Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannicacid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane. Our results provide the first ultrastructural demonstration of exocytosis in bovine chromaffin cells with the aid of transmission electron microscopy. It is also clear that exocytosis is the mechanism of release in both intact and permeabilized cells.     

A study of the acid phosphatase of Treponema denticola

This study describes some of the properties of the acid phosphatase of the potential periodontopathogen Treponema denticola. The highest enzyme activity was found in 87 h old cells. Two optimum pHs for enzyme activity were detected, one at pH 4.8 and another at pH 6.2. Divalent cations did not influence the acid phosphatase of T. denticola. The anion F- added in the form of NaF and at a level greater than 20 micrograms/ml F- diminished the activity of the acid phosphatase of intact cells of T. denticola. The addition of 10 micrograms/ml F- as SnF2 induced a statistically significant reduction of acid phosphatase activity. The apparent Km for the acid phosphatase was 7.3 mM with p-nitrophenyl phosphate as substrate. Fluoride appeared to be a noncompetitive inhibitor of the enzyme with an apparent Ki of 0.3 mM. Acid phosphatase may be released partially in osmotic shock fluids. Also, 7-diazonium-1, 3-naphthalene disulfonate, which is incapable of penetrating the bacterial permeability barrier and is known to inactivate enzymes found in the bacterial periplasmic place, suppressed the activity of the acid phosphatase in intact cells of T. denticola.     

Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes.

The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from gamma-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Na+ + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions of p-nitrophenyl phosphate and beta-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a Km for ATP of 0.6 mM. There was an absolute requirement for Mg2+, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least l0 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution of gamma[32P]ATP and 32P2 counts became cell associated. Since the experimental evidence supports the observation that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrnae enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.     

A membrane-associated neuraminidase in Entamoeba histolytica trophozoites.

Trophozoites of the parasitic amoeba Entamoeba histolytica HM-1:IMSS possess a surface neuraminidase capable of liberating N-acetylneuraminic acid (NANA) from N-acetylneuramin-lactose (alpha 2----3 or alpha 2----6) or mucin in their medium. The neuraminidase was found to be membrane associated, with more than 50% of the yield being recovered in the plasma membrane fraction. The neuraminidase specific activity of the plasma membrane fraction was six times that of internal membrane fraction enzyme. The optimum pH and temperature for this enzyme were 6.7 and 37 degrees C, respectively. Neuraminidase activity was inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and the optimum Ca2+ concentration was 2 mM. The microfilament disruptor cytochalasin D (30 micrograms/ml) inhibited motility and neuraminidase activity of intact Entamoeba trophozoites. The cytochalasin D-induced loss of surface neuraminidase activity was explained in part by a redistribution of enzyme with a loss of plasma membrane enzyme and an increase in intracellular membrane enzyme. A qualitatively similar cytochalasin D effect was observed with two other membrane-associated enzymes, calcium-regulated ATPase and acid phosphatase. Membrane-associated enzyme was minimally affected by Triton X-100 and saponin. An N-acetylneuraminic acid aldolase, optimum pH, 7.4, was found in trophozoite homogenate supernatant fractions. NANA and NANA-containing compounds stimulated trophozoite-directed motility. This motility stimulation by NANA-containing compounds did not apparently require prior release of free NANA by the trophozoite surface neuraminidase. Entamoeba neuraminidase is one of a series of enzymes that may modify the mucus blanket and target cell surface and thereby play a role in the pathogenesis of amebiasis.     

Characterization of an ecto-phosphatase activity in the human parasite Trichomonas vaginalis

We have characterized phosphatase activity present on the external surface of Trichomonas vaginalis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at a rate of 134.3+/-14.8 nmol Pi/h per 10(7) cells. This phosphatase activity decreased by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained for at least 1 h. Experiments using classical inhibitors of acid phosphatases, such as ammonium molybdate and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate, [monoperoxo(picolinato)oxovanadate(V)] (mpV-PIC) and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), showed a decrease in this phosphatase activity, with different patterns of inhibition. Cytochemical analysis showed the localization of this enzyme on the parasite surface (cell body and flagellum) and in intracellular vacuoles. Phosphatase reaction products were also observed in exocytosed membrane-bound material.     

