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The role of TNF-alpha in modulating intestinal crypt cell growth was examined, in comparison with EGF. Both significantly increased IEC-6 cell proliferation. Neither EGF nor TNF-alpha overcame the inhibitory effect on growth exerted by the tyrosine kinase inhibitor genistein. Immunoblots with phosphotyrosine antibodies showed increased tyrosine phosphorylation of IEC-6 cell proteins in response to EGF and TNF-alpha stimulation. TNF-alpha increased ERK1 and ERK2 MAPK phosphorylation. A MAPK assay confirmed the increased activity upon TNF-alpha stimulation. Selective inhibition of MAPK activation by PD98059 resulted in a dose dependent inhibition of TNF-alpha or EGF-induced IEC-6 cell growth. These findings suggest a role for TNF-alpha in the regulation of intestinal epithelial cell growth and that the mitogenic effect of TNF-alpha requires protein tyrosine phosphorylation and MAPK activation.

During the last 5 years several studies have documented an involvement of protein tyrosine kinases (PTKs) in smooth muscle contraction and Ca2+ mobilization. Most of these studies have utilized highly selective inhibitors of PTKs, genistein and tyrphostin and have shown that these inhibitors attenuated smooth muscle contraction induced by growth factors-epidermal growth factor (EGF) and platelet derived growth factor (PDGF) and several vasoactive peptides. It has also been demonstrated that inhibitors of protein tyrosine phosphatases (PTPases) such as vanadate and pervanadate mimic growth factors and vasoactive peptides in causing the contraction of smooth muscle. In this brief review, we have summarized some of the recent observations suggesting a possible link between protein tyrosine phosphorylation pathway and smooth muscle contraction.

In three experiments, we manipulated the processing demands of a concurrent task to test the hypothesis that an event-based prospective memory task satisfies a criterion of automaticity proposed by Hasher and Zacks (1979). As in the previous studies, a prospective memory task (pressing a key whenever a target word was presented) was embedded within a short-term memory task (remembering seven words). In addition, participants performed a concurrent memory task which varied in difficulty. Participants repeated either 0 to 6 randomly generated digits or a single word. In all three experiments, short-term memory performance was influenced by the concurrent memory load. Prospective performance, in contrast, was not affected by the memory load even though an attempt was made to increase the difficulty of the prospective task by manipulating the specificity of the target instructions (Experiment 2) and the number of target words (Experiments 2 and 3). The results are discussed within the framework of automatic processing.

A Myelin Basic Protein (MBP) epitope encephalitogenic for the Lewis rat (amino acid residues 68-86) was synthesized and acylated by the attachment of a palmitoyl residue. Lewis rats treated intravenously (i.v.) with the palmitoylated peptide alone were better protected against clinical manifestations of experimental allergic encephalomyelitis (EAE) than rats treated with the peptide inserted into liposomes or with the native peptide at similar doses. The administration of the acylated peptide (PAL68 86) conferred excellent protection against a challenge with the encephalitogenic peptide (p68-86) or with the intact MBP molecule, both before and after induction of active disease, and also when administered to recipients after the transfer of lymphocytes from MBP-challenged donors. Histological manifestations were also reduced to a statistically significant degree. Treatment with a palmitoylated peptide from a non-encephalitogenic region of the MBP molecule (PAL44-62) or with a palmitoylated unrelated peptide were ineffective. In vitro Ag-specific proliferative responses as well as the ability to transfer disease to syngeneic recipients, by lymph node lymphocytes from PAL68-86-treated donors, were considerably reduced. Addition of IL-2 to these cultures failed to restore either Ag-specific responsiveness or the ability of the cells to transfer disease. The results suggest that the administration of acylated peptides induces a profound state of unresponsiveness, and thus may provide an effective means for treating T cell-mediated autoimmune inflammatory disorders.

Long chain free fatty acids (FFA) are the preferred metabolic substrates of myocardium under aerobic conditions. However, under ischemic conditions long chain FFA have been shown to be harmful both clinically and experimentally. Serum levels of free fatty acids frequently are elevated in patients with myocardial ischemia. The proposed mechanisms of the detrimental effects of free fatty acids include: (1) accumulation of toxic intermediates of fatty acid metabolism, such as long chain acyl-CoA thioesters and long chain acylcarnitines, (2) inhibition of glucose utilization, particularly glycolysis, during ischemia and/or reperfusion, and (3) uncoupling of oxidative metabolism from electron transfer. The relative importance of these mechanisms remains controversial. The primary site of FFA-induced injury appears to be the sarcolemmal and intracellular membranes and their associated enzymes. Inhibitors of free fatty acid metabolism have been shown experimentally to decrease the size of myocardial infarction and lessen postischemic cardiac dysfunction in animal models of regional and global ischemia. The mechanism by which FFA inhibitors improve cardiac function in the postischemic heart is controversial. Whether the effects are dependent on decreased levels of long chain intermediates and/or enhancement of glucose utilization is under investigation. Manipulation of myocardial fatty acid metabolism may prove beneficial in the treatment of myocardial ischemia, particularly during situations of controlled ischemia and reperfusion, such as percutaneous transluminal coronary angioplasty and coronary artery bypass grafting.

The effect of chronic hypoxia on neonatal myocardial metabolism remains undefined. With a new neonatal piglet model, we determined changes in myocardial metabolism during global ischemia after chronic hypoxia. Five-day-old piglets (N = 30) were randomly assigned to two groups and exposed to an atmosphere of 8% oxygen or to room air for 28 days before they were killed. Left ventricular myocardium was then analyzed at control and at 15-minute intervals during 60 minutes of global normothermic ischemia to determine high-energy phosphate levels, glycogen stores, and lactate accumulation. Time to peak ischemic myocardial contracture was measured with intramyocardial needle-tipped Millar catheters as a marker of the onset of irreversible ischemic injury. Results showed an initially greater level of myocardial adenosine triphosphate in the hypoxic group (27 +/- 1.2 vs 19 +/- 1.8 micromol/gm dry wt, p = 0.001) and a delay in adenosine triphosphate depletion during 60 minutes of global ischemia compared with the control group. Initial energy charge ratios (1/2 adenosine diphosphate + adenosine triphosphate/adenosine monophosphate + adenosine diphosphate + adenosine triphosphate) were also greater in the hypoxic group (0.96 +/- 0.01 vs 0.81 +/- 0.04, p = 0.01) and remained so throughout global ischemia. Initial glycogen stores were greater in the hypoxic group (273 +/- 13.3 vs 215 +/- 14.7 micromol/gm dry weight, p = 0.02) when compared with the control group. Lactate levels in the hypoxic group were initially higher (19.1 +/- 6.4 vs 8.9 +/- 3.1 micromol/gm dry weight, p = 0.001) compared with control levels and remained elevated throughout 60 minutes of ischemia. Time to peak ischemic contracture was prolonged in the hypoxic group (69.5 +/- 1.8 vs 48.9 +/- 1.4 minutes, p = 0.001) compared with the controls group. These data show that chronic hypoxia results in significant myocardial metabolic adaptive changes, which in turn result in an improved tolerance to severe normothermic ischemia. These beneficial effects are associated with elevated baseline glycogen storage levels and an accelerated rate of anaerobic glycolysis during ischemia.

