[Chinese medicine as vegetative systems biology. Part I: therapeutic methods]. / Chinesische Medizin als vegetative Systembiologi. Teil I: Therapeutische Verfahren.

Chinese medicine (CM) enjoys increasing popularity in Germany despite doubts about whether the measures taken make sense and whether they are safe and effective. In quantitative terms, CM de facto has already established itself, but very often it is reduced to the level of Asian philosophy in the form of mystical philosophical remnants. In this bipartite article we present a short overview of CM as a traditional approach to stringent and complex systems biology. Only if the internal logic of CM can clearly be appreciated by the Western doctor will its increasing integration be successful.

[Chinese medicine as vegetative systems biology. Part II: the structure of TCM diagnosis]. / Chinesische Medizin als vegetative Systembiologie. Teil II: Die Struktur der TCM-Diagnose.

This section describes the main features of functional vegetative diagnosis in traditional Chinese medicine (TCM). A complete diagnosis comprises four components: constitution, agent, orb, and guiding criterion. These are based on a stringent, almost mathematically precise theoretical matrix as shown by the Heidelberg Model of Chinese Medicine. Accordingly, the core concepts of Chinese medicine, yin, yang and the phases, can be made rationally accessible to Western doctors as technical terms of vegetative regulation.

Influence of lysophosphatidylcholine on the C-apolipoprotein content of rat and human triglyceride-rich lipoproteins during triglyceride hydrolysis.

Remnants produced from rat chylomicrons in hepatectomized rats or from human chylomicrons by incubation in postheparin plasma contained much less C-apolipoproteins, but more lysophosphatidylcholine than the parent chylomicrons. A phospholipid-triglyceride emulsion absorbed C-apolipoproteins during incubation in serum, yet not in postheparin plasma, which led to lipid-hydrolysis and increased in lysophosphatidylcholine. The fraction d = 1.006-1.019 g/ml of human serum comprised more lysophosphatidylcholine and less C-apolipoproteins than the fraction d less than 1.006 g/ml. Injection of heparin induced hydrolysis with an increase in lysophosphatidylcholine and loss of C-apolipoproteins in both fractions. These inverse changes of lysophosphatidylcholine and C-apolipoproteins during lipid-hydrolysis suggest a causal relationship, which is strongly supported by the induction of loss of C-apolipoproteins from rat chylomicrons and human triglyceride-rich lipoproteins by addition of lysophosphatidylcholine in vitro. Apolipoprotein C-II was more affected than C-III. These results may elucidate a mechanism for the regulation of the termination of the triglyceride hydrolysis and the final hepatic uptake of remnants.

Increased sensitivity of gastric acid secretion to gastrin in cirrhotic patients with portacaval shunt.

We studied acid secretory responses to exogenous pentagastrin and to exogenous and endogenous gastrin in 12 stable cirrhotic subjects with portacaval shunt, 12 unshunted cirrhotics, and 12 normal subjects. Basal and stimulated serum gastrin concentrations as well as basal and maximum acid outputs were similar in the three groups. At low doses of either exogenous pentagastrin or gastrin-17 (G17), cirrhotics with portacaval shunt secreted significantly greater amounts of gastric acid than unshunted subjects. After low doses of intragastric peptone, cirrhotics with portacaval shunt secreted significantly more acid than unshunted cirrhotics and normal subjects. At each measured serum gastrin concentration after either exogenous G17 or intragastric peptone meals, cirrhotics with portacaval shunt secreted more acid than the unshunted control groups and their dose-response curve was significantly shifted to the left. Thus, in cirrhotic patients with portacaval shunt, gastric acid secretion is abnormally sensitive to both exogenously administered or endogenously released gastrin.

Donor splice site mutation in the apolipoprotein (Apo) C-II gene (Apo C-IIHamburg) of a patient with Apo C-II deficiency.

