) were also measured. In the grafted groups, there were significant elevations in serum PRL and FSH levels. In the gonad-intact, pituitary-grafted group, animals exhibited an elevation in plasma T levels, and similar levels were achieved in the castrated, androgen-replaced groups. In hyperprolactinemic mice, the weights of SV were significantly greater than in the corresponding control groups. The relative weights of the SV showed a similar pattern. An increase in ODC activity was observed in both SV and liver in hyperprolactinemic groups. In those animals in which serum T levels were held constant, an increase in the enzyme activity in SV was detected in hyperprolactinemic group whereas in liver, no significant difference was observed. Concentrations of polyamines in the SV were increased in hyperprolactinemic, castrated, TP-treated mice. The present results indicate that PRL can exert a direct stimulatory effect on the growth, ODC activity, and polyamine levels in the SV.     

Independent and interrelated regulation of ornithine decarboxylase by calcium and cAMP in fetal rat osteoblasts

The present study was undertaken to determine in fetal rat osteoblasts whether and how the intracellular messengers calcium and cAMP are involved in stimulation of ornithine decarboxylase (ODC) activity. For that purpose we used different drugs affecting [Ca2+]i and cAMP concentration. A23187 stimulates ODC activity in a biphasic way, with maximal stimulation at 100 nM A23187. At that concentration no stimulation of cAMP production was observed. Basal and A23187-stimulated (100 nM) ODC activity were inhibited by EGTA and trifluoperazine. Forskolin stimulated dose-dependently both ODC activity and cAMP production. Besides these effects forskolin (1 and 10 microM) increased the [Ca2+]i via an increased calcium influx. Addition of La3+, verapamil or EGTA, but not of trifluoperazine, significantly inhibited the forskolin-stimulated (10 microM) ODC activity. When forskolin (100 nM and 1 microM) was added together with 1 microM A23187, a synergistic stimulation of ODC activity was observed. These results implicate that calcium is involved in basal ODC activity, and that ODC activity can be stimulated via (1) a cAMP-independent calcium pathway, and (2) a calcium-dependent, cAMP pathway. It is proposed that ODC activity can be stimulated via interaction between calcium and cAMP.     

The involvement of sodium and calcium ions in the release of amino acid neurotransmitters from mouse cortical slices elicited by hyperforin

Hyperforin is currently considered to be the major active antidepressant constituent of the medicinal herb St. John's wort (Hypericum perforatum L.). The mechanism of action however, is still largely unknown, although the involvement of sodium and calcium has been recently inferred. In the present study hyperforin (5 microM) significantly potentiated the release of endogenous aspartate and glutamate from mouse cortical slices when stimulated by veratridine or potassium. Hyperforin (5 microM) also stimulated the release of aspartate, glutamate, serine, glycine and GABA when perfused on its own. Perfusion of the sodium channel blocker, tetrodotoxin (TTX) inhibited the effect of hyperforin, whereas removal of extracellular calcium potentiated the effect. Our observations suggests that hyperforin increases the overflow of neurotransmitters from mouse cerebral cortex possibly through facilitating the entry of sodium into the neurone which leads to the release of calcium from intracellular stores.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

Intrinsic and extrinsic factors in estrogen action in human breast cancer: role of polyamines and pituitary factors

Although polyamines are important in regulating proliferation of mammalian cells, their role in hormone induction of cell growth has not been delineated. In the estradiol-responsive human breast cancer cell line, T-47D clone 11, estradiol (10(-10) M) was able to stimulate cell proliferation and the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the biosynthesis of polyamines. alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ODC, blocked the estradiol-induced cell proliferation and ODC activity. Exogenous addition of putrescine, the natural product of ODC, rescued the inhibitory effect of DFMO. In addition, DFMO abolished the estradiol-induced growth of several other estrogen-responsive human breast cancer cell lines but did not affect the growth of hormone-independent cell lines. Further, a serum factor was found to be required for estradiol to exert its effect. To gain insight into the nature of this and possibly other extrinsic factors involved, the effect of estradiol on the proliferation of T-47D cells transplanted into athymic nude mouse was evaluated. In this in vivo system, estradiol alone produced only moderate growth of the human breast tumor. The simultaneous transplantation of a prolactin (PRL)- and growth hormone (GH)-secreting rat pituitary tumor or normal rat pituitary glands at a different site dramatically potentiated the effect of estradiol on the growth of the breast tumor xenograft. Purified PRL or GH were without effect, indicating that the active pituitary factor is neither PRL nor GH. Further, conditioned medium from rat pituitary tumor cells potentiated the mitogenic effect of estradiol on T-47D and several other estrogen receptor-positive human breast cancer cell lines in vitro under serum-free condition. In conclusion, we have identified both intrinsic (polyamines) and extrinsic (pituitary/serum) factors that are importance for estrogen to exert its mitogenic action. The next goal will be to elucidate the mechanisms of action of these molecules in the modulation of estrogen action.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1

Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.     

Different effects of L-, N- and T-type calcium channel blockers on striatal dopamine release measured by microdialysis in freely moving rats.