Plasma aldosterone levels increase markedly during pregnancy, but not in proportion to the rise in plasma renin activity (PRA). We have developed a reliable in vitro method to investigate aldosterone secretion during pregnancy. With this method, we have assessed the potency and effectiveness of ACTH and potassium to stimulate this secretion during pregnancy. Adrenal capsules from pregnant and nonpregnant rats were incubated in 1 ml of culture medium within wells of tissues culture plates. The cortex was transferred every 20 min to another well containing fresh medium with or without ACTH or potassium. Basal and stimulated aldosterone secretions were not significantly affected by time under our experimental conditions. The glands remained responsive to stimulants throughout the study period (360 min). Plasma aldosterone levels and PRA were increased during pregnancy. Basal aldosterone secretion in adrenal cortex suspensions from pregnant rats showed a 1.6-fold increment (P < 0.001) in comparison with nonpregnant controls. The dose-response curves of ACTH were not significantly different between pregnant and nonpregnant animals. However, sensitivity to potassium was significantly reduced during pregnancy, as demonstrated by an elevated ED50 (4.01 +/- 0.08 vs 4.71 +/- 0.07 mM for nonpregnant versus pregnant rats respectively, P < 0.001). These data indicate that adrenal cortex suspensions are a reliable and reproducible way to study aldosterone secretion during pregnancy. They reveal that, during pregnancy, sensitivity of potassium to stimulate aldosterone secretion is decreased while the response to ACTH is not affected.

The regulation of fatty acid oxidation in isolated myocytes was examined by manipulating mitochondrial acetyl-CoA levels produced by carbohydrate and fatty acid oxidation. L-carnitine had no effect on the oxidation of [U-14C]glucose, but stimulated oxidation of [1-14C]palmitate in a concentration-dependent manner. L-carnitine (5 mM) increased palmitate oxidation by 37%. The phosphodiesterase inhibitor, enoximone (250 microM), also increased palmitate oxidation by 51%. Addition of L-carnitine to enoximone resulted in a two-fold increase of palmitate oxidation. Whereas, dichloroacetate (DCA, 1 mM), which stimulates PDH activity, decreased palmitate oxidation by 25%. Furthermore, the addition of DCA to myocytes preincubated with either L-carnitine or enoximone, had no effect on the carnitine-induced stimulation of palmitate, and reduced that of enoximone by 50%. Varied concentrations of DCA decreased the oxidation of palmitate and octanoate; but increased glucose oxidation in myocytes. The rate of efflux of acetylcarnitine was highest when pyruvate was present in the medium compared to efflux rates in presence of palmitate or palmitate plus glucose. Although the addition of L-carnitine plus enoximone resulted in a two-fold increase in palmitate oxidation, acetylcarnitine efflux was minimal under these conditions. Acetylcarnitine efflux was highest when pyruvate was present in the medium. These rates were dramatically decreased when myocytes were preincubated with enoximone, despite the stimulation of palmitate oxidation by this compound. These data suggest that: (1) fatty acid oxidation is influenced by acetyl-CoA produced from pyruvate metabolism; (2) L-carnitine may be specific for mitochondrial acetyl-CoA derived from pyruvate oxidation; and (3) it is probable that acetyl-CoA from beta-oxidation of fatty acids is directly channeled into the citric acid cycle.

The role of nitric oxide (NO) as well as its interaction with prostaglandins (PG) in setting the limits of autoregulation of retinal blood flow (RBF) and choroidal blood flow (ChBF) were studied in newborn pigs (1-5 d old). Blood flows were measured by the microsphere technique. Low and high ocular perfusion pressures (OPP) were induced by inflating balloon-tipped catheters placed at the aortic root and isthmus, respectively. Animals were treated with the NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME, 1 mg/kg followed by 50 mu g/kg/min; n = 12) or NG-monomethyl-L-arginine (L-NMMA, same dose as L-NAME; n = 3), or with saline (n = 12). In separate animals (n = 42), guanosine 3',5'-cyclic monophosphate (cGMP), the second messenger for NO, and PG were measured at an average OPP of 90 mm Hg and 125 +/- 6 mm Hg; cGMP levels served as an index of NO release. The effect of the NO donor sodium nitroprusside on choroidal vessel diameter was determined using video imaging of isolated eyecup preparations. In control animals RBF was constant only within a range of 30 to 80 mm Hg OPP (r = 0.03, p > 0.9). There was no autoregulation of ChBF which increased as a function of OPP (tau = 0.58-0.72, p < 0.01). L-NAME and L-NMMA prevented a change in RBF and ChBF from 30 to 146 mm Hg [the highest OPP studied (r < 0.3, p > 0.15)] and caused an increase in retinal as well as choroidal vascular resistance as OPP was raised; these agents did not affect ocular blood flow at OPP < 30 mm Hg. Elevated OPP caused increases in cGMP, 6-keto-PGF1alpha, and PGE2 in the choroid (a vascular tissue), which were prevented by L-NAME and L-NMMA. Sodium nitroprusside caused a dilatation of choroidal vessels in isolated eyecup preparations, which was significantly attenuated by indomethacin. Data suggest a role for NO in the autoregulation of RBF and ChBF in the newborn such that a release of NO during a rise in OPP prevents adequate constriction necessary for maintaining RBF and ChBF constant; data also suggest that the vasodilator effect of NO might in part be mediated through a release of PG.

The present study was undertaken to characterize the contractile effects of vanadate on thoracic aorta rings from virgin and term-pregnant rats. Vanadate caused concentration-dependent contraction in rat aortic rings with an EC50 (concentration producing 50% maximum response) of 0.10 mM. Contractions in response to vanadate were equivalent to the ones measured with 1 microM phenylephrine. The effects of vanadate were not affected by indomethacin (up to 10 microM), an inhibitor of prostanoid cyclooxygenase, but were blocked in a concentration-dependent manner by staurosporine (0.1-1.0 microM), an inhibitor of protein kinase C. Vanadate exhibited a significant decrease of contractile responses in aorta of pregnant as compared to virgin rats. When aortic rings were bathed in presence of different concentrations of vanadate, the concentration-response curve to phenylephrine was shifted to the left, but maximum response was not affected. The potentiation of the contractions to phenylephrine by vanadate was significantly more prominent in aorta of virgin than of pregnant rats. These results suggest that the contractile effect of vanadate on rat aorta is independent of endogenous prostanoids and may be mediated by protein kinase C-dependent pathway. These results also show that the contractile response to vanadate on the rat aorta is impaired during pregnancy.

The effects of L-carnitine on 14CO2 release from [1-14C]pyruvate oxidation (an index of pyruvate dehydrogenase activity, PDH), [2-14C]pyruvate, and [6-14C]glucose oxidation (indices of the acetyl-CoA flux through citric acid cycle), and [U-14C]glucose (an index of both PDH activity and the flux of acetyl-CoA through the citric acid cycle), were studied using isolated rat cardiac myocytes. L-carnitine increased the release of 14CO2 from [1-14C]pyruvate, and decreased that of [2-14C]pyruvate in a time and concentration-dependent manner. At a concentration of 2.5 mM, L-carnitine produced a 50% increase of CO2 release from [1-14C]pyruvate and a 50% decrease from [2-14C]pyruvate oxidation. L-carnitine also increased CO2 release from [1-14C]pyruvate oxidation by 35%, and decreased that of [2-14C]pyruvate oxidation 30%, in isolated rat heart mitochondria. The fatty acid oxidation inhibitor, etomoxir, stimulated the release of CO2 from both [1-14]pyruvate and [2-14C]pyruvate. These results were supported by the effects of L-carnitine on the CO2 release from [6-14C]- and [U-14C]glucose oxidation. L-carnitine (5 mM) decreased the CO2 release from [6-14C]glucose by 37%, while etomoxir (50 microM) increased its release by 24%. L-carnitine had no effect on the oxidation of [U-14C]glucose. L-carnitine increased palmitate oxidation in a time- and concentration-dependent manner in myocytes. Also, it increased the rate of efflux of acetylcarnitine generated from pyruvate in myocytes. These results suggest that L-carnitine stimulates pyruvate dehydrogenase complex activity and enhances non-oxidative glucose metabolism by increasing the mitochondrial acetylcarnitine efflux in the absence of exogenous fatty acids.