The DNA, RNA, and protein of apo C-II have been analyzed in a patient with apo C-II deficiency (apo C-IIHamburg). Markedly reduced levels of plasma and intrahepatic C-II apolipoprotein were demonstrated by immunoblotting and immunohistochemical analysis. Northern, slot blot, and in situ hybridization studies revealed low levels of a normal-sized apo C-II mRNA. No major rearrangement of the apo C-II gene was detected by Southern blotting. Sequence analysis of apo C-II genomic clones revealed a G-to-C substitution within the donor splice site of intron II. This base substitution resulted in the formation of a new Dde I and loss of a Hph I restriction enzyme cleavage site. Amplification of the mutant sequence by the polymerase chain reaction and digestion with Dde I and Hph I restriction enzymes established that the patient was homozygous for the G-to-C mutation. This is the initial report of the DNA sequence of an abnormal apo C-II gene from a patient with deficiency of apo C-II. We propose that this donor splice site mutation is the primary genetic defect that leads to defective splicing and ultimately to an apo C-II deficiency in this kindred.

Characterization of CA 19-9 bearing mucins as physiological exocrine pancreatic secretion products.

The monoclonal antibody CA 19-9 reacts with a carbohydrate epitope (sialylated lacto-N-fucopentaose II), which was shown to be part of a ganglioside extracted from a colon carcinoma cell line as well as of a mucin isolated from gastrointestinal tract tumor patients' sera. Recently, when we compared CA 19-9 levels in pancreatic juices and corresponding serum samples from a large group of patients, we showed the high serum values to be indicative solely for a malignant disease. In contrast, the overall high CA 19-9 content in pancreatic juices from all diagnostic groups raised the question about the antigenic moieties in these samples. By means of thin layer chromatography of glycolipids with subsequent antibody overlay, gel chromatography, and density gradient analysis, we found only the mucin form in all sources investigated. Thus we conclude that the discrimination potential of the CA 19-9 assay in serum is based on an altered secretion or distribution in pancreatic tumors.

Monoclonal antibody-defined human pancreatic cancer-associated antigens.

Three pancreatic cancer-associated antigens were characterized by use of monoclonal antibodies in immunobinding studies with various cellular and soluble target antigens, in immunoprecipitation, and in immunoperoxidase staining. C54-0 represents a tumor-associated Mr 122,000 antigen, which appears to be widely distributed on various epithelial tumors and to a lower extent on normal tissue. C1-N3 antigen exhibited a more restricted distribution, reacting with pancreatic and various gastrointestinal tract tumors as well as with chronically inflamed pancreatic tissue. The most specific antigen expression was observed for C1-P83 antigen, found on all exocrine tumors of the pancreas, but not on normal or chronically inflamed pancreatic tissue.

Modulation of platelet-derived growth factor A- and B-chain/c-sis mRNA by tumor necrosis factor and other agents in adenocarcinoma cells.

Human Platelet Derived Growth Factors (PDGF) are potent mitogens for mesenchymal cells and encoded by two related genes, the A- (or 1-) and B- (or 2-) chain. The latter is known as the human homolog (c-sis) of the v-sis oncogene. We investigated the expression and cytokine-mediated regulation of PDGF A- and B-chain mRNA in endoderm-derived cells, i.e. cultured human pancreatic adenocarcinoma cells. Northern blot analysis revealed that out of 14 cells lines 11 were positive for the A-chain and 10 for the B-chain. Tumor Necrosis Factor (TNF) -alpha and -beta, but not Interferon (IFN) -gamma, drastically upregulate the mRNA levels for PDGF B-chain and for the A-chain in a dose-dependent manner in nearly every pancreatic tumor cell line investigated (n = 6). With respect to the signal pathway stimulated by TNF, no evidence emerged for an activation of protein kinase A. The inhibition of protein kinase C by staurosporine (in the absence or presence of TNF) as well as its stimulation by PMA resulted in an increased mRNA level for the B-chain, indicating a functional role of PKC in this system. Furthermore, time course experiments and Cycloheximide treatment showed that the A- and B-chain mRNA are regulated by different mechanisms in transformed epithelial cells. Irrespective of these differences, the sum of their biological functions may contribute to the phenomenon of desmoplasia in pancreatic tumors by epithelial/mesenchymal interactions.

Absence of APOBEC-1 mediated mRNA editing in human carcinomas.

