Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo.

The lipid mediator lysophosphatidic acid (LPA) regulates cell proliferation and enhances cell motility in vitro, both of which are important events in wound healing. To evaluate the effects of LPA in vivo, it was applied to a full-thickness wound of rat skin. LPA in micromolar concentrations, or solvent, was applied daily. Animals were killed at 1, 3, 6, and 9 days after wounding and processed for histological evaluation, including hematoxylin-eosin staining and histochemical markers for macrophage-histiocytes, proliferating cells, and capillary endothelial cells. LPA treatment accelerated wound closing and increased neoepithelial thickness. Cytological evaluation showed no evidence for a secondary inflammation-mediated injury, infection, or increased keloid formation. Whereas LPA caused only a modest dose-dependent increase in proliferating cells, a marked increase in the immigration of histiocyte-macrophage cells was observed as early as day 1. The peaks of several cytological features and immunohistological markers preceded those of the untreated side. Our data suggest that exogenously applied LPA in this model promotes healing and that macrophage-histiocytes are the primary LPA-responsive cells in vivo.

Rho proteins play a critical role in cell migration during the early phase of mucosal restitution.

In the intestine, several growth factors stimulate migration of epithelial cells, contributing to the maintenance of tissue integrity. The Ras-like GTPase Rho regulates a signal transduction pathway linking growth factor receptors to the formation of actin stress fibers and focal adhesions, presumed to be important for motility. Using an in vitro wound-induced migration assay, we have examined the role of Rho GTPases in the migration of IEC-6 and Caco-2 cells, and provide evidence that the Rho GTPases play an essential role in the initial phase of mucosal wound healing. Treatment of the cells with Clostridium difficile toxins A and B, inhibitors of the Rho family GTPases inhibited migration in a dose-dependent fashion. Microinjection of the inhibitory exchange factor Rho-guanine nucleotide dissociation inhibitor (GDI), or Clostridium botulinum C3 ADP-ribosyl transferase (C3) toxin, a Rho-ADP-ribosylating exoenzyme, potently inhibited migration. Microinjection of RhoT19N, a dominant negative form of RhoA, or in vitro ADP-ribosylated RhoA impaired the ability of cells to migrate. Rho-GDI and C3 exoenzyme also inhibited EGF-induced migration of IEC-6 cells. These results demonstrate that Rho is required for endogenous and EGF-induced migration of small intestinal crypt cells, and that Rho proteins are essential elements of a mechanism by which growth factors induce cell migration to restitute mucosal integrity.

Clearance receptor and neutral endopeptidase-mediated metabolism of atrial natriuretic factor.

A novel small linear C-atrial natriuretic factor receptor ligand [C-ANF-(11-15)] and phosphoramidon (PHO) were used to determine the effects of C-ANF receptor blockade alone, or in combination with inhibition of neutral endopeptidase (NEP), on the pharmacokinetics and metabolism of ANF in the rat. C-ANF-(11-15) infusion decreased apparent volume of distribution (Vss) and metabolic clearance rate (MCR) of administered 125I-ANF-(1-28) to one-third of their control values, whereas PHO alone was without effect on these parameters. In combination with C-ANF-(11-15), however, PHO further decreased MCR of 125I-ANF-(1-28) and increased plasma half time by more than threefold. High-performance liquid chromatography analysis revealed that C-ANF-(11-15) inhibited the delayed appearance of free 125I and [125I]monoiodotyrosine but had no effect on the small proportion of NEP metabolites in plasma. The combination of C-ANF-(11-15) and PHO further delayed the appearance of small metabolites, abolished the appearance of NEP metabolites, and markedly prolonged the permanence of intact 125I-ANF-(1-28) in plasma. The results demonstrate that C-ANF receptor blockade by C-ANF-(11-15) impairs clearance and metabolism of ANF, an effect which is synergistically potentiated by concomitant inhibition of NEP. C-ANF-(11-15) alone or in combination with NEP inhibitors may be a potentially useful therapeutic tool in the treatment of cardiovascular and renal diseases.