The hypothesis that Ca2+ channel function is altered during pregnancy was tested by comparing responses to potassium chloride (KCl) and phenylephrine in aortic rings of virgin and term-pregnant rats under the influence of nifedipine and Bay K 8644. Maximum response to KCl was progressively reduced by increasing nifedipine concentrations (1.0-100 nM) in both groups of tissues. Nifedipine produced a smaller inhibition of KCl-induced contraction in aortic rings of pregnant than of virgin rats. It exerted little inhibition on the concentration-response curve to phenylephrine. The Ca2+ channel antagonist (100 nM) reduced the maximum response to the alpha-adrenoceptor agonist in rings from virgin rats, but had no effect in pregnant rats. Bay K 8644, a Ca2+ channel activator, potentiated the responses to low concentrations of both phenylephrine and KCl in the tissues of both virgin and pregnant rats, but did not affect maximum responses. It also induced concentration-dependent contractions in rings of virgin but not of pregnant rats. The effects of Bay K 8644 were markedly potentiated by precontracting the aorta with 10mM KCl. Nevertheless tissues from pregnant rats were still less responsive to Bay K 8644. However, when the strips were precontracted to the same level by different concentrations of KCl, the concentration-response curves to Bay K 8644 were identical in both groups. [3H]Nitrendipine binding to membrane preparations of the thoracic aorta was similar in virgin and pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: The purpose of this study was to investigate the contribution of potential-operated calcium (POC) channels in the mechanisms of the blunted effects of vasoconstrictors on mesenteric resistance arteries during normal pregnancy., Methods: Mesenteric resistance arteries of virgin and term pregnant rats were set up under optimum passive tension in wire myograph systems. Cumulative concentration-response curves of arginine8-vasopressin (AVP) were measured in the absence and presence of nifedipine or Bay K 8644, a blocker and an activator, respectively, of POC channels. Binding studies were performed on membrane preparations of the mesenteric vascular bed of both groups of rats using saturation with [3H]nitrendipine., Results: The maximal response to AVP was statistically similar in the two groups of arteries. Pregnancy shifted the AVP concentration-response curves to the right. Nifedipine (1 mumol/L) similarly reduced the maximum response to AVP in arteries of both groups, but produced a larger increase in EC50, the concentration inducing 50% maximum response, in resistance arteries of virgin versus pregnant rats. Bay K 8644 did not affect the maximum tension reached with AVP. However, it increased the effects of small concentrations of AVP in arteries of both groups. This was more important in tissues of virgin than pregnant rats. Binding of [3H]nitrendipine to membrane preparations of mesenteric vessels was not modified by pregnancy., Conclusion: Our results suggest a reduced functional influence of POC channels in the myotropic effects of AVP on mesenteric resistance arteries in pregnancy. This decreased influence of POC channels may contribute to resistance of the vasculature to vasopressor agents during pregnancy.

MHC class-II-negative astrocytes prevented from intracellular antigen (Ag) processing induce myelin basic protein (MBP)-specific short-term T cell lines to proliferate. This process results from the ability of the T cells themselves to take up, process, and present Ag to each other. The Ag-presenting function of the T cells occurred in the absence of any conventional antigen-presenting cell (APC), was independent of their T cell receptor specificity, was sensitive to chloroquine, and was prevented by anti-class-II MHC antibody. Both native and HPLC-purified MBP were effective in stimulating T cell lines, and there was no obvious benefit in using either enzymatically digested or synthetic peptide preparations of the Ag. Furthermore, the Ag-presenting T cells could take up, reutilize, and re-present Ag adsorbed to the surface of histoincompatible astrocytes. Responding T cells activated by Ag-presenting T cells in the absence of other conventional APC were fully encephalitogenic upon transfer to syngeneic recipients. These results have relevance for understanding pathogenetic mechanisms in T cell-mediated autoimmunity in the central nervous system.

Oxygen-derived free radicals have been identified as the mediators of tissue injury during reperfusion in organ transplantation. Lipid peroxidation of cell membrane polyunsaturated fatty acids, generating conjugated dienes (CD), is a toxicity of oxygen-derived free radicals. The CD structure in fatty acyl moieties was measured by high-pressure liquid chromatography in samples of inferior pulmonary venous blood and pulmonary tissue to assess reperfusion injury and oxygen-derived free radical-mediated damage in a canine model of left lung allotransplantation. The cold ischemic preservation interval was 6 hours and the posttransplantation monitoring period was 6 hours. Twenty-eight size- and weight-matched adult male dogs underwent left lung allotransplantation and were randomized to receive pulmonary artery flush of modified Euro-Collins (EC) (40 mL/kg) or University of Wisconsin (UW) (40 mL/kg) solutions alone or with the addition of the platelet-activating factor antagonist BN 52021 (10 mg/kg). When employed, BN 52021 was administered to donors 30 minutes before harvest and recipients 30 minutes before reperfusion. Left and right inferior pulmonary venous blood samples were obtained at baseline before transplantation and at 1, 2, 4, and 6 hours after transplantation; tissue samples were obtained after euthanasia. Serum and tissue CD levels are expressed as mean fraction of the total hydroperoxide sample +/- standard error of the mean. At 6 hours after transplantation, the EC group's (n = 7) CD fraction was 0.28 +/- 0.03, whereas that of the EC + BN 52021 group (n = 7) was 0.12 +/- 0.03 (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of endothelial cells to activate helper T (Th) cells by antigen presentation was studied using the murine endothelial cell line SVEC4-10 and antigen-specific murine T cell clones. SEVEC4-10 cells constitutively express vascular cell adhesion molecule 1 but not intercellular adhesion molecule 1. Interferon gamma (IFN-gamma) treatment of these cells induced class II major histocompatibility complex (MHC) expression and antigen-presenting capabilities, but did not alter surface integrin expression. IFN-gamma-treated SVEC4-10 cells were competent at mediating antigen-dependent cytokine production and proliferation of a Th2 clone. In contrast, endothelial antigen presentation to Th1 cells did not stimulate T cell proliferation. The addition of MHC mismatched spleen cells as a source of costimulatory molecules resulted in the ability of the endothelial cells to stimulate Th1 cell proliferation in an antigen-specific manner. The failure of the endothelial cell line alone to support Th1 cell proliferation correlated with the failure to stimulate interleukin 2 (IL-2) gene expression. T cell exposure to the endothelial cells plus antigen resulted in upregulation of IL-2 receptors and an enhanced response to subsequent antigen presentation by splenic antigen-presenting cells. Despite the lack of functional costimulators for IL-2 expression, antigen presentation by the endothelial cell line did not induce Th1 cell anergy, indicating that costimulator deficiency for IL-2 expression is not obligatorily linked to anergy induction. Thus, endothelial cells are capable of presenting antigens to helper T lymphocytes, but stimulate only partial T cell responses. These partial responses may serve to selectively stimulate transmigration of antigen-specific T cells and may enhance functional responses upon subsequent, extravascular antigen exposure.

