Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats.

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.

Noradrenergic lesions differentially alter the expression of two subtypes of low Km cAMP-sensitive phosphodiesterase type 4 (PDE4A and PDE4B) in rat brain.

This study examined the effects of selective, central noradrenergic dennervation with 6-hydroxydopamine (6-OHDA) on the expression of type 4 phosphodiesterases (PDE4). Twenty-one days following i.c.v. injection of 6-OHDA (200 microg) hypothalamus, neostriatum, and cerebellum were dissected. Infusion of 6-OHDA reduced norepinephrine (NE) content in all the brain areas examined (to 17%, 76% and 16% of sham-operated controls in hypothalamus, striatum, and cerebellum, respectively). 6-OHDA injections also reduced dopamine levels in hypothalamus (53%) and neostriatum (68%). Administration of desipramine (20 mg/kg, i.p.) 30 min prior to 6-OHDA injection protected neostriatal and cerebellar noradrenergic neurons NE levels (110-122% of the control levels). Desipramine partially attenuated the 6-OHDA-mediated decrease in NE content of hypothalamus, but had little or no effect on either striatal or hypothalamic dopamine (DA) levels. Western blot analysis using a PDE4A-selective antibody revealed three major bands (109 kDa PDE4A5, 102 kDa PDE4AX and 76 kDa PDE4A1) in hypothalamus and striatum. Infusion of 6-OHDA decreased the expression of PDE4A5 and PDE4AX but not of PDE4A1 in hypothalamus, as determined by quantitative Western blotting. Pretreatment of rats with desipramine attenuated the 6-OHDA-induced down-regulation of PDE4A5 and PDE4AX bands in hypothalamus. The PDE4B selective antibody K118 labels 5 major bands in all the brain regions studied. One hundred kDa PDE4B3, 86 kDa PDE4B2 and a 78 kDa PDE4B band was identified using recombinant proteins. Treatment of rats with 6-OHDA resulted in a 52% decrease in the PDE4B3 and 58% decrease in 78 kDa PDE4B variant in hypothalamus; administration of desipramine attenuated the 6-OHDA-induced down-regulation of both PDE4B variants. Neither 6-OHDA nor desipramine altered striatal PDE4A or PDE4B isozymes. In contrast, cerebellar PDE4B3 variant is up-regulated by 6-OHDA treatment and were partially normalized to control values by desipramine pretreatment. These data demonstrate that PDE4 subtypes are differentially regulated by presynaptic noradrenergic activity and may play an important role in the maintaining homeostasis of noradrenergic signal transduction in rat brain.

Effects of noradrenergic lesions on the development of rolipram-sensitive, low-K(m), cyclic AMP specific phosphodiesterase in rat brain.

Rolipram-sensitive, low-K(m)80% loss of norepinephrine in cerebral cortex) without affecting dopaminergic systems. The lesions resulted in temporary reduction of PDE4 activity in cerebral cortex, cerebellum and brainstem. Lesions in the adult rats, on the other hand, did not alter PDE4 activity. Decreased PDE4 activity by neonatal noradrenergic lesions was due to a decrease in the V(max) of cAMP hydrolysis by PDE4, and not a change in the K(m) values. Immunoblot analysis showed that decreased PDE4 activity in cerebellum was associated with reduced expression of PDE4A5, PDE4A1, and several PDE4B variants. No change in the expression of any PDE4 subtype in cerebral cortex was detected with the antibodies used in this study. Neither the permanent loss of noradrenergic innervation in cerebral cortex, nor the permanent noradrenergic hyperinnervation in brainstem was accompanied by any permanent change in PDE4 activity. Decreasing PDE4 activity early after neonatal noradrenergic lesions might be important in maintaining constant concentrations of cAMP, which is critical for the cellular proliferation and differentiation that is active during this period.

Ontogeny of rolipram-sensitive, low-K(m), cyclic AMP-specific phosphodiesterase in rat brain.

The postnatal development of rolipram-sensitive, low-K(m), cyclic AMP-specific phosphodiesterase (PDE4) was investigated in discrete regions of rat brain using a PDE4 activity assay and immunoblot analyses with K116, a PDE4 antibody. The Vmax for cyclic AMP hydrolysis by PDE4 was lower at birth when compared to adult levels in cerebral cortex, cerebellum, and neostriatum. K(m) values for cyclic AMP hydrolysis by PDE4, in contrast, did not change throughout the observed period in any brain region tested. The developmental patterns for PDE4 were significantly different among the examined brain regions. PDE4 activity in olfactory bulb and hippocampus also was found to be lower at birth in comparison to adult levels. Immunoblot analyses showed that developmental patterns of PDE4 were significantly different for the various subtypes, and also varied substantially across brain regions. The results suggest that PDE4 might be differentially regulated by different ontogenetic events.