Structure and cytochemistry of the acinar cell in the rat maxillary gland

The ultrastructural and cytochemical properties of the maxillary gland were investigated in 24 adult Sprague-Dawley rats of both sexes. The relationship between the interstitial cell and/or excitatory cholinergic terminals with the secretory cell was discussed. The secretory material containing primarily neutral lipids, proteins, carbohydrates and mucosubstances appeared electron-opaque following staining with uranyl acetate and lead citrate. Ruthenium red precipitated strongly on zymogenic granules which showed an increasing affinity for phosphotungstic acid, paralleling a rise in pH towards 7.7. The process of fusion of the plasma membrane with the membrane of secretory granules was observed, although some membrane-bound granules were identified in the acinar and ductal lumina. Intracellular membranous structures were best revealed with phosphotungstic acid at pH 1.0–1.5, whereas ruthenium red reacted primarily with lipid inclusions and tonofilaments. Acid phosphatase activity was predominantly limited to secondary lysosomes and, to a lesser degree, to primary lysosomes and Golgi saccules. The morphological and chemical properties of secondary lysosomes in the secretory cell suggested a pronounced hydrolytic activity related to lipid inclusions and endocytotic vesicles.     

Giant cell tumor of bone

Summary The fine structural localization of acid phosphatase in the different cells in a benign giant cell tumor of bone has been studied. Stromal cells type 1 and 2 (fibroblast-like and macrophage-like, respectively) showed the presence of lead phosphate precipitate following incubation in a Gomori-type lead salt medium only in conventional lysosomes. In the multinucleated giant cells, the final product was deposited over lysosome-like organelles, and also over Golgi cisternae, vesicles, and vacuoles. Furthermore, evidence for presence of acid phosphatase was obtained in smooth-surfaced tubular, sausage-, horse-shoe-, and ring-shaped structures and over digestive vacuoles of autophagic or heterophagic origin. Finally, in these cells, many of the tubular and vacuolar elements located subjacent to areas of the plasma membrane with microvillous specializations (abortive brush borders?) were shown to carry acid phosphatase.     

Potential difference and the distribution of ions across the human red blood cell membrane; a study of the mechanism by which the fluorescent cation, diS-C3-(5) reports membrane potential.

1. The mechanism by which the fluorescent, cationic dye diS-C3-(5) responds to the membrane potential of red blood cells has been investigated. 2. The dye in aqueous solution absorbs most strongly at 650 nm. Addition of white, haemoglobin-free membranes red shifts the absorption maximum ca. 20 nm, while addition of membrane-free cell lysate results in the appearance of a new dye absorption peak at 590 nm. Thus the dye binds both to cell membranes and to cell contents. The component of the cytoplasm which binds the dye is non-dialysable, presumably haemoglobin. 3. Dye added to a suspension of intact cells shows a strong absorption at 590 nm indicating that the dye has bound to the cell contents and that the membrane is permeable to the dye. 4. The amount of dye which partitions into (and on to) the cells can be determined, as reported by Sims, Waggoner, Wang & Hoffman (1974), from the fluorescence of the dye remaining in the supernatant after the cells are centrifuged to the bottom of the suspension. In most conditions the proportion of the cell associated dye which is either free inside the cell or bound to the outside face of the membrane is negligible compared to the proportion bound to the cell contents. 5. On the assumption that the dye is not actively transported, the ratio of the equilibrium dye activities inside and outside the cell, ai/ao, is determined by the membrane potential according to the Nernst relation. Driving the membrane potenial negative then increases the cell associated dye by increasing the activity ratio and hence ai and the amount of dye bound to cell contents. 6. At the known Donnan equilibrium potential the internal dye activity can be calculated from the external activity. An empirical relation between cell associated dye and internal activity has been determined by measuring the dye partition between cells and medium at different external activities. 7. Using this empirial relation, and providing that any changes in cell composition do not affect the dye binding, the internal activity at any potential can be calculated from the measured amount of cell associated dye. The external activity can be estimated fluorimetrically. The membrane potential is then calculated from the activity ratio. 8. The membrane potenial of cells has been altered by adding valinomycin in the presence of different K gradients. Under the conditions used, the 'constant field' permeability for K-Val is 15-20 times that of Cl. 9. Dye binding to haemoglobin is influenced by pH and thus dye partitioning into cells changes with intracellular pH. Increasing intracellular pH increases the amount of dye partitioned, while decreasing pH decreases this amount. 10. When large potentials are produced with valinomycin there is no change in intracellular pH. This result indicates that in red blood cells intracellular pH is determined by the external pH and the Cl concentration ratio and not by the membrane potentials. 11. DiS-C3-(5) can be used to estimate potentials across resealed ghost membranes...     