The transgene expression of the catalytic subunit APOBEC-1 of the apo B mRNA editing enzyme-complex can cause hepatocellular carcinoma in mice and rabbits. It has been proposed that aberrant editing of mRNA may represent a novel oncogenic principle. This investigation aimed to define whether such aberrant hyperediting mediated by APOBEC-1 occurs in human carcinomas. Editing and hyperediting of apo B, NAT1 or NF1 mRNA was not identified in any of 28 resected tumor specimens, including hepatocellular, bile duct, gastric, colorectal, pancreatic adeno- and neuroendocrine, lung adeno-, medullary thyroid and breast carcinoma, soft tissue sarcoma and neuroblastoma. In most types of carcinoma, significant levels for full-length APOBEC-1 mRNA could not be detected. Low level expression of APOBEC-1 was found in colorectal and gastric carcinoma where most of the APOBEC-1 mRNA is inactivated by alternate splicing. The 'auxiliary' components of the apo B mRNA editing enzyme-complex are missing in many tumors including colorectal and gastric carcinoma, but are highly expressed in hepatocellular, lung adeno- and breast carcinoma all of which lack APOBEC-1. Taken together, either APOBEC-1 or the 'auxiliary' components of the apo B mRNA editing enzyme-complex or both are missing in human carcinomas resulting in the absence of mRNA editing. Currently, there is no evidence that aberrant editing mediated by APOBEC-1 contributes to the tumorigenesis of natural human carcinomas.

Activation and inhibition of lipoprotein lipase. Studies with artificial lipoproteins.

Human plasma very low density apolipoproteins C-I, C-II and C-III were recombined in vitro with triolein. The lipid-protein complexes were analyzed by ultracentrifugal flotation, agarose gel electrophoresis, immunoelectrophoresis and electron microscopy. Maximal protein/triolein ratios for apoprotein C-I, C-II, C-III-1 and C-III-2 were 50, 45, 95 and 55 microgram/mg, respectively. Electron micrographs exhibited spherical particles with diameters ranging from 200--2000 A comparable to native VLDL and chylomicrons. On agarose gel electrophoresis these complexes showed alpha-mobility. Kinetics of triolein hydrolysis by purified human plasma lipoprotein lipase were studied using these artificial lipoprotein substrates with different apoprotein/triolein ratios. The reaction followed the Michaelis-Menten equation. With increasing amounts of apo C-II, the apparent Km decreased from 0.60 to 0.11 mM. Incubation of the substrate with either rabbit anti-apo C-II gamma-globulins or digestion with trypsin prior to hydrolysis reversed this lowering effect on apparent Km. V was not altered significantly. Increasing amounts of apo C-I, apo C-III-1 or apo C-III-2 without apo C-II caused inhibition of triolein hydrolysis. In the presence of apo C-II, however, similar kinetic parameters were obtained as described above.

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages.

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.

In vitro modulation of the distribution of normal human plasma high density lipoprotein subfractions through the lecithin: cholesterol acyltransferase reaction.

The effect of the lecithin: cholesterol acyltransferase reaction on the chemical composition, morphology and distribution of normal human plasma high density lipoprotein (HDL) subclasses was studied in vitro. Incubation of plasma in the presence of polyenephosphatidylcholine (PPC) resulted in a 45 +/- 11% (n = 6) decrease in unesterified cholesterol after 20 h. This effect was abolished by prior heating of the plasma at 56 degrees C or by the addition of diisopropyl fluorophosphate (DIFP). Plasma triacylglycerol levels were constant. Analysis of the plasma lipoproteins by zonal ultracentrifugation and isopycnic equilibrium banding revealed a bimodal distribution of the HDL of native plasma and both heat-inactivated or DIFP-treated samples with peak maxima at d = 1.084 g/ml and d = 1.110 g/ml. Following the lecithin:cholesterol acyltransferase reaction essentially all of the HDL material had flotation characteristics typical of HDL2. The peak maximum was d = 1.086 g/ml. There were no apparent changes in the distribution of the lipoproteins of d less than 1.063 g/ml. The newly formed HDL were poor in PC and unesterified cholesterol but rich in cholesteryl ester, sphingomyelin and lyso-PC. The HDL apolipoprotein pattern was unaltered. HDL morphology was not affected by the lecithin:cholesterol acyltransferase reaction. Similar results were obtained in the absence of PPC. However, under these conditions the total phospholipid content of the HDL was reduced and lyso-PC was not demonstrable as a product of the lecithin:cholesterol acyltransferase reaction after 20 h. The results are interpreted to indicate that lecithin:cholesterol acyltransferase is involved in the transformation of HDL3 into HDL2.