Renal receptors and effects of atrial natriuretic factor in compensatory renal hypertrophy.

In the present study we investigated the in vivo and in vitro renal responsiveness to ANF, and the adaptation of ANF receptors in compensatory renal hypertrophy in the rat. One week after left nephrectomy (UNx), plasma levels of immunoreactive ANF, blood pressure (MAP), hematocrit (Hct), and urine flow rate (V) were unaltered compared to control (C) rats. Baseline GFR and potassium excretion (UKV) were significantly higher, and sodium excretion (UNaV) tended to be elevated in UNx rats. Administered ANF led to similar dose-related decreases in MAP and increases in Hct in UNx and C rats. However, at each dose of infused ANF, absolute values and the increase in GFR and UNaV were higher in UNx than in C rats. Hypertrophied (H) kidneys were removed from UNx and perfused in vitro to determine distribution and density of ANF receptors, responsiveness to ANF, and receptor-mediated organ clearance of 125I-ANF1-28. The density of ANF receptors in cortex, outer medulla, and papilla of H kidneys was not significantly different from that in C kidneys. In H isolated kidneys, ANF led to dose-related increases in GFR, V, UNaV, and UKV that were indistinguishable (P greater than 0.05) from those in C kidneys. Receptor-mediated organ clearance of 125I-ANF1-28 in isolated H kidneys was 2.8 +/- .02 ml/min, a value not significantly different (P greater than 0.05) from that in C kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamics of atrial natriuretic factor-guanylate cyclase receptors and receptor-ligand complexes in cultured glomerular mesangial and renomedullary interstitial cells.

The dynamics of the guanylate cyclase receptor of atrial natriuretic factor (GCA-ANF receptor) were investigated in cultured glomerular mesangial and renomedullary interstitial cells from the rat. In these cells, the GCA-ANF receptor did not mediate internalization and lysosomal hydrolysis of 125I-ANF1-28 and did not undergo ligand-induced endocytosis. Glomerular mesangial cells were able, however, to mediate internalization and lysosomal hydrolysis of 125I-ANF1-28 via clearance ANF (C-ANF) receptors and to promote rapid receptor-mediated internalization and lysosomal hydrolysis of 125I-(Sar1) angiotensin II. Radioligand specifically bound to surface GCA-ANF receptors was rapidly dissociated at 37 degrees C (k(off) greater than 0.8 min-1), with a Q10(30-37 degrees C) greater than 6. The dissociation was markedly slower at subphysiological temperatures (Q10(4-30 degrees C), 2-3) or in the presence of 0.5 mM amiloride. The results demonstrate that the GCA-ANF receptor, contrary to C-ANF receptors and most other polypeptide hormone receptors, is a membrane resident protein that does not mediate internalization and lysosomal hydrolysis of ligand. The termination of the interaction of ANF with GCA-ANF receptors results from a physiological process that leads to rapid dissociation of receptor-ligand complexes. The unique dynamics of GCA-ANF receptor-ligand complexes are likely to contribute importantly to stimulus-response homeostasis of ANF.

Effects of small C-ANF receptor ligands on plasma levels of atrial natriuretic factor, blood pressure, and renal function in the rat.