Objectives: Our objective in this study was to assess the basic mechanical properties of umbilical and chorionic vessels of placentas delivered after both normal and preeclamptic pregnancies., Study Design: Placentas were selected when the parturient women were admitted to the delivery room. Normal pregnancy (n = 17) was characterized by delivery at term (38 to 40 weeks) after uncomplicated pregnancy without any medication. Preeclamptic pregnancy (n = 7) was characterized by delivery after 28 weeks of pregnancy (28 to 39 weeks) after sustained hypertension and proteinuria. Arteries and veins from the umbilical cord and chorionic plate were prepared in rings for in vitro study in tissue baths. Passive and active (on stimulation by potassium chloride or serotonin) mechanical properties of these vessels were studied., Results: In vessels from normal pregnancy, responsiveness, but not sensitivity, was increased with increasing passive tension on vessels until optimal tension was reached. The passive stretch-tension curve was shifted downward in umbilical veins and upward in umbilical arteries and chorionic veins obtained from preeclamptic mothers in comparison with normal parturient women. In the absence of passive tension, contractions in response to potassium chloride were produced in all umbilical veins and some chorionic veins from preeclampsia but not from normal pregnancy. Developed wall tension curves in chorionic vessels from preeclampsia were shifted upward. In umbilical veins and arteries and in chorionic veins, the optimal passive tension was lower in tissues from preeclampsia than in tissues from normal pregnancy., Conclusion: Our results indicate that both passive and active mechanical properties of umbilical vessels are modified after pregnancy complicated by preeclampsia.

Lewis rats undergo a relapsing paralytic disease upon challenge with spinal cord emulsified in complete Freund's adjuvant (CFA). Treatment with two intracardiac injections of liposomes composed of whole myelin significantly reduced the severity of disease. Protection was disease-specific since treatment with myelin liposomes did not protect Lewis rats against adjuvant arthritis (AA), a CNS-unrelated T-cell-mediated autoimmune disease. Myelin-liposome-treated, spinal cord/CFA-immunized rats displayed borderline reduction of delayed-type hypersensitivity (DTH) (ear swelling) reactions to myelin and myelin basic protein (MBP), but significantly reduced in vitro lymphnode cell proliferation in response to these antigens. Responses to purified protein derivative of Mycobacterium tuberculosis (PPD) were not reduced, emphasizing the antigen-specific nature of the myelin-liposome-mediated suppression. Spleen cell proliferative responses were inconsistent and often poor. However, when cultured in the presence of NG-monomethyl-L-arginine (MMA), antigen-specific proliferation of spleen cells from both treated and control rats was greatly enhanced, indicating that reactive nitrogen intermediates contributed to the decrease in spleen cell proliferation. Purified splenic T cells from treated rats displayed a pattern of proliferation similar to that of unseparated lymphnode cells. Treatment of rats with a single injection of myelin liposomes after recovery from the first clinical episode significantly reduced the severity of the relapses.

Objective: The aim was to investigate vascular receptors for atrial natriuretic factor (ANF) in spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Wistar rats (WR) at different ages., Methods: Relaxation and guanylate cyclase responses of blood vessels to atrial natriuretic factor were investigated, as was the binding of 125I-ANF to vascular membranes and ANF receptor subtypes, using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in reducing conditions, after solubilisation and irreversible binding of 125I-ANF., Results: Vascular relaxation responses of aorta showed an increased sensitivity to ANF in four week old SHR [pD2 = 8.9 (SEM 0.1) v 8.5(0.1) in WKY rats, p < 0.05] while sensitivity was similar for the three strains at older ages. Production of cyclic GMP in mesenteric arteries in response to 100 nmol.litre-1 ANF was greater (p < 0.05) in SHR than in WKY rats at four weeks of age, but was similar in older rats. The density of binding sites for ANF in mesenteric arteries, however, was lower in SHR at four weeks (p < 0.01), and increased in older rats, becoming similar to that of normotensive rats at 12 weeks of age. Affinity of ANF sites was similar in all strains. The proportion of high and low molecular weight ANF binding peptides in solubilised blood vessel membranes on SDS-PAGE was similar in all strains except in four week old SHR, in which binding to the high molecular weight band (presumably the guanylate cyclase containing receptor) was increased relative to the low molecular weight band (non-cyclase-coupled receptor) in comparison to other strains and ages., Conclusions: Activity of guanylate cyclase in response to occupancy of ANF receptors may be increased in young SHR. Normal relaxation of blood vessels in response to ANF in older SHR could result in failure to counteract the increased vasoconstrictor activity present in these rats, which could play a role in the increase in blood pressure.

Platelet activating factor (PAF) is a potent phospholipid mediator of the immune and inflammatory responses, which causes physiologic effects similar to post-transplant pulmonary dysfunction. This study investigates the hypothesis that the use of a specific PAF antagonist (PAFA), BN 52021, in canine lung transplantation improves lung preservation. Twelve pairs of canines underwent left lung allotransplantation after pulmonary artery flushing with modified Euro-Collins (EC) solution (40 ml/kg). The experimental group (N = 6) received EC with BN 52021 (10 mg/kg). BN 52021 was administered to donors prior to harvest and to recipients prior to reperfusion. The preservation interval was 20 hr and the study period was 12 hr post-transplant. Differential pulmonary function and hemodynamics were monitored, comparing the transplanted left lung and the native right lung. Recipients were ventilated on 100% O2. Administration of the platelet activating factor antagonist, BN 52021, was associated with improvement in transplant lung oxygenation, pulmonary vascular resistance, and compliance. At 12 hr, transplant lung pulmonary venous oxygen tension in the treatment group (EC + BN 52021) was 154 +/- 21 mm Hg versus 87 +/- 10 mm Hg in the control group (EC) (P less than 0.05). Pulmonary vascular resistance of the transplant lung at 12 hr was 146 +/- 24 Dynes.sec.cm-5 in the EC + BN 52021 group as compared to 320 +/- 51 Dynes.sec.cm-5 in the EC group (P less than 0.05). Dynamic pulmonary compliance of the transplant lung at 12 hr was 32 +/- 2.9 ml/cm H2O in the EC + BN 52021 group versus 13 +/- 2.0 ml/cm H2O in the EC group (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cryopreserved allograft valves are increasingly being used as valvular replacements. Leaflet fibroblast viability has been suggested to influence clinical durability. The warm ischemic time is thought to be a critical determinant of this cell viability. The purpose of this study was to apply quantitative morphometric methods to characterize, by transmission electron microscopy, valvular cellular injury resulting from progressive warm ischemic time. Porcine aortic valves were harvested with a spectrum of warm ischemic times (40 minutes and 2, 6, 12, 24, and 36 hours; five valves per warm ischemic time; n = 30) and processed by standard electron microscopic methods. To ensure randomized tissue selection within each warm ischemic time interval, we randomly selected one thin section from each leaflet. The first ten cells in each thin section were photographed and cellular injury was assessed (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes). Nine hundred micrographs have been analyzed by Cochran-Mantel-Haenszel statistics to determine if a significant association between warm ischemic time and cellular injury exists. Our findings indicate a significant association between reversible cell injury through 24 hours of warm ischemic injury (p less than 0.0001). Furthermore, a significant association between irreversible cell injury and progressive warm ischemia through 36 hours was also found. These findings indicate that the ischemic interval after donor death is associated with progressive leaflet cell injury. Cellular damage begins shortly after donor death and continues incrementally throughout 36 hours. After 2 hours of warm ischemic injury 37% of the cells had morphologic evidence of injury. After 6 hours of warm ischemic injury the number of injured cells increased to 73%. By 36 hours 22% of the cells appeared normal. Irreversible cell injury increases with prolonged ischemia and becomes quantitatively impressive at 24 hours, by which time 26% of cells are so affected. Conversely, some cells are resistant to irreversible injury for a prolonged ischemic interval.

