Cell transplantation as future therapy for cardiovascular disease?: A workshop of the National Heart, Lung, and Blood Institute.

Despite the development of improved therapies and the significant advances in the understanding of the basis of disease pathogenesis, millions of Americans continue to live with life-threatening cardiovascular diseases. Recent breakthroughs suggest exciting directions that are likely to produce more effective therapies for the treatment of cardiovascular disease. One such area, cell transplantation (grafting of healthy cells into the diseased heart), holds enormous potential as an approach to cardiovascular pathophysiology. Once thought to be a scientific long shot, cell transplantation is becoming recognized as a viable strategy to strengthen weak hearts and limit infarct growth. The technology could also be used for the long-term delivery of beneficial recombinant proteins to the heart, which is a strategy to complement molecular biology advances and provide an alternative strategy for gene therapy. On August 24, 1998, the National Heart, Lung, and Blood Institute convened a workshop to discuss the current status of this fast-moving line of research and to explore its promise for treating cardiovascular disease. The participants included basic and clinical researchers, with representatives from academic and commercial research settings. The workshop was designed to establish the state-of-the-art and to equate current research with practical clinical application. The group recommended short- and long-term goals to assist in realizing, in the most expedient manner, the potential utility of cell transplantation for the treatment of cardiovascular disease. A summary of the meeting discussions and recommendations for future areas of research is presented.

Report of the National Heart, Lung, and Blood Institute Special Emphasis Panel on Heart Failure Research.

The SEP identified priorities to support in future basic and clinical research and pointed out directions likely to result in advances against heart failure. The list is not intended to be all-encompassing and does not address, for example, exciting lines of work already under way. Rather, the recommendations are designed to point out gaps in current knowledge not being adequately addressed and highly promising new directions. Although the incidence of heart failure continues to grow, emerging lines of research provide hope that research advances will eventually lead to more effective treatment and ultimately to prevention. This research will be well served by bringing the latest multidisciplinary approaches and the best investigators to focus on the problems of heart failure. It is hoped the efforts of distinguished expert entities such as the task force and SEP will be a useful guide in addressing the needs of the biomedical community and assisting in its success.

Strychnine-binding proteins in intestinal cells: novel brucine binding site with binding affinities for alkaloids.

The purpose of this work was to define the pharmacology of an intestinal epithelial [3H]strychnine binding site. Strychnine, brucine, verapamil and desmethoxyverapamil bind to small intestinal mucosal homogenates with nanomolar affinity at a site not related to the strychnine receptor, which is in the spinal cord. The antidiarrheal agents, fluperamide and loperamide, and several alkaloids have an order of magnitude lower affinity. Agents that bind to cytochrome P450IID6 also displace [3H]strychnine binding, which implies that the binding site may have some properties similar to the catalytic site of this cytochrome P450 enzyme.

Monitoring of intracellular free calcium in perfused rat liver.

Fluorescent calcium indicators have been widely used to assess cytoplasmic calcium concentration in cells. To examine the role of calcium ions on different physiological functions (e.g. in case of liver; bile secretion, glucose metabolism, etc.) there is a need for whole organ studies. We have developed a technique to estimate intracellular free calcium changes in perfused rat liver. Krebs-Henseleit perfused livers were loaded with 7 microM or 35 microM Indo-1/AM. An area 3 mm in diameter and approximately 300 microns in depth was illuminated at 340 nm. Fluorescence was monitored with photomultiplier tubes at 3 wavelengths (400 nm for Ca-bound dye, 504 nm for free dye and 464 nm for NADH). The viability of liver preparations was assessed by measurement of the concentrations of lactate dehydrogenase and alanine aminotransferase in the effluent. Loading of the livers with 7 microM Indo-1/AM via the portal vein resulted in a 5-fold increase of fluorescence at 400 nm. However the dye 'leaked' out of the liver with a half-time of 18 min. Probenecid (a specific anion carrier blocker) inhibited loss of dye in a dose dependent fashion (2.5-10 mM). Transient calcium elevations were observed in response to vasopressin (5-50 nM) at physiological levels, ethanol (0.3-0.8 M) and the calcium ionophore, ionomycin. Certain limitations were apparent with this approach: (1) it was necessary to use an anion carrier blocker to maintain a relatively steady dye concentration; (2) endogenous NADH fluorescence interfered with the calcium signal; and (3) absolute values of calcium concentration could not be determined.

Chronic exposure of cerebellar granule cells to ethanol results in increased N-methyl-D-aspartate receptor function.