In this article, after a very brief review on ANF receptors, we report our study on the effects of small C-ANF receptor ligands in the rat. Two small ligands were synthesized: 2-naphthoxyacetyl-isonipecotyl-rANF11-15-NH2 (5 aa), containing 5 amino acids; and Ala7-rANF8-17-NH2 (10 aa), containing 10 amino acids from the ring structure of ANF1-28. After control periods, 5 aa or 10 aa were infused i.v. at a dose of 10 micrograms.min-1.kg-1 body weight for 70 min in anesthetized rats, followed by a 60-min recovery period. The 5 aa and 10 aa peptides significantly and reversibly increased plasma levels of endogenous immunoreactive ANF by 106 +/- 29 and 52 +/- 24 pg/mL, respectively. Infusion of the 5 aa peptide significantly decreased mean arterial blood pressure from 113 +/- 1 to 100 +/- 3 mmHg (1 mmHg = 133.32 Pa) and increased glomerular filtration rate from 1.6 +/- 0.2 to 2.3 +/- 0.2 mL/min, sodium excretion from 0.6 +/- 0.3 to 3.4 +/- 0.4 mumol/min, and potassium excretion from 0.5 +/- 0.2 to 1.2 +/- 0.2 mumol/min. Similar results were obtained with the 10 aa peptide. The effects of both peptides on blood pressure and sodium excretion persisted throughout the recovery period. The results confirm and extend previous observations showing that C-ANF receptors mediate the removal of ANF from the circulation. The shortening of the minimal peptide length necessary to bind to C-ANF receptors markedly enhances the possibility of developing orally active C-ANF receptor ligands for the treatment of cardiovascular and renal diseases.

[Right-sided mechanical circulatory support in acute right ventricular failure in the dog]. / Pravostranný mechanický podporný obeh pri akútnom zlyhaní pravej komory u psa.

The technique of surgically induced acute progressive right ventricular failure in experimental animals is described. It sumultates the hemodynamic situation of right ventricular failure in some patients after termination of extracorporeal circulation applied for carrying out procedures on the left ventricle. The described technique consists of rightsided longitudinal ventriculotomy, destruction of the tricuspid valve, and ligation of the right coronary artery. Nine control dogs died within two hours after induction of failure due to low stroke volume caused by low pulmonary and left atrial pressure. The use of rightsided support appliance draining blood from the right atrium in systole and pumping it into the trunk of the pulmonary artery in diastole by means of a membrane pump resulted in further 12 dogs in the restoration of left ventricular diastolic pressure, significant increase of aortal pressure (p less than 0.003) and stroke volume (p less than 0.003) and in a decrease of right atrial pressure (p less than 0.003). The study demonstrated that by using the described mechanical support the circulation can be adequately assisted so that the failing right side of the heart can get restored. (Tab.1,Fig.5,Ref.25.).

[The effect of diltiazem (Lachema) on transmural non-homogeneity of histochemical changes due to the Ca-paradox]. / Transmurálna nehomogénnost' histochemických zmien po Ca-paradoxe a po jeho ovplyvnení diltiazemom (Lachema).

Calcium paradox was induced by 2.5 min perfusion of the rat heart (Langedorf's system) at 37 degrees C with calcium-free Krebs--Henseleit solution and subsequent 10 min reperfusion with the same solution containing calcium. Ca2+ depletion itself did not affect the activities of the histochemically studied enzymes (succinate dehydrogenase, lactate dehydrogenase, beta-hydroxybutyric dehydrogenase, phosphorylase, and ATPases). Ca paradox was histochemically characterized by decrease and even disappearance of activities of the given enzymes and by marked transmural nonhomogeneity of these changes exhibiting a decreasing trend towards the endocardium. Diltiazem in the concentration of 0.4 and particularly of 4.0 mumol.l-1 exerted a protective effect manifested by a better preservation of the enzymatic activities.

[Transmural differences between damaged cardiomyocytes due to post-ischemic reperfusion and calcium paradox]. / Transmurálne rozdiely medzi poskodením kardiomyocytov postischemickou reperfúziou a kalciovým paradoxom.