Selective association of lipoprotein cholesteryl esters with liver plasma membranes.

High-density lipoprotein (HDL) cholesteryl esters are taken up by hepatocytes without parallel uptake of HDL apolipoproteins. This selective uptake of HDL cholesteryl esters is mediated by a non-endocytotic mechanism. Recently, selective uptake of cholesteryl esters also from low-density lipoprotein (LDL) was demonstrated. In this study, the role of the plasma membrane in selective uptake by the liver was investigated. Plasma membranes were prepared from rat liver or from human Hep G2 hepatoma cells. Human HDL3 (d = 1.125-1.21 g/ml) was either radioiodinated or labeled with [3H]cholesteryl oleate. Human low-density lipoprotein (d = 1.019-1.05 g/ml) was labeled in its protein and in its lipid moiety as well. Labeled lipoproteins, unlabeled lipoproteins and membranes were incubated. After separation by ultracentrifugation, apparent lipoprotein particle association with membranes was determined. Plasma membranes from rat liver and Hep G2 cells bound 125I-HDL3, indicating specific HDL3 particle binding. With both types of membrane, apparent HDL3 particle association according to [3H]cholesteryl oleate-labeled HDL3 was in significant excess on that due to 125I-HDL3. This indicates selective, i.e., particle binding independent, association of cholesteryl esters with the membrane. Excess unlabeled HDL3 competed for selective association, indicating a specific process. Selective association of HDL3 cholesteryl esters was concentration-, time-, temperature-dependent; however, parameters differed from HDL3 particle binding. HDL3 was modified by nitration; this modification inhibited HDL3 particle binding in contrast to unchanged selective association. These results suggested distinct membrane sites for HDL3 particle binding and selective cholesteryl ester association. Regulation of selective association was investigated. Hep G2 cells were cholesterol-loaded or cholesterol-depleted. Cellular cholesterol-loading down-regulated selective association of HDL3 cholesteryl esters with isolated membranes prepared from these cells. In parallel, selective uptake of HDL3 cholesteryl esters by Hep G2 cells was down-regulated in cholesterol-loaded cells. This parallel regulation of selective association with membranes and selective uptake by cells suggests a functional relationship. LDL, radiolabeled in its protein and in its lipid moiety, was incubated with liver plasma membranes. Besides LDL holo-particle receptor binding, also LDL cholesteryl esters were selectively associated with membranes. These data showed that selective association with membranes is not restricted to HDL but can occur from LDL as well. It is concluded that HDL3 as well as LDL cholesteryl esters can selectively be associated with hepatic plasma membranes, i.e., independent from particle binding. Results suggest an important role of the plasma membrane in the mechanism of selective cholesteryl ester uptake by the liver.

Purification of human plasma lipoprotein lipase.

Human plasma lipoprotein lipase was purified in a highly active form. Addition of the non-ionic detergent Triton X-100 led to stabilization of enzyme activity during the purification procedure. Antithrombin III, the major contaminant after affinity chromatography with heparin-Sepharose 4B, could be removed by gel filtration on Bio-Gel A-5m. The application of Tris-glycine buffer in the absence of denaturating agents allowed identification of the protein band corresponding to lipoprotein lipase activity on polyacrylamide gels.

Quantitative determination of apolipoprotein A-I in high-density lipoproteins and 'free' apolipoprotein A-I by two-dimensional agarose gel lipoprotein-'rocket' immunoelectrophoresis of human serum.

