Physiological effects and biochemical properties of a serum protein that produces positive inotropic and chronotropic effects on isolated guinea pig atria

Isolated left and right guinea pig atria were used as a bioassay for the detection of an endogenous cardioactive substance in bovine serum. Serum, buffer exchanged to Krebs-Henseleit solution, produced positive inotropic and chronotropic effects on the isolated guinea pig atria. The cardiotonic effects were unaffected by the combined presence of propranolol and methysergide (both 10(-6)M) and were also dissimilar in time course from other known cardiotons such as catecholamines and cardiac glycosides. Following ultrafiltration (using XM100A Amicon membranes), activity was found solely in the retentate fractions and was therefore probably due to a large molecular weight (> 100 kDa) substance or a small molecule bound to a large protein. The cardioactive factor (CF) in the whole serum was heat labile, sensitive to acidification, exposure to potassium bromide and equilibration to physiological buffers of a low ionic strength. Isolation by conventional protein purification techniques was unsuccessful due to the labile nature of the active molecule(s) when exposed to non-physiological experimental conditions. Physical and biochemical properties of the CF which may help avoid inactivation are discussed for future experiments aimed at elucidating the nature and identity of the cardiotonic principle.     

Cardiotonic activities of pumiliotoxin B, pyrethroids and a phorbol ester and their relationships with phosphatidylinositol turnover

The cardiotonic activities of pumiliotoxins, pyrethroids and sodium and calcium channel activators were assessed in vitro with spontaneously beating guinea pig atria. The ability of these compounds to stimulate phosphoinositide turnover was assessed in guinea pig cerebral cortical synaptoneurosomes. The activity of pumiliotoxins for both cardiotonic activity and phosphoinositide breakdown was strongly dependent on the structure and configuration of the side chain and there was a correlation between structure and activity in the two systems. Pyrethroids that had cardiotonic activity also induced phosphoinositide breakdown. Other sodium channel and calcium channel activators that induced phosphoinositide breakdown were also cardiotonic. It is suggested that phosphoinositide breakdown leading to inositol phosphates and diacylglycerols may represent a mechanism underlying the cardiotonic effects of certain agents. A phorbol ester, phorbol 12-myristate 13-acetate, that mimics the activation of protein kinase C elicited by diacylglycerols, had cardiotonic activity.     

Rosette formation between rat thymocytes and guinea pig erythrocytes requires "active" fetal calf serum. I. Characteristics of "active" serum factors and receptors on thymocytes and erythrocytes.

Rosette formation between rat thymocytes and guinea pig erythrocytes is dependent on at least two factors present in non-heat treated fetal calf serum. One factor is a high molecular weight, heat stable substance and the other factor is a heat labile substance(s). The rosette formation process is divalent cation dependent and seems to involve the sequential binding to thymocytes of the high molecular weight, heat stable substance, the heat labile substance (s), and then guinea pig erythrocytes. Thymocytes appear to bear a receptor which is dependent on hexose monophosphate shunt metabolism, is not removed by many digestive enzymes, but is blocked with phytohemag-glutinin and pokeweed mitogen. Erythrocytes appear to bear a receptor which is dependent on hexose monophosphate shunt metabolism, removed by pronase treatment, and blocked by phytohemagglutinin and concanavalin A.     

Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule I. Mechanism of inhibition

Isotonic reabsorption by the rat kidney proximal tubule was drastically inhibited after less than 2 min intraluminal perfusion with fresh sera from rat (both homologous and autologous), cat, rabbit and human, but not with sera from mouse and guinea pig. The inhibitory factor in serum in a heat (56° C for 30 min) and storage (4°C for 2–5 days) labile macromolecule (mol. wt 50 000) and requires Ca²⁺ for its effect. The cellular electrical potential difference of the proximal tubular cells was irreversively destroyed and intraluminally perfused trypan blue dye incorporated into the tubular cells after the intraluminal perfusion with serum for 2 min. These observations suggest that lysis of the proximal tubular cells is the mechanism for serum-induced inhibition of proximal tubular isotonic reabsorption.     