Normothermic 3 min lasting perfusion of the isolated rat heart by Krebs--Henseleit solution in which Ca2+ was replaced by EDTA and subsequent perfusion with a Ca2+ containing medium induced structural and metabolic changes demonstrated electron microscopically and histochemically. In contrast to the ischemic reperfusion damage, in calcium paradox, the histochemically studied enzymes alpha-glucan-phosphorylase, lactate dehydrogenase, succinic dehydrogenase, beta-hydroxybutyric dehydrogenase, and ATPases were better preserved in the subendocardial region of the left ventricle. Ultrastructural analysis of this phenomenon showed good correlation with histochemical findings. A large portion of cardiomyocytes in the subendocardial layer exhibited but small changes. On the other hand, myocytes in the subepicardial region and in the midmyocardium were markedly damaged and all characteristic signs of calcium paradox were present, including hypercontraction bands with myofilament fusion, extrusion and accumulation of edematous mitochondria with occurrence of electron dense material in mitochondrial cristae, ruptures of the sarcolemma in all its layers, separation of intercalated discs, etc. The better preservation of the subendocardial region in experiments with calcium paradox is attributable to inadequate perfusion of this region by calcium free medium due to transmural anatomic inhomogeneity of capillary supply whose insufficiency in the subendocardial region results in a better protection of these myocytes from Ca2+ paradox.

[The effect of diltiazem manufactured by Lachema on the calcium paradox in the isolated rat heart]. / Ovplyvnenie vápnikového paradoxu v izolovanom srdci potkana diltiazemom Lachema.

The calcium paradox known as irreversible damage of the metabolism, structure, and function of the heart was used as model for testing the efficacy of diltiazem (Lachema) in the concentrations of 0.4 and 4.0 mumol.l-1. On the isolated rat heart perfused by Langendorff's method, the calcium paradox induced by 2.5 min depletion and subsequent 10 min repletion of calcium was manifested by loss of electric and contractile activity, by drop of coronary flow, and by significant decrease of ATP and ADP content, as well as of the total content of adenine nucleotides in the tissue. Diltiazem (Lachema) in the tested concentrations of 0.4 and 4.0 mumol.l-1 effectively reduced the damage of cardiac function. Its effect was demonstrated by improvement of the hemodynamic and metabolic parameters studied.

Bilateral acute heart atrial auriectomy reduced diuresis and natriuresis following hypertonic sodium load in anaesthetized dogs.

Anaesthetized dogs were deprived of a portion of the atrial natriuretic factor producing tissue by bilateral acute heart atrial auriectomy. Their ability to respond by diuresis and natriuresis either to the expansion of extracellular fluid volume with isotonic saline (3% b.w.) or to hyperosmolality induced by hypertonic saline loading (0.13% b.w. of 20% NaCl solution) was subsequently reduced by about 50%. It is thus suggested that atrial natriuretic system may also play a role in osmoregulation by taking part in the promotion of renal sodium excretion.

Protective effect of oestradiol on the heart of rats exposed to acute ischaemia.

Sex-related differences in mortality from ischaemic heart disease are attributed chiefly to difference in the incidence of atherosclerosis. Little attention has been paid to the influence of sex hormones on resistance of the myocardium itself to acute ischaemia. Experiments on rats showed that isolated female hearts were more resistant than male hearts. A period of eight weeks spent at an altitude of 1,350 m raised heart resistance only in males. Conversely, gonadectomy abruptly reduced the resistance of the male heart to ischaemia, especially under conditions of mild altitude hypoxia. The administration of oestradiol to gonadectomized male rats largely abolished the disturbance caused by isolated gonadectomy. Since coronary vasoconstriction and vasospasm lead to temporary ischaemia and even to infarction, the above effect of the sex hormones may play a role in the increased incidence of heart attacks after the gonads have ceased to function.

The influence of exaprolol upon the ischaemic rat heart and its interaction with sarcolemmal (Na+ + K+)-ATPase.