A method has been developed for quantitative analysis of 'free' apolipoprotein A-I and apolipoprotein A-I associated with high-density lipoprotein (HDL) in serum. The method utilizes the difference between the rate of electrophoretic migration of apolipoprotein A-I associated with HDL (alpha) and 'free' apolipoprotein A-I (pre-beta) in agarose gel. Apolipoprotein A-I is subsequently quantitated by electrophoresis in a second dimensional gel containing anti-apolipoprotein A-I antibodies. Using this method all apolipoprotein A-I of normal fasting serum was found associated with HDL (n = 16). By contrast, 'free' apolipoprotein A-I accounted for up to 12% of the total in the serum of patients with isolated hypertriglyceridemia (n = 8) or mixed hyperlipoproteinemia (n = 8). Between 30 and 35% of 'free' apolipoprotein A-I was found in one patient afflicted with the apolipoprotein C-II deficiency syndrome. Also, 'free' apolipoprotein A-I could be detected in normal postabsorptive serum. 30 and 90 min following heparin-enhanced lipolysis 'free' apolipoprotein A-I accounted for 23 and 20%, respectively, of the total apolipoprotein A-I of serum. Apolipoprotein A-I associated with HDL remained unaltered. It appears, therefore, that 'free' apolipoprotein A-I is liberated from triglyceride-rich lipoproteins during lipolysis.

Selective uptake of low-density lipoprotein-associated cholesteryl esters by human fibroblasts, human HepG2 hepatoma cells and J774 macrophages in culture.

High-density lipoprotein-(HDL) associated cholesteryl esters (CE) are taken up by hepatic and extrahepatic cells at a higher rate than HDL apolipoproteins. This selective uptake of HDL CE is independent from HDL particle uptake. For low-density lipoprotein (LDL), receptor-mediated endocytosis by cells is well established. In this study, the question was addressed if LDL-associated CE are also taken up by cells independently from LDL particles, i.e., selectively. Human LDL (d = 1.02-1.05 g/ml) was doubly radiolabeled with intracellularly trapped tracers: [125I]Tyramine-Cellobiose ([125I]TC) traced apolipoprotein B, [3H]cholesteryl oleyl ether ([3H]CEt) traced CE. The uptake of doubly radiolabeled LDL by normal and LDL receptor-negative human skin fibroblasts, human HepG2 hepatoma cells and murine J774 macrophages was investigated. Each cell type took up LDL particles as indicated by [125I]TC. However, in fibroblasts, HepG2 cells and J774 macrophages the rate of uptake for LDL-associated [3H]CEt was greater than that according to [125I]TC. These results indicate that extrahepatic and hepatic cells selectively take up LDL CE and this uptake is independent from LDL receptor-mediated endocytosis. Loading cells with cholesterol down-regulated selective uptake of LDL CE. In summary, human skin fibroblasts, human HepG2 cells and murine J774 macrophages selectively take up LDL CE, i.e., CE are taken up independently from LDL particles.

Stimulation of beta 2-adrenergic receptors suppresses sterol synthesis in human mononuclear leukocytes.

The beta-adrenergic receptor mediating the inhibition of sterol synthesis by catecholamines in freshly isolated human mononuclear leukocytes was defined pharmacologically by using selective beta 1- and beta 2-agonists and -antagonists. Incubation of cells for 6 h in a medium containing lipid-depleted serum resulted in a 3-fold increase in the incorporation of [14C]acetate or tritiated water into sterols. The beta-agonist (-)-isoproterenol was approximately equipotent with (-)-epinephrine and (-)-norepinephrine in suppressing sterol synthesis, yielding a sigmoidal log-dose-effect curve. Accordingly, the effects of the catecholamines were reversed by the beta-antagonist (+/-)-propranolol. The beta 2-agonists terbutaline and salbutamol inhibited sterol synthesis by 42 and 26%, respectively, at a concentration of 0.1 mmol/l. Contrary to that, the beta 1-agonists prenalterol and dobutamine had no effect. In accordance with the influence of the agonists, the beta 2-antagonist butoxamine, but not the beta 1-antagonists atenolol, metoprolol and practolol, reversed the catecholamine action on sterol synthesis. The results provide evidence that catecholamines may regulate sterol synthesis by stimulating beta 2-adrenergic receptors.