Synthesis and biological activities of leukotriene F4 and leukotriene F4 sulfone

Leukotriene F₄ (LTF₄ and LTF₄ sulfone have been synthesized and their biological activities determined in the guinea pig. LFT₄ displayed comparable activity to LTD₄ on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF₄ induced a bronchoconstriction (ED₅₀ 16 μg Kg⁻¹) which was blocked by indomethacin and FPL-55712 and was 50–100 X less potent than LTD₄ in this assay. LTF₄ sulfone was approximately 2–5 times less active than LTF₄ and . When injected into guinea pig skin with PGE₂ (100 ng); LTF₄ and LTF₄ sulfone (10–1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE₄ sulfone = LTD₄ = LTD₄ sulfone > LTE₄ > LTF₄ = LTF₄ sulfone.     

Participation of capsaicin-sensitive neurons in the cardiovascular effects of neurotensin in guinea pigs

Intravenous (IV) infusions of neurotensin (NT) in anesthetized guinea pigs elicited dose-dependent pressor effects and tachycardia. Both effects were significantly reduced or abolished in guinea pigs given a chronic treatment with the neurotoxin capsaicin. In guinea pig isolated atria NT evoked a positive inotropic and chronotropic effect. Both effects were completely abolished in atria derived from capsaicin-treated guinea pigs. The positive inotropic and chronotropic effects of NT in guinea pig atria were mimicked by capsaicin and calcitonin gene-related peptide (CGRP). These results were interpreted as an indication that NT produces its cardiovascular effects in guinea pigs by activating capsaicin-sensitive sensory neurons.     

Preparation and characterization of a biologically active spin-labeled sea anemone toxin

A derivative of the polypeptide cardiostimulant anthopleurin-B(AP-B) labeled with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide has been prepared and characterized. The product was found by mass spectrometry to be labeled at a single site, which amino acid sequencing showed to be the N-terminus. It also retained positive inotropic activity when assayed on isolated guinea pig atria. The spin-labeled (SL) product was found to exist in two distinct conformations by reversed-phase HPLC and in at least two conformations by electron spin resonance spectroscopy (ESR) over thepH range 2–9. The ESR data also show evidence for multimetric states of SL-AP-B over thepH range 2–9, with maximum aggregation at pH 4.5–5, and a slow disaggregation when thepH is adjusted to 8–9. The presence of multiple conformers of SL-AP-B and its tendency to aggregate render it unsuitable for high-resolution NMR structural studies of the isolated ligand, but the retention of activity may make it useful for studies of the sodium-channel-bound form of the molecule.Abbreviations AP-A anthopleurin-A - AP-B anthopleurin-B - ATX Ia toxin Ia fromAnemonia sulcata - Sh I neurotoxin I fromStichodactyla helianthus - TFA trifluoroacetic acid - SL-AP-B AP-B labeled at the N-terminus with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide     

Anti-peptide antibodies specific for the gastrin/cholecystokinin-B receptor

Summary Seven synthetic peptides, between 7–22 residues long, corresponding to six different parts of the gastrin/CCK_B receptor molecule which are conserved among the species, were used for raising antibodies. The peptides were coupled to keyhole limpet hemocyanine and injected into rabbits. ELISA analysis demonstrated that all peptides produced an immune response after three to six injections given at biweekly intervals. The titer ranged from 1:10⁴ to 1:10⁵. All antibodies recognized a 78 kDa protein on immunoblots of NIH 3T3 cells stably transfected with human gastrin/CCK_B receptor cDNA, as well as human and guinea pig stomach mucosal extracts. Preincubation of the sera with the corresponding peptides abolished the staining. Indirect immunofluorescence staining revealed that four antibodies out of the seven tested recognized the receptor in fixed COS-7 cells transiently transfected with human gastrin/CCK_B receptor cDNA. The reactive antibodies were raised against the peptides corresponding to receptor residues 40–58, 153–160, 288–294 and 356–372. Immunohistochemical staining of guinea pig stomach using these antisera resulted in intense staining of parietal cells in the fundus and cardia regions.Abbreviations CCK cholecystokinin - GR gastrin/CCK_B receptor - TFA trifluoroacetic acid - BSA bovine serum albumin - PBS phosphate-buffered saline - PAGE polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay - EDTA ethylenediamino tetraacetic acid - CLSM confocal laser scanning microscopy - KLH keyhole limpet hemocyanine     