Pregnancy is associated with decreases of blood pressure and vascular sensitivity to vasopressor agents. We have hypothesized that the increased liberation of endogenous vasodilator(s) by the vascular endothelium or other structures could mediate these blunted responses. Thoracic aorta rings of nonpregnant, 21 days pregnant, and first day post partum rats respond similarly to acetylcholine, an endothelium-dependent vasorelaxant. In contrast, the potency of the response to sodium nitroprusside, an endothelium-independent vasorelaxant, is unchanged in tissues of pregnant rats and increased (p less than 0.05) in those of post partum animals. In the presence of indomethacin (10 mumol/L) the three groups of tissues show a decreased potency. The effects of phenylephrine on aortic rings of both nonpregnant and pregnant rats are markedly increased in the presence of Ng-monomethyl-L-arginine. Indeed, the concentration producing 50% of the maximum response of phenylephrine decreases (p less than 0.001) from 50.7 to 8.02, from 93.8 to 37.6, and from 60.4 to 5.97 nmol/L with the use of Ng-monomethyl-L-arginine (0.1 mmol/L) in rings from nonpregnant, pregnant, and postpartum rats, respectively. Simultaneously, the maximum response to phenylephrine increases markedly in the three groups of tissues. In the presence of Ng-monomethyl-L-arginine, indomethacin does not influence the response to phenylephrine. Our results do not support the possible involvement of an endogenous vasodilator (prostaglandin-like or endothelium-derived) in the blunted responses to vasoconstrictors during pregnancy.

SJL/J mice challenged with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) developed only mild chronic-relapsing experimental allergic encephalomyelitis (EAE) with very low incidence. However, treatment of challenged mice with anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb) determined severe disease in all cases. Similarly, in passive EAE, the addition of anti-IFN-gamma to the in vitro MBP-activated cells at the time of transfer led to significant disease exacerbation in all recipients. The disease enhancing effect was observed only when the mAb was given at the time of active challenge or of passive transfer, but not at later times. Anti-interleukin-2 (IL-2) antibody had only a marginal effect in the active induction, but drastically reduced the manifestations of passive EAE, even when mixed with a disease-enhancing dose of anti-IFN-gamma. These findings support the notion that IL-2 is required for disease induction whereas IFN-gamma plays a disease-limiting role early in the development of EAE.

Ross' first homograft replacement of the aortic valve was reported in 1962. The homograft has been in continuous use around the world ever since. Much has been learned about how to handle homografts, both before and during their implantation. Homografts have special advantages, which make them an appropriate choice in a number of settings. This first successful case by Donald Ross set the stage for the growth in homograft valve use and the subsequent development of many ways of using allograft cardiovascular tissue for optimal cardiac reconstructions.

Numerous experimental studies have reported that common antihypertensive drugs such as diuretics, beta-blockers, and methyldopa have adverse effects on plasma lipids and lipoproteins. The present study was designed to define the effect of clentiazem (10 mg/kg/day) an antihypertensive drug, on hyperlipidemia in rabbits on a cholesterol-rich diet (1%) for 12 weeks. Compared with controls, clentiazem treated rabbits had lower plasma concentrations of triglycerides (55%), total cholesterol (24%), free cholesterol (27%), esterified cholesterol (23%) and phospholipids (24%). The decrease in cholesterol was accounted for by a reduction of VLDL-cholesterol (13%), IDL-cholesterol (24%) and primarily LDL-cholesterol (45%). Neither HDL-cholesterol nor chemical composition of VLDL, IDL, LDL and HDL was altered. When the aortic atherosclerotic involvement was evaluated by computerized planimetry, a 24% reduction of lesions was noted in clentiazem treated animals (P less than 0.05). Similarly, cholesterol content extracted from aortic wall was decreased. Our data therefore demonstrated that clentiazem is a potential antiatherosclerotic agent capable of decreasing plasma lipids and atherogenic lipoproteins as well as aortic fatty streaks.

Blood pressure and sensitivity of blood vessels to vasoconstrictors are decreased in term-pregnant rats (20-21 days). To determine if changes in receptors for vasoactive peptides could account for these observations, receptor kinetics were measured for Arg8-vasopressin (AVP), angiotensin II (Ang II), and atrial natriuretic peptide (ANP) in the mesenteric vascular bed of the rat throughout pregnancy. Receptors for AVP were statistically similar in the five groups of animals (nonpregnant; pregnant 9, 15, and 21 days; and postpartum). The dissociation constant (KD) for [3H]AVP varied from 0.41 to 0.52 nmol/L (NS), while receptor density (Bmax) varied from 310 +/- 110 to 455 +/- 135 fmol/mg protein for six experimental measurements. Similar observations were made for Ang II receptors where KD of 125I-labelled Sar1, Ile8-Ang II was between 0.60 and 0.97 nmol/L and Bmax between 215 +/- 30 and 250 +/- 40 fmol/mg protein in the different groups. 125I-labelled ANP (101-126) receptors were markedly modified in terms of number of sites. Bmax was significantly increased during pregnancy (9 days, 429 +/- 86; 15 days, 541 +/- 54; 20 days, 438 +/- 72) and decreased in the postpartum period (133 +/- 21) by comparison with the nonpregnant group (245 +/- 35 fmol/mg protein), while KD was similar in the different experimental groups (57 to 82 pmol/L). Despite these increases in receptor density, the vasorelaxant effects of ANP was only increased at 9 days of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

The clinical use of cryopreserved allograft valves is rapidly increasing. Viability of valve leaflet fibroblasts has been proposed to be critical to durability. Harvesting of allograft valves involves variable warm ischemia times, defined as the time from cessation of donor heart beat to initial cooling for transport. This ischemic period has been implicated as one of the more critical periods of injury to leaflet cell, even though adequate characterization of this potentially injurious phase has never been accomplished. The present study was undertaken to characterize the metabolic response to warm ischemia in a porcine valve leaflet model. Valve handling was similar to clinical valve harvest and transport protocols. Injury was assessed by protein (1H) and phosphorus (31P) magnetic resonance spectroscopy of 224 porcine semilunar valves. Leaflets were analyzed over time for lactate accumulation and ATP degradation. A radiolabelled incubation assay (48 valves) was used to measure proline accumulation by fibroblasts. Electron microscopy was performed on 36 valves with varying warm ischemia times. ATP stores were entirely depleted after 2 h hypoxia (p less than 0.05). However, lactate continued to accumulate over 24 h. Although aerobic metabolism ceased after 2 h warm ischemia, anaerobic metabolism continued for up to 24 h, which may represent an extended window for harvesting fresh tissue for allograft valve implantation.

