Intramural nerve-mediated inotropic responses of left atria of rats and guinea pigs: demonstration of alpha adrenergic and nonadrenergic noncholinergic responses in guinea pigs

The effects of transmural nerve stimulation (TNS) on contractile responses of rat and guinea pig atria were analyzed pharmacologically. Isolated left atria were electrically driven through AgAgCl field electrodes and TNS was performed by brief introduction of defined stimulation patterns through the same electrodes. Step elevations in stimulating voltage induced biphasic inotropic responses in the left atria of both species: an initial negative component which was usually overwhelmed by a subsequent positive one. The transient negative inotropic response was induced by parasympathetic cholinergic nerve excitation, inasmuch as it was abolished by atropine. In the left atrium of the rat, the TNS-induced positive inotropic response was due exclusively to adrenergic nerve excitation through activation of beta-1 adrenoceptors. In contrast, analysis of the time course of responses in guinea pig left atria after nerve stimulation at 10 Hz revealed a positive inotropic response consisting of two phases; rapid and delayed phases were superimposed upon each other. The rapid phase was reduced by atenolol, a beta-1 antagonist, and attenuated further by prazosin, an alpha-1 antagonist. In the presence of both atenolol and prazosin, TNS of guinea pig left atria still induced a positive inotropic response but it had a slow onset and decay. This is termed the delayed phase response. TNS induced a similar delayed inotropic response in atria from surgically sympathectomized or reserpine-pretreated guinea pigs, from which catecholamine-fluorescence nerves and responses to tyramine were absent. These results demonstrate that TNS excitated adrenergic, cholinergic and nonadrenergic noncholinergic nerves in guinea pig left atria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative chronotropic and positive inotropic actions of phencyclidine on isolated atrial muscle in guinea pigs and rats

Chronotropic and inotropic actions of phencyclidine were studied in spontaneously beating right atrial muscle and electrically paced left atrial muscle preparations isolated from guinea-pig or rat hearts. In right atrial muscle preparations, phencyclidine (10-100 microM) decreased the frequency of spontaneous beating. Guinea-pig and rat heart preparations had similar sensitivities to this action of phencyclidine. The negative chronotropic effect was not altered by atropine. A high concentration of naloxone failed to affect the chronotropic effect of phencyclidine in guinea-pig muscle, but significantly reduced the effect in rat heart muscle preparations. Phencyclidine (1-100 microM) caused positive inotropic effects in both guinea-pig and rat heart left atrial muscle electrically stimulated at 1.5 Hz; rat heart preparations had a higher sensitivity to the positive inotropic action of phencyclidine. The positive inotropic effect was reduced by verapamil, nifedipine and relatively high concentrations of diltiazem, but was not affected by propranolol, phentolamine, tripelennamine, atropine or ryanodine, indicating that the effect is not mediated by adrenergic, histaminergic or cholinergic systems or does not involve ryanodine-sensitive calcium pools. Inactivation of the fast sodium channels by partial membrane depolarization, and subsequent restoration of the contraction by raising the extracellular Ca++ concentration, did not abolish the positive inotropic action of phencyclidine. These results suggest that the negative chronotropic effect of phencyclidine is not mediated by a stimulation of the muscarinic receptor. The positive inotropic effects of phencyclidine seem to result from an increase in Ca++ influx through the slow channels of the cardiac cell membrane.     

Comparison of contractile responses of the guinea pig ileum longitudinal muscle to ethanol and GABAA agonists

The guinea pig ileum myenteric plexus contains GABAA receptors linked to chloride ion channels which are pharmacologically similar to those in the central nervous system. The present study examined the reported ability of acute ethanol treatment to directly activate GABAA receptors or to increase GABAA agonist-mediated activation of the GABAA receptor in the myenteric plexus. Direct addition of ethanol to preparations of the guinea pig ileum longitudinal muscle had two effects. Immediately after ethanol (10-300 mM) was added to the tissue bath a concentration-related contractile response was observed which became maximal within 10 sec and then decayed over the next 60 sec. Contractile responses to higher concentrations of ethanol (greater than 100 mM) also were followed by a sustained reduction of longitudinal muscle tone. Contractions evoked by gamma-aminobutyric acid (GABA) and GABAA agonists, 3-aminopropane sulfonic acid (APSA) (3-100 microM) or muscimol (0.3-30 microM) developed maximally and decayed within 20 sec. Acetylcholine (0.01-10 microM) induced contractions were sustained over several minutes. Preincubation of tissue strips in ethanol (30 mM) for 1 min did not alter concentration relationships for GABA, muscimol or APSA contractile responses. Furthermore, addition of ethanol (10-100 mM) simultaneously with APSA, or 0.5, 2 or 5 min before the addition of APSA, also failed to consistently enhance contractile responses. Ethanol (30 mM) also did not alter desensitization-induced reductions in contractile responses to muscimol (3 microM) caused by preincubation of tissues with muscimol (1 microM). Finally, contractile responses to ethanol and APSA were completely blocked by atropine (0.1 microM) and tetrodotoxin (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane current changes responsible for the positive inotropic effect of OPC-8212, a new positive inotropic agent, in single ventricular cells of the guinea pig heart