The capability of cyclohexylphenol exaprolol of protecting the ischaemic myocardium during ischaemic cardiac arrest was assessed in the isolated working rat heart. Exaprolol added to the perfusion medium in a dose of 10(-7) mol.l-1 only minimally influenced the left ventricular function (reduced the stroke volume by 18.84% and cardiac output by 14.63%). The hearts were subjected to global ischaemia for 75 min at 26 degrees C and subsequently reperfused for 60 min at 37 degrees C. The recovery of left ventricular function following reperfusion, expressed as a percentage of preischaemic functional performance was used as an indicator of the ischaemic tolerance of the heart. The effect of exaprolol on sarcolemmal (Na+ + K+)-, Mg2+- and Ca2+-ATPase activities was also examined. Exaprolol-pretreated hearts revealed better postischaemic recovery of the left ventricular dP/dt max and stroke volume as well as improved efficiency in the transformation of chemical energy to mechanical work. Exaprolol in 10(-4) mol.l-1 concentration significantly stimulated the specific activity of sarcolemmal (Na+ + K+)-ATPase. Possible mechanisms of the salutary effect of exaprolol on the ischaemic heart are discussed.

Differences in transmural distribution of cardiomyocyte injury Ca paradox versus postischemic reperfusion phenomenon.

Normothermic, 3 min lasting perfusion of isolated rat heart with Krebs-Ringer-Henseleit solution in which Ca was replaced by EDTA, followed by successive perfusion of Ca2+ containing medium resulted in structural and metabolic derangement of myocardial cells; this could be demonstrated electronmicroscopically and histochemically. Unlike in ischemia, Ca paradox left the enzymes LDH, SDH, beta-HBDH as well as alpha-glucan phosphorylase and ATP-ases better preserved in the subendocardial layer of the left ventricle. Ultrastructural analysis of this phenomenon showed good correlations with the histochemical findings. A large portion of cardiomyocytes in the subendocardial layer showed only slight changes. On the other hand, myocytes in the subepicardial layer were severely injured and all characteristics of the calcium paradox were present including hypercontraction bands with fusion of myofilaments, extrusion and accumulation of oedematous mitochondria containing electron dense material intracristally, sarcolemmal ruptures, separation of intercalated discs etc. The better preservation of the subendocardial region in experiments with calcium paradox could be explained by inadequate perfusion of this layer with Ca2+ free medium due to transmural anatomical inhomogeneity of the capillary supply, resulting in a better protection of these myocytes from Ca paradox. The heterogeneity in transmural distribution of injuries is a multifactorial phenomenon. In addition to factors such as intramural pressure gradient, transmural pressure, enddiastolic intraventricular pressure etc., the most important factors in both types of injuries should be regarded the amount of vascular supply, blood flow and perfusate volume.

Partial prevention of calcium paradox in isolated perfused rat hearts by diltiazem.

It is well known that excessive calcium entry into the myocardial cells may contribute considerably to damage of the heart caused by postischemic reperfusion. The effect of increased calcium entry on hemodynamics, energy metabolism and histochemically estimated enzyme activities in isolated, perfused (Langendorff) rat heart preparation was investigated using calcium paradox (CaPX) as a model. After a 15 min period of stabilized perfusion of the heart, CaPX was induced at 37 degrees C by 2.5 min lasting calcium depletion (calcium-free perfusion) and subsequent calcium repletion (10 min). Changes induced by CaPX concerned loss of electrical and mechanical activities of the heart, significant decreases in coronary flow and ATP, ADP and the total content of adenine nucleotides in tissue as well as considerable depression in ATPases, SDH, beta-HBDH, LDH and glycogen phosphorylase activities in the myocardium. Diltiazem in concentration of 4.0 mumol.l-1 applied prior to calcium depletion and during calcium repletion prevented partially the deterioration of cardiac function by improving contractility and electrical activity of the heart as well as the coronary flow. The effect of diltiazem in concentration of 0.4 mumol.l-1 was less expressed. After both concentrations of diltiazem used, a better preserved ultrastructure, higher activities of the enzymes investigated, significantly higher ATP and total adenine nucleotide levels were seen in the myocardium as compared to the untreated controls.