Chronic-relapsing experimental allergic encephalomyelitis (CR-EAE) in the Lewis rat, induced by the injection of spinal cord tissue in complete Freund's adjuvant (SC/CFA), was studied in vivo by treatment with liposomes containing central nervous tissue antigens, and in vitro by lymphocyte proliferation assays. Intracardiac administration of myelin basic protein (MBP) liposomes, galactocerebroside (GC) liposomes, or MBP + GC liposomes substantially reduced the clinical severity and/or delayed the onset of the initial phase of disease. Liposomes prepared from whole myelin provided even greater protection, and were effective at suppressing both the first disease episode and the relapses. These results indicate that while GC and MBP may play significant roles in the development of CR-EAE in the Lewis rat, immune responses to other antigens are probably also involved. Splenic and lymph node lymphocytes from MBP-GC liposome-treated rats, and splenic lymphocytes from cytochrome-GC (CYT-GC) liposome-treated rats, showed drastically reduced abilities to proliferate in response to MBP in culture. Spleen cells from both the MBP-GC- and CYT-GC-liposome-treated donors were able to actively suppress antigen-induced proliferation of MBP-primed lymphocytes. These findings suggest participation of both clonal anergy, and active suppressor cells in the liposome-mediated suppression of CR-EAE in the Lewis rat.

Responses to adrenergic agents were measured on rings of mesenteric, artery of nonpregnant and pregnant rats at 21 days' gestation. In tissues from pregnant rats, norepinephrine, and phenylephrine were less sensitive than in rings from nonpregnant animals; maximum responses were not affected. The effect of isoproterenol was not modified in tissues from pregnant rats. The presence of endothelium in tissue rings was evaluated as fractional relaxation to acetylcholine. Sensitivity to phenylephrine and norepinephrine was inversely related to the effect of acetylcholine. This relationship was similar in slope in both groups but different in intercept. The sensitivity to isoproterenol was directly related to the presence of endothelium (curves similar in both slope and intercept). This demonstrates that the responses of mesenteric artery rings depends on the presence of endothelium. Furthermore, vascular endothelium is not involved in the blunted responses to adrenergic vasoconstrictor during pregnancy.

In previous experiments, we showed that administration of myelin basic protein (MBP) inserted into phosphatidyl-serine liposomes, to susceptible animals suppressed the clinical manifestations of both acute and chronic-relapsing EAE. In this report we compare the effectiveness of treatment with MBP-liposomes and with MBP-coupled syngeneic spleen cells in EAE protection. Lewis rats treated with 150 micrograms MBP-liposomes or with 160 micrograms (35 x 10(6] MBP-coupled spleen cells, given 7 days before and 7 days after encephalitogenic challenge were equally protected against clinical EAE, when compared to untreated controls. In addition to clinical protection, in vitro proliferative responses of lymphocytes from treated rats were significantly reduced, but delayed hypersensitivity (DTH) reactions remained unaffected. Proliferation of lymphocytes from MBP-sensitized donors was inhibited by the addition of spleen cells but not of lymph node cells from treated donors. The inhibitory effect was observed with spleen cells regardless of whether the donors were treated or not, was antigen nonspecific, and localized in a radio-resistant, adherent cell population. Adoptive transfers of spleen cells from treated donors, after a 48-hr in vitro incubation with concanavalin A, showed that the cells from donors treated with MBP-coupled spleen cells, but not with MBP-liposomes, suppressed the disease in recipients, following challenge with MBP-complete Freund's adjuvant (CFA). These results suggest that two distinct mechanisms operate in the protection by MBP-coupled cells and MBP-liposomes, respectively.

Recent evidence suggests that angiotensin II (ANG II) and vasopressin (AVP) act on the liver via specific receptors. We have examined the binding properties of these receptors in isolated rat hepatocytes and studied the regulation of the biological responses to ANG II and AVP during pregnancy in the rat. In contrast to [3H]ANG II, 125I-labeled-[Sar1-Ile8]ANG II was markedly resistant to degradation by isolated liver cells. Displacement and saturation experiments with this iodinated antagonist revealed the presence of a single class of binding sites [2 x 10(5) sites/cell, dissociation constant (KD) = 1.0 nM]. The potency of ANG II analogues to displace 125I-[Sar1-Ile8]-ANG II agrees closely with data reported for vascular smooth muscle cells. Isolated hepatocytes have approximately 8 x 10(4) [3H]AVP binding sites/cell (KD = 1.0 nM) based on saturation experiments. AVP analogues selectively displaced [3H]AVP, suggesting the presence of V1-AVP receptor subtype. The maximum response of [Sar1]ANG II-induced glycogenolysis in the cells was decreased during gestation, whereas the effective concentration producing 50% of maximum response (EC50) was significantly increased (0.15-0.28 nM) when compared with cells from nonpregnant animals. In pregnancy, receptors for 125I-[Sar1-Ile8]ANG II were not changed in affinity (KD) or in density (Bmax). The maximum response and EC50 of AVP on liver glycogenolysis were not significantly decreased during pregnancy, whereas an increased number of AVP binding sites (from 5.0 +/- 0.5 x 10(4) to 11.0 +/- 1.7 x 10(4)) with similar KD was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

To understand the regulation of vasopressin (AVP) receptors in spontaneous hypertension, we investigated the pressor response of AVP in the perfused mesenteric vasculature, AVP binding sites in the membrane preparation of the same vascular bed, and the production of inositol trisphosphate (InsP3) stimulated by AVP in the aorta of spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and Wistar rats (WR) at different ages (4-16 weeks). Plasma AVP concentrations were similar in SHR, WKY, and WR at all ages. The density of AVP vascular binding sites was significantly higher in WKY than in SHR and WR at 12 weeks. Receptor affinity was similar in all strains. The pressor response of the mesenteric vasculature to AVP was similar in the three strains of rats at 4 weeks (prehypertensive stage) and increased progressively in SHR compared with WKY and WR at 8 and 12 weeks of age by 43 and 35%, respectively, and by more than 80% at 16 weeks of age (established hypertensive stage). There was no difference in vascular sensitivity to AVP. A significantly increased pressor response to a supramaximal dose of norepinephrine was also found at 16 weeks in SHR, but not in younger rats. InsP3 production in the aorta in response to AVP was increased in SHR at 8, 12, and 16 weeks, compared with WKY and WR. These results suggest that the vascular response to AVP is increased in SHR, in spite of decreased or normal density of binding sites compared with WKY or WR.(ABSTRACT TRUNCATED AT 250 WORDS)

Human myelin basic protein (MBP) was inserted into phosphatidyl-serine liposomes and Hartley guinea pigs were treated with 1 or 2 injections of MBP-liposomes mixture by the intracardial route before an encephalitogenic challenge with MBP in CFA. Other groups of guinea pigs were pretreated with equivalent amounts of MBP in saline or of MBP in IFA. Of 24 untreated animals, 22 developed EAE and died or had to be sacrificed within 15 days after challenge. Only 4 of 29 guinea pigs treated with either 75 microgram or 112 microgram MBP-liposomes developed clinical signs of the disease. In the group pretreated with MBP in saline, 11 of 15 animals died, whereas in the MBP-IFA group, only 4 out of 10 died. Histologic modifications were also decreased in the MBP-liposomes and MBP-IFA groups, but in many instances, clinical normal guinea pigs showed disseminated perivascular infiltrates in the brain and spinal cord. Delayed hypersensitivity reactions to MBP were positive in all but 1 animal tested. Early T-rosette levels followed essentially the clinical course of the disease: they were drastically decreased 7 days after challenge in the untreated and MBP-saline-treated groups, but remained essentially normal in the MBP-liposomes group throughout the experiment. Lymphocyte transformation tests carried out in parallel with soluble MBP and with MBP-liposomes indicated that animals in certain groups responded preferentially to 1 form or the other of the antigen. Compared with prevailing procedures, the single i.v. injection of a relatively small amount of liposome-associated MBP appears to represent a promising approach for the antigen-specific suppression of EAE.