Effects of OPC-8212, a new positive inotropic drug, and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, on membrane currents were examined in single ventricular cells of the guinea pig heart. Single ventricular cells were prepared by the collagenase dispersion procedure. Both OPC-8212 (0.1 mM) and IBMX (0.1 mM) augmented the plateau and increased the duration of the action potential without affecting the resting membrane potential. Under voltage clamp condition, OPC-8212 (0.1 mM) increased the inward calcium current and decreased the delayed outward and the inward-rectifying potassium current. IBMX (0.1 mM) increased not only the inward calcium but also the delayed outward current. The isolated inward calcium current obtained by intra- and extracellular perfusion with Cs+, was increased by both drugs. When the inward calcium current was abolished by superfusion with D600 (10 microM) or Co++ (0.9 mM), OPC-8212 (0.1 mM) decreased the delayed outward and the inward-rectifying potassium current. On the other hand, IBMX (0.1 mM) increased the delayed outward current. From these results it can be concluded that OPC-8212 augments the plateau and increases duration of the action potential not only by increasing the inward calcium but also by decreasing both the delayed outward and the inward-rectifying potassium current, and the effects can be a cause of the positive inotropic effect of this drug.     

Cardiorespiratory, sympathetic and biochemical responses to T-2 toxin in the guinea pig and rat

The cardiorespiratory, sympathetic and biochemical effects of T-2 toxin were examined in conscious rats and guinea pigs. The pithed rat preparation was also used to evaluate possible direct effects of T-2 on the heart and vasculature. Injection of T-2 (0.5-2.0 mg/kg i.v.) into conscious rats produced prolonged (6-8 hr) hypertension and tachycardia, followed by hypotension. Total peripheral resistance was increased and cardiac output decreased. In guinea pigs, a steady decrease in pressure and rate occurred. Intravenous administration of T-2 to pithed rats did not alter blood pressure or heart rate at a time when, in conscious rats, both blood pressure and heart rate were increased. Significant elevations of arterial plasma norepinephrine, epinephrine and dopamine occurred after T-2, with metabolic acidosis, hypocarbia and hyperoxemia in both conscious rats and guinea pigs. In the rat, increase in plasma vasopressin and prostacyclin were elevated, but thromboxane and leukotriene C4-immunoreactivity were not changed. In pithed rats, T-2 did not increase basal or stimulated plasma catecholamines but produced the same changes in blood gases, pH and lactate. The LD50 values for i.v. T-2 in the rat and guinea pig were 0.74 and 1.30 mg/kg, respectively. The data are consistent with the hypothesis that T-2 toxin disrupts cellular aerobic metabolism, resulting in lactic acidosis, sympathoadrenomedullary activation, variable initial circulatory responses and eventual cardiovascular collapse.     

Evidence for leukotriene D4 receptors in guinea pig left atria.