In primary cultures of cerebellar granule cells, activation of the N-methyl-D-aspartate (NMDA) receptor leads to Ca2+ influx. Previous work showed that this response is selectively inhibited by acute exposure to low concentrations of ethanol. The present results demonstrate that the response to NMDA (measured as an increase in intracellular Ca2+ concentration, using fura-2 fluorescence) is significantly enhanced after chronic in vitro exposure of the cells to ethanol (100 mM for 2-4 days; 20 mM for 3 or more days). This enhancement is consistent with an increased number of NMDA receptors, with no change in receptor properties. Specifically, there was no change in the EC50 values for NMDA and glycine or in the magnitude of inhibition of the NMDA response by competitive or uncompetitive antagonists. There was also no change in the ability of acute ethanol to inhibit the NMDA response after chronic exposure of the cells to ethanol. Furthermore, chronic ethanol exposure did not alter depolarization-dependent increases in intracellular Ca2+ observed after exposure of the cells to 30 mM KCl. The data suggest that chronic ethanol exposure produces a selective up-regulation of NMDA receptor function. In the intact animal, such a change may be associated with particular symptoms of ethanol withdrawal, i.e., withdrawal seizures.

Abnormal secretagogue-induced intracellular free Ca2+ regulation in cystic fibrosis nasal epithelial cells.

These studies identify a further abnormality in cystic fibrosis (CF). The increase in intracellular free calcium concentration ([Ca2+]i) after exposure to histamine and PGE1 is demonstrated to be abnormally low in nasal cells, studied in short-term culture, from patients with CF compared with control subjects. [Ca2+]i is measured by using the Ca(2+)-sensitive fluorescent dye fura-2 and a fluorescence microscope imaging system. The percentage of CF cells that increase [Ca2+]i in response to histamine is decreased compared with controls, and, even in those CF cells that increase [Ca2+]i, the magnitude of the increase in [Ca2+]i in response to histamine is smaller than in controls. When exposed to PGE1, a similar number of control and CF cells responded with an increase in [Ca2+]i, but again the magnitude of the response was smaller in the CF cells. The mechanism of the PGE1-induced increase in [Ca2+]i is not mediated by cAMP, since 8-bromo-cAMP failed to increase [Ca2+]i in these cells. This abnormality in [Ca2+]i response did not apply to all secretagogues, with the response to carbachol being similar in CF and normal cells. How the abnormal CF gene product accounts for the abnormality in intracellular Ca2+ response to some but not all secretagogues is unknown.

Anti-class II MHC antibody induces multinucleated giant cell formation from peripheral blood monocytes.

Multinucleated giant cells (MGCs) are an integral part of the host immune response to infectious disease and are seen in granulomas induced by pathogens and inorganic substances. We have developed a novel system for the production and study of MGCs: Peripheral blood monocytes, when cultured in the presence of anti-class II major histocompatibility complex monoclonal antibodies (MHC mAb's) and lymphocyte-conditioned medium form MGCs within 48 h. MGC formation was strictly dependent on the presence of anti-class II MHC mAb's and lymphocyte-conditioned medium. MGC formation was not induced by mAb's to other monocyte surface proteins. None of the previously identified macrophage fusion factors (calcitriol, interleukin 4, interferon-gamma) were able to substitute for the lymphocyte-conditioned medium in our assay; however, the conditioned medium could be replaced by the phorbol ester phorbol 12-myristate 13-acetate. We have also demonstrated that the induction of MGCs by anti-class II MHC antibody and phorbol ester requires protein kinase C activity, because MGC formation was totally inhibited by the protein kinase C inhibitors staurosporine and H-7. In analyzing the signal induced by anti-class II MHC mAb's we have demonstrated that cross-linking of the class II MHC antigens with intact mAb's, or with F(ab')2 fragments of anti-class II MHC mAb's and F(ab')2 fragments of rabbit antimouse (RAM) immunoglobulin G, produced an intracellular calcium rise. Furthermore, using the calcium channel blocker verapamil, it was demonstrated that calcium channel activity is necessary for MGC formation. These data support the view that MGC formation is a tightly regulated differentiative pathway of peripheral blood monocytes that is dependent on protein kinase C second messenger systems and involves an increase in intracellular calcium concentration.

Uncoupling of phospholipase C from receptor regulation of [Ca2+]i in T84 colonic cells by prolonged exposure to phorbol dibutyrate.