Using a microdialysis method, we have investigated effects of the voltage-dependent calcium channel blockers, verapamil, nicardipine, omega-conotoxin and flunarizine on the dopamine release and metabolism in the striatum of freely moving rat. Perfusion of verapamil (1-300 microM) and nicardipine (1-100 microM), an L-type calcium channel blocker, into the striatum through the dialysis membrane showed a dose-dependent decrease of dopamine release in the dialysate and slight increase of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. Treatment of omega-conotoxin (0.1, 1 microM), an N-type channel blocker, decreased about 50% basal dopamine release and slightly decreased DOPAC and HVA levels. Treatment with flunarizine (10 microM), an T-type channel blocker, did not affect the dopamine release and metabolism. From these data, it appears that treatments of the L- and N-type voltage-dependent calcium channel blockers in rat striatum suppress basal dopamine release, but T-type blocker does not suppress it, suggesting that L-, N- and T-type calcium channels regulate in vivo dopamine release in a different mechanism.     

Excitatory Amino Acid-Induced Phosphoinositide Turnover in Guinea Pig Cerebral Cortical Slices: Selective Enhancement by Spermine of the Response to DL-1-Aminocyclopentane- trans-1, 3-Dicarboxylate

In the presence of 1 mM spermine, accumulations of 3H labelled inositol phosphates elicited by quisqualate (100 microM) and 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD, 300 microM) were significantly enhanced by 21 and 26%, respectively, without a significant alteration in the accumulation elicited by L-glutamate (10 mM) or DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionate (10 microM). Analysis of concentration-response data indicated that the presence of spermine led to an increase in the maximal response to t-ACPD without altering the EC50 value. The stimulatory effect of spermine on the accumulation of t-ACPD-elicited 3H-inositol phosphates was not reversed by ifenprodil or diethylenetriamine (putative polyamine site antagonists), by agents that activate or inhibit protein kinase C, or by calcium channel blockade, but was abolished in the presence of elevated extracellular calcium ion concentration. We conclude that spermine enhances the phosphoinositide turnover in guinea pig cerebral cortical slices elicited by the "metabotropic" excitatory amino acid receptor. The site through which the action of spermine is mediated remains to be defined, but it is apparently distinct from that suggested to modulate N-methyl-D-aspartate receptor activity.     

Prolactin, an activator of epithelial Na+ channel, inhibits basolateral K+ channels in adult tree frog skin

Adult amphibian skin actively transports Na+ from its apical to basolateral side while in turn, K+ is recycled through Na+, K+-ATPase and K+ channels located in the basolateral membrane. We previously found that PRL stimulates Na+ transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na+ channel (ENaC). If PRL also activates basolateral K+ channels, this activation would help to stimulate Na+ transport, too. Whether PRL does indeed stimulate basolateral K+ channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K(ATP) channel blocker, and tetrapentylammonium (TPA), a KCa channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K+ channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K+ current, suggesting that the (Na+)-H+ exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K+ channels in Hyla.     

Studies on the prolactin-releasing mechanism of histones H2A and H2B

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.     

Ca2(+)-independent secretory mechanism of thyrotropin-releasing hormone (TRH) involves protein kinase C in rat pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates biphasic prolactin (PRL) secretion from rat pituitary GH3 cells. The pretreatment of cells with EGTA (100 microM) plus arachidonic acid (15 microM), a condition which decreased TRH-responsive intracellular Ca2+ pools, eliminated the activity of TRH on burst PRL secretion (2 min) but did not alter that on sustained PRL secretion (30 min). However, the treatment of cells with EGTA, arachidonic acid and H-7 (300 microM), a potent inhibitor of protein kinase C (PKC), almost completely suppressed the activity of TRH for sustained PRL secretion. In cells down-modulated for PKC, TRH abolished this Ca2(+)-independent sustained PRL secretion. These results suggest that TRH acts through a separate, Ca2(+)-independent secretory mechanism, besides by modulating the Ca2(+)-dependent mechanism and that PKC is involved in this Ca2(+)-independent secretory pathway.     

Prolactin-stimulated ornithine decarboxylase induction in rat hepatocytes: coupling to diacylglycerol generation and protein kinase C

The trophic effects of prolactin (PRL) in rat liver have been linked to activation of protein kinase C (PKC). Since alterations in PKC activity imply its activation by 1,2-diacylglycerol (DAG), we tested whether PRL treatment stimulated DAG generation coupled to induction of a growth response in primary hepatocytes. Addition of PRL to hepatocyte cultures significantly increased [3H]-glycerol incorporation into DAG within 5 minutes which was followed by a loss of cytosolic PKC activity by 10 minutes. Prolactin also significantly enhanced radiolabel incorporation into triacylglycerol and phospholipids within 10 minutes and induced ODC activity at 6 hours. Therefore, prolactin-stimulated alterations in PKC activity are preceded by enhanced DAG generation. Moreover, these events appear to be coupled to PRL-stimulated entry of hepatocytes into cell cycle.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.