The effects of peptidoleukotrienes (LTs) on electrically driven guinea pig left atria (GPLA) were investigated. LTD4 produced a positive inotropic response; however, rapid desensitization required the construction of noncumulative dose-response curves to naive tissues. The maximal inotropic response to LTD4 was 24 +/- 3% of isoproterenol and the EC50 = 267 +/- 77 nM. The functional response was corroborated by the demonstration of specific and rapid [3H]LTD4 binding to GPLA membranes with low affinity (Kd = 212 +/- 80.2 nM), in a saturable (Bmax = 20 +/- 1.1 pmol/mg protein) manner. In tissues pretreated with acivicin, which inhibits conversion of LTC4 to LTD4, the response to LTC4, but not LTD4, was abolished. Selectivity towards LTD4 was demonstrated by the inability of propranolol, prazosin, atropine, pyrilamine, capsaicin or indomethacin (all tested at 1 microM) to alter the functional response to LTD4. Similarly, none of the tested compounds (100 microMs) was inhibitory in the binding assay. Structurally diverse LTD4 antagonists SKF102922 (pKb = 6.42) and ICI 198.615 (pKb = 8.74) were able to inhibit the functional response as well as [3H]LTD4 binding to GPLA membranes. The calcium channel antagonist, verapamil, inhibited the functional response but did not alter [3H]LTD4 binding. These data support the existence of specific LTD4 receptors in GPLA which evoke a modest, rapidly desensitized, increase in the force of myocardial contraction.     

Positive inotropic effect of carcinine in the isolated perfused guinea pig heart

Carcinine (beta-alanylhistamine) is a recently discovered compound that is present in the hearts of several mammalian species, including man. Although the function of carcinine is unknown, its structural similarity to histamine, a compound known to have profound effects on the mammalian heart, and to carnosine (beta-alanylhistidine), a compound which we have previously shown to serve as a histamine source, led to the hypothesis that carcinine may play a role in mammalian cardiac physiology. We therefore administered several doses of carcinine (10, 25, 50, 75, and 100 micrograms) to isolated, perfused guinea pig hearts in a Langendorff apparatus. Carcinine exerted a dose-dependent positive inotropic effect, similar to that of histamine. Comparable doses of carnosine yielded no measurable change in contractility. We conclude that carcinine appears to be a positive inotrope in the mammalian heart, and may play a role in cardiac physiology via its metabolic link to histamine.     

Ontogeny of cortical muscarinic receptor subtypes and muscarinic receptor-mediated responses in rat

To determine the ontogenetic relationship of muscarinic receptor and effector systems in the central nervous system, the developmental time courses for binding sites with high (M1) and low (M2) affinity for pirenzepine as well as muscarinic receptor-mediated stimulation of phosphoinositide breakdown and inhibition of cyclic AMP accumulation were examined in rat cortex. M1 sites were 30% of adult levels at 1 week, 70% at 2 weeks, 90% at 3 weeks and equal to adult levels at 4 weeks postpartum. M2 sites, on the other hand, did not show a significant change between the ages of 1 and 6 weeks. Acetylcholine-stimulated phosphoinositide breakdown was detected at all ages tested (1, 2, 3, 4 and 6 weeks). The percentage of conversion of [3H] phosphoinositides to [3H]inositol phosphates, stimulated by 1 mM acetylcholine, increased with age until 3 weeks after birth and then decreased slightly at ages 4 and 6 weeks. Carbachol inhibition of [3H]cyclic AMP accumulation, on the other hand, was undetectable in tissues from 1- and 2-week-old rats, whereas in tissues from 3- and 4-week-old rats, the responses were at 6-week level. Thus, for carbachol inhibition of cyclic AMP accumulation, a time in development existed (2 weeks after birth) at which receptors appeared to be present but response to stimulation was absent. To examine indirectly the coupling of binding sites to second messenger systems via guanine nucleotide-binding regulatory proteins, the density of binding sites for the muscarinic receptor agonist, [3H]oxotremorine-M, was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of the positive inotropic action of the alpha adrenoceptor agonist, phenylephrine, in isolated rat left atria.

Alpha adrenoceptor agonists have been reported to increase contractile force and to stimulate Na+/H+ exchange in the heart. We studied the influence of hexamethylamiloride (HMA), a selective inhibitor of Na+/H+ exchange, on the positive inotropic action of phenylephrine in isolated, paced rat left atria (3 microM propranolol). HMA (10 microM) blocked the ouabain-induced contracture, an event dependent on Na+ uptake via the Na+/H+ exchanger. The same concentration of HMA prevented 50% of the positive inotropic effect of phenylephrine (10 microM), but had no effect on base-line developed force. HMA reduced the maximal effect (234 +/- 19 vs. 117 +/- 20% increase of base-line), but not the EC50 (4.4 +/- 1.0 vs. 3.6 +/- 2 microM) of phenylephrine. Phenylephrine (100 microM) caused both a leftward and upward shift of the Ca++ concentration-effect curve, but only a leftward shift, in the additional presence of HMA (3 microM). It is known that lithium, but not choline, will exchange for H+ via the Na+/H+ exchanger: phenylephrine's (100 microM) positive inotropic effect in choline-substituted solutions averaged 37% of that in lithium-substituted solutions. The positive inotropic effect of phenylephrine was amplified by ouabain (200 microM). These results are consistent with the hypothesis that alpha adrenoceptor agonists produce their positive inotropic effects, in part, via stimulation of Na+/H+ exchange. Such stimulation could cause an intracellular alkalinization and (in the presence of ouabain), elevated intracellular Na+.     