The T84 colonic cell line, a cultured Cl- secretory cell, elevates intracellular free Ca2+ [( Ca2+]i) in a concentration-dependent manner when exposed to carbachol or histamine. As determined with a fluorescence microscope imaging system, exposure of T84 cells to 100 microM carbachol or histamine resulted in an immediate [Ca2+]i rise of approximately 50-80 nM in all cells. Preincubation of monolayers for 1 h or longer with 0.4 microM phorbol 12,13-dibutyrate (PDB) reduced the number of cells which responded to histamine or carbachol and reduced the magnitude of the increase in the responding cells. This effect reached its maximum after 2 h and persisted for at least 24 h of PDB incubation. Binding of quinuclidinyl benzilate, a cholinergic receptor antagonist, indicated that down-regulation of external receptors was not an explanation for this effect. Examination of phospholipase C activity in T84 cell membranes showed increased basal activity in PDB-treated compared with control cells. Measurement of inositol phosphates generated by intact cells using myo-[3H]inositol incorporation or receptor binding assays showed that 2 h of incubation with PDB elevated basal levels of inositol 1,4,5-trisphosphate and prevented any further carbachol-induced generation of inositol trisphosphate. Probably as a consequence, both total cell calcium and Ca2+ ionophore-releasable calcium were decreased after 2 h of PDB incubation. Membrane-associated protein kinase C activity was elevated after a 2 h exposure to PDB but was below the level of detection after 24 h with PDB. Protein kinase C antagonists neither duplicated nor blocked the uncoupling of carbachol receptors induced by long term treatment with PDB. The results suggest that prolonged PDB incubation caused uncoupling and elevation of phospholipase C activity from cholinergic and histaminergic receptor regulation resulting in increased basal levels of inositol 1,4,5-trisphosphate. Protein kinase C apparently is not involved directly in the mechanism that leads to these effects.

Hyperthermia effects on cytosolic [Ca2+]: analysis at the single cell level by digitized imaging microscopy and cell survival.

Digitized video-intensified fluorescence microscopy with the Ca2(+)-sensitive fluorescent dye fura-2 was used to measure cytosolic free Ca2+ [( Ca2+]f) in HT-29 human colon cancer cells. At 37 degrees C, the [Ca2+]f of individual cells ranged between 50 and 150 nM, with a mean of 120 nM. Raising the temperature to 41 degrees C for 1 h resulted in a slight reversible decrease (10-20%) in the mean [Ca2+]f. At 44 degrees C for 1 h, most (greater than 80%) cells exhibited a [Ca2+]f greater than 200 nM. This heat-induced rise in [Ca2+]f was not immediate but commenced after a lag time of 30 min. Postincubation at 37 degrees C for 2-6 h after heating, for 1 h at 44 degrees C resulted in a recovery of the basal [Ca2+]f in some but not all cells. A linear relationship was determined between percentage of cell killing and the number of cells with [Ca2+]f of greater than 200 nM after 37 degrees C post-heating incubation. Manipulation of extracellular [Ca2+] between 0.1 and 10 mM during heating did not modify the heat-induced changes in [Ca2+]f. No significant differences in survival at 37 degrees C or 44 degrees C were observed with cells incubated at 10, 1.0, and 0 [plus 1.0 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] mM extracellular Ca2+. The Ca2+ channel blockers verapamil and nifedipine did not protect cells from heat treatment. These results suggest that irreversible heat-induced changes in intracellular Ca2+ homeostasis mechanisms may be a critical factor in heat cytotoxicity.

Ethanol-induced increases in [Ca2+]i and inositol (1,4,5) triphosphate in rat hepatocytes.

Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.

Rabbit ileal villus cell brush border Na+/H+ exchange is regulated by Ca2+/calmodulin-dependent protein kinase II, a brush border membrane protein.

Ileal brush border membranes contain an endogenous Ca2+/calmodulin (CaM)-dependent protein kinase activity that modulates the activity of the apical membrane Na+/H+ exchanger. To further characterize this kinase, synapsin I, a substrate for Ca2+/CaM-dependent protein kinases, was added to preparations of ileal brush border membranes. In the presence of Ca2+/CaM, synapsin I was phosphorylated. Phosphopeptide mapping demonstrated that the addition of Ca2+/CaM to brush border membranes stimulated the phosphorylation of sites in synapsin I specific for Ca2+/CaM-dependent protein kinase II. Immunoblots containing brush border and microvillus membrane proteins were probed with an antibody that recognizes the 50-kDa subunit of rat brain Ca2+/CaM-dependent protein kinase II. This antibody labeled major and minor species of 50 and 53 kDa, respectively, with more labeling of the brush border than the microvillus membranes. Right-side-out ileal villus cell brush border vesicles were prepared containing CaM, ATP, and 350 nM free Ca2+. Na+/H+ exchange was inhibited by the presence of Ca2+/CaM/ATP within the vesicles. A 21-amino acid peptide inhibitor of CaM kinase II was enclosed within some vesicle preparations by freeze-thaw. The effect on Na+/H+ exchange of Ca2+/CaM/ATP was partially reversed by the inhibitor peptide. These studies demonstrate the presence of Ca2+/CaM-dependent protein kinase II in rabbit ileal villus cell brush border membranes. Based on the effect of a specific inhibitor peptide of Ca2+/CaM kinase II, it is concluded that this kinase inhibits brush border Na+/H+ exchange, which participates in the regulation of ileal Na+ absorption.

Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry.

Changes in cytosolic free Ca2+ ([Ca2+]i) in response to the secretagogue carbachol have been characterized in the human colon cancer cell line T84, a model Cl- secretory cell. In this study, [Ca2+]i was determined with the fluorescence indicator fura-2 at the single-cell level with a fluorescent microscope-imaging system. Basal [Ca2+]i in T84 cells in Ringer-HCO3 solution was 76 +/- 4 nM and was decreased by exposure to Ca2+ free solution or 25 microM verapamil. The cholinergic agonist carbachol caused a concentration-dependent rise in [Ca2+]i with a Km of 4 microM and a peak increase in [Ca2+]i of approximately 50 nM. The onset of the [Ca2+]i increase was within 3 s, occurred uniformly among cells, and peaked at 10-15 s. The increase in [Ca2+]i was heterogenous in the length of time the [Ca2+]i remained elevated above basal, and cell responses could be divided into at least two groups on that basis. Blocking the contributions of intracellular Ca2+ with dantrolene inhibited the increase in [Ca2+]i as early as could be determined, whereas blocking the extracellular contribution with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), verapamil, or nifedipine inhibited a slightly later increase in [Ca2+]i. In conclusion, the initial detectable increase in [Ca2+]i caused by carbachol is due to the release of Ca2+ from internal stores, whereas the contribution of extracellular Ca2+ occurs later and at least partially involves a nifedipine- and verapamil-sensitive process.

[Ca2+]i independent mitogenesis in cultured human fibroblasts revealed by single cell microfluorimetry.

A transient rise in intracellular free Ca2+ concentration ([Ca2+]i) has been implicated in mitogenic induction of cell division. Individual human foreskin fibroblasts in confluent cultures examined with the Ca2+ indicator Fura-2 and a fluorescence microscope-imaging system had a basal [Ca2+]i which varied markedly from cell-to-cell. A transient serum-induced rise in [Ca2+]i was demonstrated the magnitude of which was directly correlated with the basal [Ca2+]i level. In contrast to serum-induced increase in [Ca2+]i, exposure to an elevated level of extracellular Ca2+, which is at least equally mitogenic for fibroblasts, did not alter the basal [Ca2+]i of single subconfluent cells or confluent cells. Elevated extracellular Ca2+ does not exert its mitogenicity via a transient rise in [Ca2+]i.

Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles.

High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2,000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control; with both techniques, the concentration of dextran after being taken up into the vesicles was similar to that in the incubation medium, suggesting attainment of equilibrium. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca2+-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with [32P]ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.

Phosphorylation of the Ca2+-pumping ATPase of heart sarcolemma and erythrocyte plasma membrane by the cAMP-dependent protein kinase.

The Ca2+ ATPase of heart sarcolemma was stimulated by the exposure of sarcolemma vesicles to ATP and the catalytic subunit of the cAMP-dependent protein kinase. The effect of the phosphorylation system was primarily on the Km(Ca2+) of the pumping ATPase. The ATPase purified from heart sarcolemma or erythrocytes became phosphorylated under the conditions mentioned above. Hydroxylamine treatment of the labeled ATPase has shown that the phosphorylation was additive to be acylphosphate formed on the ATPase during the reaction cycle. The stoichiometry of the kinase-promoted phosphorylation (i.e. the fraction of the ATPase molecules that became labeled) approached 30% with both the heart and the erythrocyte enzyme.

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma.

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca++-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca++-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10 microns Ca++, free, but does not change the Km for Ca++. A possible physiological role for the activator is discussed.

Charge movements during the Na+-Ca2+ exchange in heart sarcolemmal vesicles.

The Na+-Ca2+ exchange was studied in a highly purified vesicular preparation derived from heart sarcolemma. The initial velocity of the Na+-driven Ca2+ influx, which was monitored continuously with a specific electrode, was 15 nmol/mg of protein per sec; the total Ca2+-accumulation capacity was 80 nmol/mg of protein. The Na+-Ca2+ exchange generated a current that was compensated for by the uptake of tetraphenylphosphonium in (Ph4P+) (when the latter was present in the medium), the influx of K+, and the efflux of Cl-. The movements of Ph4P were followed with a specific electrode. Ca2+ in the concentration range 3-50 microM induced an increase in the permeability of the sarcolemmal membrane to K+. Under conditions of optimal charge neutralization by K+ (i.e., in the presence of valinomycin), the Km (Ca2+) of the exchanger was 1.5 microM. The Na+-Ca2+ exchange was inhibited by chlorpromazine and was not inhibited by vanadate.