Dietary Protein and Central Monoamine Concentrations in the Rat.

In a previous report we demonstrated that rats that consumed a high-protein diet (HP; 50% casein) for 36 weeks were hyperactive and hyperresponsive to nociceptive stimuli, compared to rats that consumed normal (NP; 20% casein) or low-protein (LP; 8% casein) diets. In addition, we have also previously, reported that dopamine concentrations in the nigrostriatal system of the rats were decreased and increased, respectively, with a decrease and increase in dietary protein. In the present study, rats were maintained on the HP, NP and LP diets and regional changes in the concentrations of norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) were assessed. Concentrations of 5-HT in the medial raphe, dorsal raphe, and several of their target tissues, revealed no consistent effect of manipulating dietary protein over the range of 5-HT levels measured. NE concentrations in most of the brain regions innervated by neurons of the locus coeruleus and lateral tegmentum showed no significant differences among the diet groups. However, NE concentrations in the parietal cortex were significantly increased in rats that consumed the HP diet. The present study indicates that the brain NE pathways, particularly that innervating the parietal cortex, is susceptible to dietary protein manipulation.

Noradrenergic activity differentially regulates the expression of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat brain.

In a previous study, it was observed that the activity of rolipram-sensitive, low-Km, cyclic AMP phosphodiesterase (PDE4) was decreased in vivo with diminished noradrenergic stimulation. The results of the present experiments indicated that the reduction in the activity may be associated with down-regulation of PDE4 protein. Immunoblot analysis using PDE4-specific, subfamily-nonspecific antibody (K116) revealed four major bands of PDE4 in rat cerebral cortex; those with apparent molecular masses of 109 and 102 kDa are variants of PDE4A. Diminished noradrenergic activity, produced by intracerebroventricular infusion of 6-hydroxydopamine (6-OHDA) or chronic subcutaneous infusion of propranolol, decreased the intensities of the protein bands for the 109- and 102-kDa PDE4A variants in rat cerebral cortex but not of the 98- or 91-kDa PDE4 forms. 6-OHDA-induced noradrenergic lesioning also decreased the content of 102-kDa PDE4A in hippocampus as labeled by PDE4A-specific antibody (C-PDE4A). Enhanced noradrenergic stimulation up-regulated PDE4 in cerebral cortex. This was indicated by the finding that repeated treatment with desipramine increased the intensity of the protein band for the 102-kDa PDE4 but not for the other variants of PDE4. These results suggest that PDE4 subtypes are differentially regulated at the level of expression, as evidenced by an apparent change in the amount of PDE4 protein, following changes in noradrenergic activity. These observations are consistent with the notion that PDE4s, especially the PDE4A variants with molecular masses of 109 and 102 kDa, play an important role in maintaining the homeostasis of the noradrenergic signal transduction system in the brain and may be involved in the mediation of antidepressant activity.

Degenerative changes in spermatogonia are associated with loss of glucose transporter (Glut 3) in abdominal testis of surgically induced unilateral cryptorchidism in rats.

Expression of facilitative glucose transporters (Glut 1, 2 and 3) was examined by Western blot analyses 10, 20 and 30 days following surgically induced unilateral abdominal cryptorchidism. The cryptorchid testes exhibited marked degenerative changes in the seminiferous tubules and spermatogonia, impaired and incomplete spermatogenesis and lack of spermatozoa in the lumen. Immunoblotting of testis proteins with Glut transporter antibodies revealed only the presence of Glut 2 and 3 proteins. Glut 2 expression in abdominal testis was increased (45%, 67%, and 40% at 10, 20 and 30 days, respectively) but no significant change was observed in contralateral scrotal testis. Glut 3 expression was reduced by 85-95% compared with contralateral scrotal testis. A significant decrease in Glut 3 levels in abdominal testis was accompanied by an increase in scrotal testis Glut 3 content (80%, 144% and 212% at 10, 20 and 30 days, respectively) compared to age matched control rats. These results suggested that the degenerative changes in abdominal testis may be associated with decreased Glut 3 mediated glucose transport in seminiferous tubules and spermatogonia.

Changes in monoamines and their metabolite concentrations in REM sleep-deprived rat forebrain nuclei.