Involvement of endothelium-derived NO in the basal tone and in the vasodilator responses to muscarinic agonists in the rat isolated mesenteric arterial bed

Summary— To investigate the involvement of nitric oxyde (NO) derived from endothelial cells in the control of vascular tone in the rat mesenteric vascular bed, the effects of different procedures known to interfere with the NO-cyclic GMP pathway were evaluated both on the basal tone and on the vasodilatory responses to four muscarinic agonists. To this aim, rat isolated mesenteric vascular beds were perfused at constant pressure. Water infusion significantly increased the resting perfusion pressure whereas L-NOARG, L-NAME and methylene blue were devoid of effect. In noradrenaline-preconstricted vascular bed, the perfusion pressure was significantly increased after water or L-NAME infusion. The vasodilator response induced by subsequent addition of acetylcholine in bolus was not significantly modified by pre-treatment with indomethacin but was significantly reduced by water infusion. Reponses to acetylcholine and to three other muscarinic agonists -carbachol, oxotremorine or McNeil A 343- were assessed. Incubation with L-NAME did not modify the initial peak falls of the agonists except for Mc Neil A 343, whereas it significantly reduced the area under the pressure trace for all the substances. The latter effect was reversed after a subsequent incubation with L-Arginine. Finally, L-NAME strongly and significantly increased the drop in perfusion pressure and the area under the pressure trace following bolus of glyceryl trinitrate. These results suggest that in the mesenteric arterial bed of the rat, which can be considered as a resistant arteries preparation, basal tone appears to be controlled by a factor other than NO. Moreover, the vasodilator responses of muscarinic agonists are affected by L-NAME in their second late sustained phase only, which probably relies on a de novo synthesis of endothelium derived-NO. Finally, endothelium derived-NO exerts inhibitory effects both on the sensitivy of the vascular smooth muscle to glyceryl trinitrate and on the magnitude of its contraction in the presence of noradrenaline, two types of effects which are sensitive to L-NAME.     

Compared peripheral vascular responses to intravenous and intra-arterial administrations of positive inotropic agents in conscious dogs

In addition to their direct effects on cardiac contractility, a number of positive inotropic agents also induce, through direct peripheral vasodilation, a reduction in afterload which is of major importance in their beneficial effects in the treatment of congestive heart failure. However, the induced increase in cardiac output can indirectly improve perfusion of peripheral vessels through a flow-mediated mechanism. Thus, the goal of the present study was to compare the direct peripheral vasomotor effects assessed in the iliac vascular bed of four positive inotropic agents: DPI 201-106, ouabain, milrinone and dobutamine, in the presence and absence of simultaneous changes in cardiac function. These drugs were administered either through intravenous or intra-arterial (aorto-iliac catheter) routes in 6 conscious dogs, chronically instrumented for the measurement of heart rate, arterial pressure, left ventricular dP/dt, iliac artery blood flow and iliac artery diameter (sonomicrometry). Intravenous doses were selected as those inducing equipotent positive inotropic responses whereas intra-arterial doses were below those required to induce any significant change in systemic hemodynamics. Ouabain decreased and milrinone increased both iliac blood flow and diameter after either intravenous or intra-arterial administrations. In contrast, iliac blood flow did not change after intra-arterial administration of DPI 201-106 and dobutamine whereas iliac diameter was not modified by DPI 201-106 and even decreased with dobutamine. After intravenous administration, DPI 201-106 but not dobutamine, increased both iliac blood flow and diameter. Thus, this experimental preparation can differentiate inotropic agents with direct vasodilating (milrinone) or constricting (ouabain) properties and those (DPI 201-106 and dobutamine) with indirect vasodilating effects most likely mediated by the improvement in cardiac function.     