The effect of treatment with estrogens on the biological activity of arginine8 vasopressin (AVP) in the in vitro perfused mesenteric vascular bed and on the binding characteristics of [3H]AVP on membranes prepared from the same vascular bed was studied. Female rats treated with estradiol (400 micrograms/24 h sc), compared with ovariectomized rats, had an increase in the maximum response to AVP (from 128 +/- 3 to 153 +/- 3 mmHg) in the perfused preparation and an increase in the density of AVP binding sites (from 402 to 732 fmol/mg protein) in the membrane preparation. In male rats, the injection of estradiol increased the maximum response to AVP (from 109 +/- 4 to 137 +/- 3 mmHg) and the density of AVP binding sites (from 289 to 519 fmol/mg protein). The effective concentration producing 50% of maximum response of AVP in the perfused preparation was higher in male than in female rats, while the Kd in the binding experiments was similar in the four experimental groups. Our results show that estrogens upregulate the number of AVP binding sites, leading to an increase in the pressor response to AVP in the rat mesenteric vascular bed.

Polyunsaturated fatty acids of the omega-6 and the omega-3 series have been shown to lower arterial pressure in humans and in various models of experimental hypertension by uncharacterized mechanisms. The objectives of our study were to compare the antihypertensive properties of linoleic acid (omega-6 series) and of fish oil fatty acids (omega-3 series) in a model of hypertension induced by the continuous subcutaneous infusion of angiotensin II in the rat and to determine whether or not their antihypertensive effects were mediated by the biosynthesis of vasodilator prostaglandins of classes 2 or 3. Linoleic acid and fish oil fatty acids (administered by subcutaneous injections) were equally potent in reducing, by half, the rise in systolic arterial pressure induced by the chronic infusion of angiotensin II. These antihypertensive effects were observed in the absence of any significant influence of either linoleic acid or fish oil fatty acids on the systemic and the renal synthesis of PGI2 or on the renal formation of PGE2 in vivo. Indomethacin caused a profound inhibition of the biosynthesis of PGI2 but not of PGE2 and could only partially neutralize the antihypertensive effects of linoleic acid and of fish oil fatty acids. These results suggest that, in this model of angiotensin II-induced hypertension, linoleic acid and fish oil fatty acids exert equipotent antihypertensive effects which are mainly independent of the prostaglandin system.

The present experiments were designed to elucidate the pharmacological mechanism of the relaxing action of the ovarian hormone, relaxin, upon KCl-contracted rat uteri in vitro. Rat uterine segments were made to contract by superfusion with a physiological solution, in which 15% of the normal NaCl content had been replaced by an equimolar amount of KCl. The effect of relaxin, in reversing this contraction, was achieved in part by a direct action upon the tissue and in part (about 45% at the concentration of relaxin used) by the liberation of endogenous catecholamines which relax the uterus via beta-adrenergic activity. The effect of relaxin was reduced by propranolol, and it was not blocked by indomethacin, cimetidine or diphenhydramine. The effect of the ovarian peptide on uterine segments was potentiated in the presence of theophylline and indomethacin in the superfusing fluid. This effect of theophylline was shown to be insensitive to beta-adrenergic blockade. The results suggest that relaxin relaxes the KCl-contracted rat uterus in part by a direct inhibitory action upon the uterine muscle and in part by facilitating the release of intramural catecholamines. It is also suggested that relaxin may act via the production of cyclic AMP.

Eight patients with myelodysplastic syndromes (MDS) were treated with bone marrow transplantation (BMT). Median age was 34.5 years and ranged between 3 and 45. FAB diagnosis was refractory anaemia (RA) in three, RA with excess of blasts (RAEB) in four and RAEB in transformation (RAEB-t) in one case. Four patients were prepared with cyclophosphamide and total body irradiation whilst the other four received busulphan and cyclophosphamide. Engraftment was documented in seven of eight patients. Two patients died from complications related to the procedure. One had early veno-occlusive disease of the liver whilst the other died 46 months after BMT from pulmonary fibrosis. One patient died from recurrent disease 11 months after BMT. Five patients are alive and in complete remission 9-35 months post-transplantation. Four of these patients have a Karnofsky score greater than or equal to 90%. These results suggest that BMT can induce prolonged disease-free survival in patients under 50 years of age. If a compatible donor is available, marrow transplantation should be seriously considered in the treatment of MDS.

We have recently demonstrated specific binding for 3H-arginine8-vasopressin (3H-AVP) to high affinity sites on membranes of rat mesenteric arteries. We have now measured the biological activity of this peptide (AVP) and analogues on the perfused rat mesenteric artery. There was a close relationship between the ED50 of agonists or the pA2 of antagonists on the perfused tissue and the relative potency (IC50) of analogues for displacing 3H-AVP from the membrane preparation. The ED50 measured was 67 +/- 7 ng for AVP and 7.2 +/- 1.1 microgram for oxytocin. In sodium-depleted rats we have observed an increase (27%) of the maximal response to AVP with no significant change in ED50 (from 2.8 +/- 1.0 X 10(-8) M to 1.3 +/- 0.2 X 10(-8) M). On the membrane preparation, the number of binding sites for 3H-AVP was increased from 71 +/- 17 fmole/mg protein (Kd 3.5 +/- 0.5 nM) to 115 +/- 10 fmole/mg protein (Kd 4.8 +/- 0.3 nM) in the sodium-depleted rat by comparison to control animals. These results suggest that AVP and its analogues interact in a similar manner in the in vitro perfused rat mesenteric artery and with the membrane receptors isolated from the same tissue. Receptors for AVP are increased in the mesenteric vascular bed by sodium depletion.

To investigate the role of atrial natriuretic peptide (ANP) in pregnancy, we measured, in cyclic and pregnant female rats (9- and 21-days pregnant), the vascular responsiveness to ANP using helical strips of the thoracic aorta, the binding characteristics of 125I-labeled ANP in a membrane preparation of the mesenteric vascular bed, and the plasma level and the atrial content of immunoreactive ANP (IR-ANP). On aorta strips, concentration-response (C-R) curves to phenylephrine (PE) were measured and were slightly displaced to the right in the aorta of both groups of pregnant rats in comparison with the cyclic rats. There was a potentiation of the relaxant response of ANP on the PE-precontracted aortic strips of 9-day pregnant rats but it was not statistically modified in tissues of 21-day pregnant rats in comparison with strips from cyclic rats. The number of binding sites (Bmax) for ANP in the mesenteric vascular bed was similar in cyclic rats and the two groups of pregnant rats. The dissociation constant (KD) of ANP was lower in 9-day pregnant rats than in cyclic and 21-day pregnant ones. Plasma IR-ANP was not different in 9-day pregnant rats and cyclic rats but was markedly decreased at the end of gestation. Atrial content of IR-ANP increased at the end of gestation, but not in midpregnancy in comparison with cyclic rats. These results indicate that despite the reported important increase in blood volume during gestation the secretion of ANP is not increased and suggest that the ANP-volume relationship is reset during pregnancy in the rat.