Rapid eye movement sleep deprivation (REMSD) is a potent stressor in rats. Behavioral abnormalities such as passive and active avoidance, locomotor activity, problem solving, sensory information processing, and the development of adaptive copping strategy in response to repeated stress are among the earliest obvious symptoms of REMSD, the mechanism for which remain largely unknown. The aim of this study was to determine whether 96 h of REMSD causes changes in monoamine neurotransmitters concentrations in rat forebrain regions (frontal cortex, FC; parietal cortex, PC, and striatum) that are involved in mediating higher brain functions such as attentional mechanisms, sensory information processing, and locomotor activity, which are severely affected in REMSD conditions. Rats were subjected to 96 h of REMSD using inverted flower pot water tank technique. To account for the stress associated with water tanks, a tank control group (TC) was included where the animals could reside comfortably on a large pedestal in the water tank. Regional brain concentrations of norepinephrine (NE), dopamine (DA), dihydroxyphenyacetic acid (DOPAC), L-3,4-dihydroxyphenylalanine (L-DOPA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (HIAA) were determined by electrochemical detection using high-performance liquid chromatography. The concentrations of serotonin and its metabolite, HIAA, was reduced in the frontal and parietal cortexes of REMSD rats compared with TC or cage control (CC) group. NE, DA, DOPAC, and HVA concentrations in FC and PC of REMSD animals were remained unchanged compared with TC or CC rats. A significant increase in the concentrations of DA metabolites was observed in the striatum of REMSD rats when compared with CC and TC rats. There was a 29 and 31% increase in the concentration of striatal DA in REMSD group compared to the TC and CC groups, respectively; however, these percentages were not statistically different. Striatal NE, 5-HT, and HIAA concentrations were not significantly different among the three groups. These results suggest that 96 h of REMSD alters dopaminergic and serotonergic systems in different locations in rat brain. The effect of REMSD on the serotonergic systems are localized in the cerebral cortex, whereas dopaminergic metabolism is increased in the striatum.

Abnormal behaviors in the stargazer rat are maladaptive, but not anxiety related.

The stargazer rat is an autosomal recessive mutant (homozygous stg/stg) that displays abnormal behavior, including profound hyperactivity. Heterozygous stg/+ littermates are unaffected (i.e., are nonmutants), and display normal spontaneous behaviors. Abnormal spontaneous behavior in the stargazer rats suggest that they may be more responsive than their normal littermates to external stimuli and more likely to display anxiety-related behavior in tests of emotionality. To test these hypotheses, the reactive behavior of stargazers and unaffected littermates were compared with regard to their responsiveness to nociceptive stimuli (tail flick test), in the open field test, the elevated plus-maze, and in the swim test of Porsolt. In the open field test, the stargazers spent a greater percentage of the observation period in the open area (p < 0.05), and demonstrated a significantly higher level of locomotor activity (p < 0.05). In the elevated plus-maze, stargazers spent a significantly greater percentage of their total time in the open arms (p < 0.05), but the number of open-arm entries as a percentage of total entries into either arm was not different among the two groups. The stargazers were unable to complete the Porsolt test, perhaps owing to their being ineffective swimmers. No differences among the groups were observed with the tail flick test. Contrary to the original hypothesis, these data suggest that the mutant rats demonstrated less anxiety-related behavior than their nonmutants siblings in tests of emotionality.

Stress-related behavior and central norepinephrine concentrations in the REM sleep-deprived rat.

Rapid eye movement sleep deprivation (REMd) is a potent stressor in the rat. Behavioral abnormalities are among the earliest overt symptoms of REMd, the mechanisms for which remain largely unknown. The phenomena of hyperphagia and weight loss that are associated with REMd may contribute to its later morbidity; however, little is known about the onset of these phenomena or the neurotransmitter mechanisms that are involved. The aim of this study was to determine whether the earliest effects of REMd on consumatory behavior in the rat and its performance in the swimming cylinder of Porsolt are related to changes in norepinephrine (NE) concentrations in the cerebral cortex and selected areas of the hypothalamus. Sprague-Dawley rats were divided into three groups (n = 6): the REMd group resided in a water tank on 6.5-cm diameter pedestals for 96 h; the tank control (TC) group resided in the water tank on 15-cm pedestals for 96 h; the cage controls (CC) remained in their home cages for the duration of the study. In the first series of experiments, body weights and caloric intake were recorded daily, along with the performance of all animals in the swimming cylinder of Porsolt. In the second series of experiments, body weights and caloric intake were recorded, but the Porsolt test was not employed and the brains were dissected after 96 h for NE analysis by HPLC. It was observed that the REMd group had lower immobility times (p < 0.05) in the Porsolt test after only 24 h, compared to groups TC and CC.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential modulation of dopaminergic systems in the rat brain by dietary protein.