Hyperosmolarity-induced dilation and epithelial bioelectric responses of guinea pig trachea in vitro: role of kinase signaling

Exercise-induced airway obstruction is thought to involve evaporative water loss and hyperosmolarity of the airway surface liquid. Hyperosmolar challenge of the epithelium of isolated, perfused guinea pig trachea rapidly alters transepithelial potential difference (V(t)), and it elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). In many cell types, protein kinases mediate responses to hyperosmolarity and regulatory volume increase. In this study, inhibitors were used to investigate the involvement of kinases and phosphatases in bioelectric responses of epithelium to hyperosmolarity and their possible relationship to EpDRF-mediated relaxation. After contraction of the perfused trachea with extraluminal methacholine, D-mannitol applied intraluminally (< or = 80 mosM) increased V(t) and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased V(t). In tracheas exposed to 30 mosM D-mannitol (approximately EC(50)), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) and SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole] (p38 inhibitors) potentiated the dilation, whereas SP 600125 [anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone] and dicumarol [c-Jun NH(2)-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO(2) (mitogen-activated protein kinase stress inducer) and Na(3)VO(4) (protein tyrosine phosphatase inhibitor) inhibited the hyperpolarization. Large increases in the phosphorylation of p38 and JNK occurred at concentrations higher than those needed to elicit functional responses. The phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and Na(3)VO(4) did not affect the V(t) responses, but they inhibited methacholine-induced constriction; SP 600125 and dicumarol potentiated, and chelerythrine inhibited, methacholine-induced epithelial hyperpolarization. These results suggest that JNK, PKC, and phosphatase(s) are involved in hyperosmolarity-induced hyperpolarization of the tracheal epithelium but that p38 is involved in EpDRF-mediated relaxation.     

Glucocorticoid modulation of muscarinic and β-adrenergic receptors in guinea pig lung

Summary— We investigated the effect of the in vivo treatment of guinea pigs with methylprednisolone, 10 mg/kg daily, on lung muscarinic and β-adrenergic receptors. Receptor densities were assessed by saturation experiments of tritiated N-methylscopolamine and dihydroalprenolol binding to lung membranes. After 3 h of treatment, methylprednisolone induced a decrease of 19.2% ( P < 0.05) of muscarinic receptors but was without effect on β-adrenergic receptor density. After 24 h, an increase of 39.7% ( P < 0.01) and 16.9% ( P < 0.05) was observed for muscarinic and β-adrenergic receptors, respectively. For muscarinic receptors, this increase reached 53.4% ( P < 0.01) within 48 h and stayed at this level until 96 h. The increase of β-adrenergic receptors was maximal (24.9%) after 72 h and returned to the control value after 96 h. The dissociation constant (K_d) values of both ligands were not affected by the glucocorticoid treatment. Functional studies showed that the 96 h treatment did not affect the contractile response of guinea pig lung parenchymal strips to carbachol since the 50% concentration value (EC₅₀) and the maximal contraction value (E_max) were not significatively different from control values. These data show that glucocorticoids control the expression of both muscarinic and β-adrenergic receptors in guinea pig lung but with different time courses and to a larger extent for muscarinic receptors. The glucocorticoid treament did not modify the contractile response of lung strips to carbachol, confirming the absence of effect on the affinity of muscarinic receptors and suggesting that the receptor reserve exceed the increase of their density by the steroid.     

Contractile responses in bladder body, bladder neck and prostate from rat, guinea pig and cat

Lower urinary tract smooth muscle displays marked heterogeneity in pharmacologic responsiveness to contractile agents. The present study details differences among species with regard to muscarinic, adrenergic, histaminergic and serotonergic agonists in the bladder body, bladder neck and prostate from guinea pig, rat and cat. Under in vitro conditions, all smooth muscle preparations contracted to potassium chloride. The muscarinic agonist, carbamylcholine, produced maximal contraction, whereas alpha receptor agonists exerted only minimal, if any, effect in bladder body preparations from all three species. In contrast, alpha receptor-mediated responses predominated relative to muscarinic responses in bladder neck preparations from all three species. Prostatic contractility was examined in tissue from guinea pig and rat and contraction occurred to both alpha and muscarinic receptor agonists. Contractile response to norepinephrine in bladder neck and prostate was potentiated by neuronal uptake inhibition but not by beta receptor blockade. Serotonin and histamine exhibited more diverse effects among species and tissues. In general, histamine contracted all three tissues from guinea pig with minimal contraction occurring in tissues from rat or cat. On the other hand, serotonin markedly contracted the cat bladder body and rat prostate, but exerted no effect on tissues from the guinea pig. These data reinforce and detail the heterogeneity of pharmacologic contractile responses in lower urinary tract smooth muscle. Furthermore, the studies document the relative similarity among species in cholinergic and adrenergic responsiveness and the dissimilarity among species in serotonergic and histaminergic responsiveness of lower urinary tract smooth muscle.     