Regulation of matrix vesicle metabolism by vitamin D metabolites

Matrix vesicles are membrane organelles found in the extracellular matrix of calcifying cells. Vitamin D-responsive alkaline phosphatase specific activity has been localized to matrix vesicles in chondrocyte and osteoblast cultures. The effect of hormone is both metabolite and cell specific. Alkaline phosphatase in matrix vesicles produced by resting zone chondrocytes is stimulated by 24,25(OH)2D3 whereas alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is responsive to 1,25(OH)2D3. However, mesenchymal cell cultures, which exhibit a chondrogenic phenotype when exposed to bone inductive proteins in vitro, produce vesicles with alkaline phosphatase activity that is unaffected by either 1,25(OH)2D3 or 24,25(OH)2D3. Incorporation and release of arachidonic acid into phosphatidylethanolamine is also differentially regulated by 1,25(OH)2D3 and 24,25(OH)2D3 in chondrocytes. These data suggest that vitamin D metabolites may regulate endochondral ossification by altering matrix vesicle enzyme activities, perhaps through changes in membrane phospholipid metabolism.     

Regulation of Ca2+ channel and phosphatase activities by polyamines in intestinal and vascular smooth muscle--implications for cellular growth and contractility

Polyamines added extracellularly to intestinal and vascular smooth muscle cells cause relaxation through inhibition of Ca2+ channel activity. Intracellularly applied polyamines also affect Ca2+ channel properties. Polyamines do not readily pass over the plasma membrane because of their positive charges but in permeabilized smooth muscle preparations they have free access to the cytoplasm. In this system they increase sensitivity of the contractile machinery to Ca2+ through inhibition of myosin phosphatase activity. The magnitude of Ca2+ channel and phosphatase inhibition depends on the number of positive charges on the polyamine molecule. Polyamines have an obligatory, but yet undefined, role in regulation of cell growth and proliferation. Several groups of protein kinases, such as tyrosine and mitogen activated protein (MAP)-kinases transmit the growth signal from the plasma membrane to the cell nucleus where mitosis and protein synthesis are initiated. The data reviewed here show that polyamines may affect such signal transmission via inhibition of phosphatase activity.     

Alterations in nonspecific cross-reacting antigen localization during cell culture. An immunoelectron microscopic study using a human lung adenocarcinoma cell line

Nonspecific cross-reacting antigen (NCA), a constituent of the carcinoembryonic antigen family, was localized ultrastructurally in a human lung adenocarcinoma cell line, PC-9. NCA was distributed predominantly on the plasma membrane in the early phases of cell culture. Deletion of fetal bovine serum (FBS) from the culture medium suppressed cell division without significantly altering cell viability, and induced a dramatic but reversible change in NCA localization. Under these conditions, NCA was localized to membrane degradation products within cytoplasmic vesicles and vacuoles. Acid phosphatase activity was also present in some of these intracellular structures. Similar changes in NCA localization were seen in cells cultured with FBS at day 6 when the cells reached a plateau stage of growth. These findings strongly suggest that plasma membrane degradation is accelerated by the cessation of cell growth. Cytoplasmic reactivity for NCA in cancer cells may therefore reflect degradation of plasma membrane-associated NCA and may not necessarily be correlated with increased systhesis of this glycoprotein.     

Human peripheral blood dendritic cells concentrate in contrast to monocytes intracellular HLA class II molecules in a juxtanuclear position

The localization of histocompatibility antigens of the HLA-D locus in dendritic cells (DC) and monocytes (Mo) isolated from human peripheral blood was investigated. Functionally DC were characterized by their capacity to act as strong stimulators in an allogeneic mixed leucocyte reaction. In cytospins, DC were differentiated from Mo by dendritic morphology, strong HLA class II and moderate RFD1 expression on the plasma membrane and acid phosphatase activity in a juxtanuclear spot. Ultrathin cryosections showed that DC had a heavily labelled plasma membrane for HLA-D. In addition, these antigens were found in intracellular vesicles predominantly located in the juxtanuclear zone. This pattern of labelling was not seen in Mo. Obviously, DC concentrate intracellular class II antigens in the same area as lysosomal activity. These results may indicate that this juxtanuclear area is a center of antigen processing in DC.     

Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga

Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion system.     

Resistance of leishmanial phosphatases to inactivation by oxygen metabolites.

Leishmania donovani promastigotes produce large quantities of two distinct acid phosphatases; a tartrate-resistant enzyme is localized to the external surface of the plasma membrane, and a tartrate-sensitive enzyme is secreted into the growth medium. It was shown previously that preincubation of human neutrophils and macrophages with the tartrate-resistant phosphatase markedly reduced the ability of these host cells to produce superoxide anions in response to stimulation with the activator formyl-methionyl-leucyl-phenylalanine. The possibility that the cell surface acid phosphatase or the phosphatase that is secreted into the extracellular fluid might compromise other host cell functions, especially intracellular ones, depends on the ability of the enzyme to resist exposure to toxic oxygen metabolites (e.g., superoxide anion, hydrogen peroxide, hypochlorite) generated by phagocytic cells. In the present report, we show that both leishmanial acid phosphatases were relatively resistant to inactivation by oxygen metabolites. At pH 5.5, the activity of the tartrate-resistant phosphatase was reduced 50% by incubation for 1 h with each of the following: 30 mM O2-, 500 mM hydrogen peroxide, and 6 mM hypochlorite ion. These concentrations are many fold greater than the concentrations of these substances that are generated by stimulated polymorphonuclear phagocytes. The tartrate-sensitive acid phosphatase differed markedly from the tartrate-resistant phosphatase in that the former was essentially insensitive to even very high concentrations of superoxide anion and hydrogen peroxide. Furthermore, 50% inactivation of the tartrate-sensitive leishmanial phosphatase required exposure to 35 mM hypochlorite for 30 min. These results indicate that the catalytic potential of these two leishmanial acid phosphatases probably survives exposure to toxic oxygen metabolites generated by neutrophils and macrophages.     

Alterations in Nonspecific Cross-reacting Antigen Localization during Cell Culture

Nonspecific cross reacting antigen (NCA), a constituent of the carcinoembryonic antigen family, was localized ultra-structurally in a human lung adenocarcinoma cell line, PC 9. NCA was distributed predominantly on the plasma membrane in the early phases of cell culture. Deletion of fetal bovine serum (FBS) from the culture medium suppressed cell division without significantly altering cell viability, and induced a dramatic but reversible change in NCA localization. Under these conditions, NCA was localized to membrane degradation products within cytoplasmic vesicles and vacuoles. Acid phosphatase activity was also present in some of these intracellular structures. Similar changes in NCA localization were seen in cells cultured with FBS at day 6 when the cells reached a plateau stage of growth. These findings strongly suggest that plasma membrane degradation is accelerated by the cessation of cell growth. Cytoplasmic reactivity for NCA in cancer cells may therefore reflect degradation of plasma membrane-associated NCA and may not necessarily be correlated with increased systhesis of this glycoprotein. Acta Pathol Jpn 39: 772 778, 1989.     

Nondiffusional release of allergens from pollen grains of Artemisia vulgaris and Lilium longiflorum depends mainly on the type of the allergen

BACKGROUND: Upon contact with a wet surface, mature pollen grains hydrate and release proteins including allergens. Knowledge of the release mechanism of allergens that are mainly localized intracellularly may allow the design of strategies for inhibition of allergen release and the consequent sensitization process. METHODS: An improved pollen chromatography was performed with Artemisia vulgaris and Lilium longiflorum pollen. Using three elution media of different pH, osmolality and salt concentration mimicking various types of wet surfaces, the time-dependent elution profiles of total protein, a cell wall-bound acid phosphatase activity (acPase), allergenic (profilin, Art v 1) and nonallergenic molecules (14-3-3 protein, actin) were monitored. RESULTS: The release kinetics of total protein and cell wall-bound acPase followed an exponential decrease in both pollen species indicating a diffusion-based protein release, whereas the elution profiles of profilin, Art v 1 and 14-3-3 protein showed nondiffusion characteristics. No general dependence on pH, osmolality or salt concentration of the elution media was observable in the elution profiles. Under the applied conditions, actin was not released indicating that the pollen grains remained intact during the elution. CONCLUSION: The elution profiles of pollen allergens indicated that substantial amounts of these proteins do not diffuse from the cell wall or are released from intracellular compartments during imbibitional leakage. Instead, a mechanism seems to operate that involves translocation from the pollen cytoplasm to the extracellular environment by crossing an intact plasma membrane. Such a mechanism would probably allow the use of pharmaceuticals for inhibition of allergen release.     