Rats that consume a diet 50% rich in protein exhibit hyperactivity and hyperresponsiveness to nociceptive stimuli, in which facilitation of dopaminergic activity has been implicated. We studied the regional changes in the concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brains of rats that were maintained on high-protein (HP, 50% casein), normal-protein (NP, 20% casein), and low-protein (LP, 8% casein) diets for 36 weeks. Brain nuclei that represented different DAergic systems were punch-dissected and analyzed using HPLC. In the substantia nigra, the striatum, and the dentate gyrus, DA concentrations decreased and increased, respectively, with a decrease and increase in dietary protein (p < 0.05 compared to the NP diet). Similar trends in the effect of the HP diet were observed in the ventral tegmental area, amygdala, frontal cortex, subiculum, centromedial nucleus (CM) of the thalamus, and inferior colliculi (IC), although the differences in DA concentrations were not statistically significant. These brain areas also showed a pattern of decreased DA concentration in association with the LP diet, and the differences were statistically significant (p < 0.05) in the CM and IC. DA concentrations in most regions of the midbrain and brainstem were not different between the diet groups, nor were consistent trends observed in those regions. Also, there were no consistent relationships between DOPAC/DA and HVA/DA ratios and dietary protein level. These data suggest that only discrete dopaminergic neuronal circuits in the rat forebrain were sensitive to changes in dietary protein level.

Induction of adenylate cyclase sensitive dopamine D2-receptors in retinoic acid induced differentiated human neuroblastoma SHSY-5Y cells.

Dopamine D2 receptor (D2-receptor) expression and its coupling to Gi sensitive adenylate cyclase was investigated in human neuroblastoma SHSY-5Y cells. Incubation of SHSY-5Y cells in the presence of 100 nM retinoic acid (RA) for 24 hours resulted in phenotypic differentiation accompanied by a 47% increase in D2-receptor mRNA and a significant increase in the specific binding of a D2-receptor antagonist, [3H]YM09151-2. Stimulation of D2-receptors in differentiated cells by LY171-555, a D2-agonist, attenuated cellular cAMP levels by 30%. The effect of LY171-555 on cAMP levels was blocked by the D2-antagonist, (-)sulpride. Application of these drugs to control undifferentiated cells or differentiated cells incubated with vehicle only had no effect on cellular cAMP levels. These studies suggest that differentiated SHSY-5Y cells express functional D2-receptors and will provide a useful model for future studies on the regulation of expression and function of D2-receptors in cellular differentiation of neuronal cells.

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.

Post-transcriptional regulation of loss of rat striatal D2 dopamine receptor during aging.

The mechanism(s) underlying age-associated diminutions in the rat striatal D2 dopamine receptor (D2-receptor) number was investigated. The levels of D2-receptor mRNA in 4-, 12- and 18-month-old rat striata were found not to change. In contrast, the levels of 110 kDa protein, labeled with a D2-receptor specific antibody, decreased in parallel with [3H]YM-09151-2 binding to striatal membranes. These data suggest a role for post-transcriptional mechanism(s) in age-associated decrease in D2-receptor.

An antibody to dopamine D2 receptor inhibits dopamine antagonist and agonist binding to dopamine D2 receptor cDNA transfected mouse fibroblast cells.

A polyclonal antibody to dopamine D2 receptor (D2-receptor) has been used to examine the immuno-inhibition in the binding of a D2 antagonist, [3H]YM09151-2 and an agonist, PPHT-fluorescein to dopamine receptor DNA transfected mouse fibroblast cells. The specific activity of the [3H]YM09151-2 binding to transfected (Ltk-RGB) cells is 4-5 fold higher than untransfected (Ltk-) cells. The antibody is able to inhibit the [3H]YM09151-2 binding to the cell membranes from Ltk-RGB cells (Bmax 110.56 +/- 5.26 and 76.20 +/- 5.18 fmoles/mg protein in the presence of preimmune and immune sera, respectively, with no change in the Kd). The flow cytometric analysis of the PPHT-fluorescein labeled Ltk- and Ltk-RGB cells indicated that ligand specific fluorescence is associated only with small Ltk-RGB cells (second peak) and autofluorescence with large cells (first peak). Preincubation of the Ltk-RGB cells with antibody, reduced the fluorescence intensity of the PPHT-fluorescein by 20-25% without changing the auto-fluorescence. These results suggest that peptide antibody recognize D2-receptor in both membranes and in intact cells and interact at or near the ligand binding site of the receptor.