Reserpine-induced supersensitivity to the chronotropic and inotropic effects of calcium in rabbit atria

Supersensitivity to the chronotropic and inotropic effects of calcium was demonstrated in spontaneously beating paired atria from reserpine-pretreated (0.1 mg/kg/day, 7 days) rabbits. Supersensitivity to the inotropic effects of calcium in electrically driven left atria was also demonstrated. Atria were driven at 80, 100 and 120 beats/min. At each frequency the reserpine-pretreated atria were more sensitive than controls. Atria were tested under diastolic tensions of 1,2 and 4 g. As the tension was increased the sensitivity to calcium increased. The sensitivity of reserpine-pretreated atria was greater at each tension than that of control atria. Atria tested at 37 degrees C were less sensitive than those tested at 30 degrees C; however, the reserpine-pretreated atria were more sensitive than control atria at both temperatures. This study demonstrates that reserpine-induced supersensitivity to the inotropic effects of calcium can be obtained and that the ability to demonstrate this phenomenon does not appear to be altered by the frequency, diastolic tension or temperature at which each experiment is performed.     

Analysis of preformed xenoreactive antibodies in the discordant guinea pig to rat model using a guinea pig fibroblast-like cell line

In the discordant guinea pig (gp) to rat model of xenotransplantation, circulating xenoreactive natural antibodies (XNA) recognizing gp antigens are usually determined by an ELISA using membrane extracts of gp platelets. We analysed the lung-derived, fibroblast-like cell line JH 4 to detect XNA by ELISA or immunoblot, which was compared to primary gp cells, i.e. platelets, liver- and spleen cells. All membrane extracts proved to be useful to detect rat XNA directed against gp antigens by ELISA. In general, IgM responses of Lewis or C6 deficient PVG rats (PVG/C6-) were higher as compared to IgG responses. However, we observed great inter-individual variabilities. The strongest IgM response of Lewis rat sera was observed when the JH 4 cell line or gp liver cells were used as antigen. JH 4 cells also showed the strongest xenoreactivity with sera from PVG/C6- rats. These data demonstrate that JH 4 cells prove useful as antigen source for XNA ELISA. In immunoblot, individual sera of the two different rat strains showed the same antigen patterns using a gp membrane extract of one particular cell type. However, the different gp cell types showed a distinct pattern of antigen expression. Whereas the JH 4 cells, platelets and spleen cells express xenoreactive proteins of the same size, a unique pattern of proteins was detected in liver cells.     

Analysis of preformed xenoreactive antibodies in the discordant guinea pig to rat model using a guinea pig fibroblast-like cell line

In the discordant guinea pig (gp) to rat model of xenotransplantation, circulating xenoreactive natural antibodies (XNA) recognizing gp antigens are usually determined by an ELISA using membrane extracts of gp platelets. We analysed the lung-derived, fibroblast-like cell line JH 4 to detect XNA by ELISA or immunoblot, which was compared to primary gp cells, i.e. platelets, liver- and spleen cells. All membrane extracts proved to be useful to detect rat XNA directed against gp antigens by ELISA. In general, IgM responses of Lewis or C6 deficient PVG rats (PVG/C6-) were higher as compared to IgG responses. However, we observed great inter-individual variabilities. The strongest IgM response of Lewis rat sera was observed when the JH 4 cell line or gp liver cells were used as antigen. JH 4 cells also showed the strongest xenoreactivity with sera from PVG/C6- rats. These data demonstrate that JH 4 cells prove useful as antigen source for XNA ELISA. In immunoblot, individual sera of the two different rat strains showed the same antigen patterns using a gp membrane extract of one particular cell type. However, the different gp cell types showed a distinct pattern of antigen expression. Whereas the JH 4 cells, platelets and spleen cells express xenoreactive proteins of the same size, a unique pattern of proteins was detected in liver cells.