The inhibitory effect of atrial natriuretic peptide on the myotropic action of phenylephrine on superior mesenteric artery and thoracic aorta rings was studied to test the hypothesis that this peptide interferes with the mobilization of intra- or extra-cellular calcium produced by vasoconstrictor agents. In the absence of calcium in the bathing solution, phenylephrine (10(-6) M) produced a residual effect, which was antagonized in a concentration-dependent manner by the atrial peptide in both mesenteric artery and aorta rings. When calcium (2.5 mM) was added to the bathing solution after the response to phenylephrine in the absence of calcium, a further increase in the tonus of the tissue was observed. This effect was also antagonized by atrial natriuretic peptide in a dose-dependent manner in the two tissues. These results suggest that atrial natriuretic peptide inhibits the effect of vasoconstrictor agents by functionally interfering with the mobilization of intra- and extra-cellular calcium produced by these vasoconstrictors.

The plasma concentration of atrial natriuretic peptide (ANP) and the density and affinity of binding sites for ANP in platelets was investigated in patients with essential hypertension. Severe hypertensives were studied whilst still on medication. All subjects were ambulatory and were investigated after 3 days on a 135 mmol/day sodium intake. Plasma ANP levels were significantly increased from 13 +/- 1 fmol/ml in healthy normotensive controls to 39 +/- 5 fmol/ml (P less than 0.01) in moderate or severe hypertensives uncontrolled by treatment. Platelet binding sites varied in a non-linear inverse relationship to plasma concentration of ANP (r = -0.76), from 14 +/- 1 fmol per 10(9) platelets in healthy subjects to 8 +/- 1 fmol per 10(9) platelets in severe hypertensives, uncontrolled by treatment, (P less than 0.05). The latter group with elevated plasma ANP and reduced density of ANP platelet sites, had a high incidence of left ventricular hypertrophy and increased left ventricular mass index by echocardiography. When a diuretic was added or stopped, plasma ANP and ANP sites in platelets varied inversely, with lower ANP concentration in plasma and higher density of ANP receptors in platelets when receiving the diuretic. Plasma concentrations of ANP are increased and the density of ANP receptors is decreased in moderate to severe uncontrolled essential hypertensives with left ventricular hypertrophy, but neither parameter differs from those of healthy age-matched volunteers in mild essential hypertension.

Guinea pig myelin basic protein (MBP) was inserted into phosphatidylserine liposomes and Lewis rats were injected by the intracardiac (ic) route with 75 microgram doses of MBP-liposomes according to various schedules. After challenge with 75 microgram guinea pig MBP in complete Freund's adjuvant, the rats were followed for clinical signs, were tested for delayed hypersensitivity (DTH) and lymphocyte transformation (LT) to MBP. The animals were sacrificed 30 days after challenge and the central nervous system tissue was examined for histological modifications. Rats treated with two injections of MBP-liposomes, 7 days before and 7 days after challenge, showed the highest degree of protection from clinical manifestations. Histological lesions were not significantly reduced. DTH reactions to MBP were all positive, regardless of treatment. LT assays were positive overall in only 50% of the animals tested. The response to rat MBP was significantly lower than to guinea pig MBP, especially in the groups treated with MBP-liposomes. Adoptive transfer of spleen cells from MBP-liposome-treated donors reduced the clinical scores of actively induced EAE in syngeneic recipients by 40-50%. These results suggest that at least one mechanism responsible for antigen-specific protection in EAE by MBP-liposomes operates through active suppression transferable by spleen cells.

Vasorelaxant effects of different atrial natriuretic peptides (ANP) were measured on rat aortic strips and mesenteric artery rings. These results were compared with the potency of the same peptides to displace 125I-labelled ANP (101-126) on membrane preparations of aorta and of mesenteric vascular bed. In aortic strips and mesenteric artery rings precontracted with phenylephrine (3 X 10(-8) and 10(-6) M, respectively), the order of potency of ANP was as follows: ANP (99-126) greater than ANP (101-126) greater than ANP (103-126) = ANP (103-125) much greater than ANP (103-123). In the displacement binding assays, the order of potency of ANP peptides was similar to that of the relaxation experiments: ANP (99-126) = ANP (101-126) greater than ANP (103-126) = ANP (103-125) much greater than ANP (103-123). When the vessels were precontracted by a smaller concentration of phenylephrine (10(-7) M in mesenteric artery and 10(-8) M in aorta), the IC50 of ANP (101-126) was significantly lower than when the higher concentration of phenylephrine was used. These results show that ANP receptors in the mesenteric artery and in the aorta have similar structural requirements, according to the order of potency of different length ANP, both for binding and myotropic responses.

Relaxin is an ovarian polypeptide hormone which is present in large amounts in the rat during the second half of gestation. During this period, blood pressure declines markedly, especially in spontaneously-hypertensive rats (SHR). To test the hypothesis that relaxin might be implicated in this decrease in blood pressure, we infused the hormone in female non-pregnant rats by means of an osmotic mini pump. Our results show that intravenous infusion of purified rat relaxin (1.8 micrograms/day) markedly reduced systolic blood pressure for at least 5 or 6 days in SHR. This decrease was highly significant from 24 hours after the beginning of the infusion and remained significant after 5 days. Rat relaxin was ineffective in control Wistar-Kyoto rats (WKY). Infusion of purified porcine relaxin (3.0 micrograms/day) also diminished blood pressure in SHR, but the effect was less pronounced and developed more slowly, reaching statistical significance on the fourth day of infusion. SHR not receiving relaxin maintained their original systolic blood pressure throughout the experiment. These results indicate that relaxin is involved in the regulation of blood pressure during gestation.

Relaxin (RLX), an ovarian polypeptide hormone that is particularly associated with gestation in viviparous species, has recently been shown to decrease blood pressure in virgin spontaneously hypertensive rats (SHR) upon chronic infusion. In this investigation, vascular reactivity to angiotensin II, arginine-vasopressin, and norepinephrine was studied in the perfused mesenteric artery and isolated portal vein of control and RLX-treated virgin spontaneously hypertensive rats. The latter received an intravenous infusion of 75 ng/hr purified rat RLX for 2 days, whereas the controls were given an equal infusion of saline. All of the animals were then killed and their tissues processed for in vitro study. In the perfused mesenteric artery, the concentration-response curves for arginine-vasopressin and norepinephrine were shifted to the right by a factor of about 2 (P less than 0.05 and P less than 0.005, respectively) after RLX treatment. In the isolated portal vein, the response to angiotensin II was not affected; the effect of norepinephrine was slightly displaced to the right (increase in EC50) and the maximum response remained unchanged. These results demonstrate that RLX treatment for 42 hr blunted the vascular response to vasoconstrictor agents in the mesenteric vasculature and are consistent with similar observations reported previously in the same tissue of 20-day-old pregnant rats. It is concluded that RLX may be involved in the blunted response to vasoconstrictor agents during gestation in the rat.