Antibodies against synthetic peptides predicted from the nucleotide sequence of D2 receptor recognize native dopamine receptor protein in rat striatum.

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)

The murine haemopexin receptor. Evidence that the haemopexin-binding site resides on a 20 kDa subunit and that receptor recycling is regulated by protein kinase C.

Haemopexin receptors from mouse hepatoma (Hepa) cells were affinity-labelled by cross-linking to haem-125I-haemopexin complexes using two homo-[disuccinimidyl suberate (DSS) and 3,3'-dithiobis(succinimidyl propionate) (DTSSP)] and one hetero-[sulphosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulpho-SMPB)] bifunctional cross-linking agents. Analysis of the cross-linked products by SDS/PAGE in the absence of reducing agents revealed that 125I-haemopexin was cross-linked specifically to a protein of apparent molecular mass 85-90 kDa. Upon reduction, haemopexin remained cross-linked to a protein of 20 kDa, suggesting that the murine haemopexin receptor has a subunit structure. Two subunits were identified: alpha (p65) and beta (p20). Furthermore, because haemopexin was cross-linked by all three agents to p20, the shortest cross-linker arm being 1.1 nm (11 A), we propose that the haem-haemopexin-binding site resides on this subunit. In addition, a cysteine residue of p20 is located near the haemopexin-binding site, since haemopexin, which has no free thiol groups, is cross-linked to this subunit by the hetero-bifunctional agent sulpho-SMPB. Exposure of Hepa cells to the tumour-promoting phorbol ester 4 alpha-phorbol 12-myristate 13-acetate (PMA) causes a rapid redistribution of haemopexin receptors from the cell surface to the cell interior. Within 2-4 min of incubation with 100 nM-PMA, there was an approx. 50% decrease in cell-surface haemopexin receptors, as judged by ligand binding at 0 degrees C and affinity labelling of the receptor. This time- and dose-dependent down-regulation was fully reversible within 60-90 min after removal of PMA, and the affinity of the remaining receptors was unaltered by PMA. The specificity of PMA was demonstrated by comparison with the non-tumour-promoter 4 alpha-phorbol, which did not affect any of the parameters examined. The amine H-7, a specific inhibitor of protein kinase C, antagonised the receptor redistribution effect of PMA, suggesting that the down-regulation of haemopexin receptors on the cell surface was a consequence of protein kinase C activation. The PMA-induced decrease in surface haemopexin receptors was due to a 2-fold increase in the rate of internalization (from 0.73 min-1 to 1.32 min-1), whereas the rate of exocytosis (0.6 min-1) was unchanged. PMA treatment, like binding of the natural ligand, haem-haemopexin, results in a lower steady-state level of surface haemopexin receptors independent of receptor synthesis, and the receptors were not degraded but were recycled back to the cell surface.

Effect of cell shape, membrane deformability and phospholipid organization on phosphate-calcium-induced fusion of erythrocytes.

Fusion of bovine and goat erythrocytes was studied using the phosphate-calcium protocol. Both bovine and goat red cells are resistant to fusion with phosphate and calcium, under conditions that promote fusion of normal human erythrocytes. Fusion resistance is not related to decreased (5%) membrane deformability of erythrocytes of these species, since chicken erythrocytes which are 40% less deformable than human erythrocytes undergo fusion with efficiency similar to human red blood cells. Incorporation of either phosphatidylcholine or phosphatidylserine into bovine erythrocytes mediated by lipid exchange/transfer protein, caused fusion of these erythrocytes. Fluorescence analysis of merocyanine 540 dye labeled erythrocytes, by flow cytometry, showed that the frequency of cells which exhibit dye binding was much less (35%) in dimyristoylphosphatidylcholine (DMPC) incorporated compared to untreated bovine erythrocytes (80%), indicating that incorporation of DMPC caused closed packing of lipids in the external leaflet of the bilayer. These studies show that fusion of bovine erythrocytes, mediated by phosphate and calcium, has a requirement for either specific phospholipids such as phosphatidylcholine, phosphatidylserine, or closed packing of lipids in the external leaflet of the bilayer.

Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes.

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12-16-fold for angiotensin-converting enzyme activity and 8-10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na+ + K+)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC6 and XVD2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD2 reacted with an antigen, indicating that the antibody XIVC6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets.(ABSTRACT TRUNCATED AT 400 WORDS)