Biotinyl-high-density lipoproteins as a probe for the determination of high-density lipoprotein turnover in humans.

A simple and reliable method has been developed for the determination of high-density lipoprotein (HDL) turnover in humans. In this method, complex formation of [14C]methylavidin with biotinyl-HDL3 and subsequent precipitation of excess [14C]methylavidin with biotinyl-silica-gel is utilized for the detection of as little as 0.1 microgram of biotinyl-HDL3 (by protein)/ml of serum with high precision and reproducibility, at 7.9 +/- 0.9% (n = 7) of the peptidyl lysine modified. In serial dilutions and quadruplicate determinations the intra- and interassay variations were less than 4.7% and 5.2%, respectively (n = 5). Recovery of biotinyl-HDL3 averaged 92 +/- 5.7% throughout the working range of the assay (n = 4). Variations in the levels of HDL or very-low-density lipoprotein (VLDL) did not interfere with the measurement of biotinyl-HDL3 in serum. Also, results were not affected by storage of serum samples at -80 degrees C for up to 4 weeks. After reinjection of autologous biotinyl-HDL3 (0.12 mg protein/kg body wt.) into five normolipidemic male volunteers, typical decay curves were obtained. The mean half-life of biotinyl-HDL3 was 5.1 +/- 0.5 days, no different from that reported for radiolabeled HDL or radiolabeled HDL apolipoproteins. Routine immunobinding analysis did not reveal formation of antibodies with specificity towards HDL 4 weeks after the reinjection studies. From this it appears that biotinyl-HDL3 is a suitable probe and a safe and reliable alternative for the determination of HDL turnover in humans when application of radiolabeled HDL is not desirable.

The prostacyclin analogue iloprost and prostaglandin E1 suppress sterol synthesis in freshly isolated human mononuclear leukocytes.

The effects of the stable prostacyclin analogue iloprost, prostaglandin E1 and prostaglandin F2 alpha on sterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for 6 h in a medium containing lipid-depleted serum led to a 3-fold rise in the rate of sterol synthesis from [14C]acetate or tritiated water. Iloprost and prostaglandin E1 added in increasing concentrations at zero time resulted in an inhibition of the synthesis of sterols, the suppression being 50 and 55% at a concentration of 1 mumol/1, respectively. Both prostaglandins yielded a sigmoidal log dose-effect curve. In contrast, prostaglandin F2 alpha had no influence on sterol synthesis up to a concentration of 1 mumol/1. The action of the prostacyclin analogue and prostaglandin E1 on the relative rate of sterol synthesis was not immediate, since the prostaglandins had no effect when given at 6 h to the incubation medium, and the incorporation of [14C]acetate into sterols was measured thereafter. The results suggest that prostacyclin and prostaglandin E1 affect cholesterol synthesis and therefore may play a role in the regulation of cellular cholesterol homeostasis and in the development of atherosclerosis.

Characterization of CCK receptors in stomach smooth muscle: evidence for two subtypes.

Cholecystokinin (CCK) and related peptides such as gastrin are important regulators of gastric smooth muscle contraction. Several studies have shown that these effects of CCK and gastrin are mediated by CCK(B) receptors. However, recent studies suggest the expression of an additional CCK receptor subtype distinct from CCK(B) receptors in this tissue. This study was designed to distinguish between CCK(A) and CCK(B) receptors on guinea-pig stomach smooth muscle cells and to evaluate these cells for additional receptor subtypes. We cloned these receptors by hybridization screening of a guinea-pig smooth muscle cDNA library using 32P random primed labeled cDNA probes from the recently cloned rat CCK(A) and CCK(B) receptor coding regions. In addition to clones representing the CCK(B) subtype, clones of CCK(A) receptor subtype, but no additional CCK receptor subtypes, could be identified. All isolated clones displayed highly homologous nucleotide sequences in comparison to previously characterized CCK(A) and CCK(B) receptors from different species. The results of cDNA hybridization at different levels of stringency and Southern blot analysis using guinea-pig genomic DNA suggest that it is unlikely that additional CCK receptors despite CCK(A) and CCK(B) receptors exist in stomach smooth muscle.