Ultrastructural localization of alkaline phosphatase activity in the developing thyroid gland of the rat

The localization of alkaline phosphatase activity during morphogenesis of the thyroid gland was studied at the fine structural level in Holtzman rats from the fifteenth day of foetal life till the first day after birth. The present work deals with the formation of thyroid follicles in the median thyroid primordium only. Since the rat thyroid does not develop synchronously, the three stages described may overlap during a given day of development. In the precolloid stage that extends roughly from the fifteenth to the nineteenth day of development, alkaline phosphatase activity is localized in Golgi saccules and vesicles and also in smooth membrane tubules and vesicles found in the cytoplasm near the lateral plasma membranes. At the end of this stage the lateral plasma membranes become strongly reactive and a cluster of positive vesicles and tubules appears immediately under the junctional zone. The second phase, the early colloid stage (17–19 days), is characterized by the formation of the colloid cavity in center of the disc-like junctional zone: at this moment the newly formed apical plasma membrane bearing the microvilli shows the reaction product. During the third stage (18 days onwards), which starts with a gradual increase in the diameter of follicular lumina, a drastic fall in alkaline phosphatase activity is observed. In one day old rats, follicular cells are completely negative. These findings are briefly discussed in connection with thyroid cell differentiation.     

Characterization of a stable spheroplast type L-form of Proteus mirabilis D 52 as cell envelope mutant II. Electronmicroscopic investigations

The ultrastructural analysis of a stable spheroplast type L-form of Proteus mirabilis D 52 revealed characteristic alterations in the organization of the cell envelope including defective changes in the cell envelope structure as for instance the loss of a coherent murein layer, the loss of some components in the outer cell wall layer, the formation of small membraneous vesicles at the tips of loose extensions of the cell wall, a decrease in associations and bindings between wall and membrane, an extension of the periplasmic space, an increase in membrane defects, as well as a disturbed cell division causing unusual modes of multiplication and the formation of various intracellular structures like membrane complexes, characteristic sheet-like membraneous bodies, typical inclusion bodies, and defective phage structures, which all could not be observed in normal rod-shaped cells. The results of these investigations and of those given in a previous paper (GUMPERT and TAUBENECK 1975) show, that the stable spheroplast type L-form LD 52 B must be considered as a true cell envelope mutant in which the biosynthesis and structure of the cell envelope is altered genetically by one or several mutations whereas the main biochemical activities are the same like those of the parent bacterium. The profound alterations in the cell envelope system, however, lead to some changes in the whole cell organization, which apparently in turn cause disorders even in metabolic and biosynthetic processes not directly involved in the biosynthesis of the cell envelope.     

Regulation of Ca2+ channel and phosphatase activities by polyamines in intestinal and vascular smooth muscle – implications for cellular growth and contractility

Polyamines added extracellularly to intestinal and vascular smooth muscle cells cause relaxation through inhibition of Ca²⁺ channel activity. Intracellularly applied polyamines also affect Ca²⁺ channel properties. Polyamines do not readily pass over the plasma membrane because of their positive charges but in permeabilized smooth muscle preparations they have free access to the cytoplasm. In this system they increase sensitivity of the contractile machinery to Ca²⁺ through inhibition of myosin phosphatase activity. The magnitude of Ca²⁺ channel and phosphatase inhibition depends on the number of positive charges on the polyamine molecule. Polyamines have an obligatory, but yet undefined, role in regulation of cell growth and proliferation. Several groups of protein kinases, such as tyrosine and mitogen activated protein (MAP)‐kinases transmit the growth signal from the plasma membrane to the cell nucleus where mitosis and protein synthesis are initiated. The data reviewed here show that polyamines may affect such signal transmission via inhibition of phosphatase activity.