Distribution, origin and sensitivity to capsaicin of primary afferent substance P-immunoreactive nerves in the heart

This report is intended as an overview of the distribution, origin and sensitivity to capsaicin of substance P-immunoreactive (SP-I) primary afferent cardiac nerves. Immunohistochemical and physiological methods were employed to compare the presence and density of these nerve fibers in the guinea pig and rat hearts. SP-I fibers are numerous in the guinea pig heart including the parietal pericardium, atria, ventricles, valves, coronary arteries and around intrinsic cardiac ganglion cells. The rat heart contains few SP-I fibers. Vagotomy does not influence the number of intensity of immunoreactive fibers in the guinea pig heart. By stimulating the atrium or ventricle and recording from the second or third thoracic dorsal roots Ad1, Ad2 and C fibers were demonstrated in the atria, but only Ad fibers in the guinea pig ventricle; in addition, only Ad fibers were recorded from the vagus nerves. Only Ad1 fibers were demonstrated in the rat heart. Treatment with capsaicin depletes the SP-I and decreases the conduction velocity of C-fibers and some Ad2 fibers in the guinea pig heart. We suggest that SP-I primary afferent nerve fibers are unmyelinated (C-type) or small myelinated (Ad2-type) nerves in the guinea pig heart and that their cell bodies of origin are predominantly in dorsal root ganglia.     

Quantitation of catalase activity by microspectrophotometry after diaminobenzidine staining

Summary The absorbance of the reaction product of catalase staining with diaminobenzidine is linearly proportional to enzyme activity. This is shown in semithin Epon sections of model systems containing serum albumin and catalase from bovine or guinea pig liver. Absorbance measurements were also performed on semithin sections of guinea pig liver, and from these, the activity of cytoplasmic (extraperoxisomal) catalase has been derived.I.W.O.N.L. bursaal 1977–1979     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

A Quantitative study of histamine H2-receptor bockade by burimamide in isolated artica.

The onset of burimamide inhibition of histamine stimulation of rabbit atria is rapid, and a near steady-state blockade occurs at approximately 15 min ( larger than or equal to 90% complete). The blockade is reversible but requires several washings suggesting the disassociation is slow. The administration of histamine may accelerate the decay of the burimamide effect. Reciprocal plots (rate response versus histamine concentration) of dose-response curves are linear for both rabbit and guinea pig atria. In the presence of low concentrations of burimamide; (2.4 times 10-5 M), the displacement of curves suggests a competitive type of inhibition both for rabbit and guinea guinea pig atria. The apparent association constants calculated from these curves are: K1 (rabbit) 3.7 times 10-6M and K-1 (guinea pig) 6.7 times 10-6M. These results for guinea pig atria are in satisfactory agreement with the value obtained in another laboratory (2). At higher concentrations of burimamide, inhibition curves showed distinct evidence of departure from competitive character for both guinea pig and rabbit atria.     

Concentration estimation via curve fitting: Quantification of negative inotropic agents by using a simple mathematical method in guinea pig atria

We created a simple method based on curve fitting in order to assess the concentration of pharmacological agonists or antagonists in the microenvironment of the receptors. We tested our method in electrically driven guinea pig left atria by estimating the concentration of N⁶-cyclopentyladenosine (CPA; A₁ adenosine receptor agonist), acetyl-β-methylcholine (muscarinic receptor agonist) and verapamil (L-type Ca²⁺ channel inhibitor) added previously to the atria in known amounts. Our results validated the fitness of the model under specified conditions. In addition, our data suggest a relatively slow elimination of CPA in isolated, practically bloodless guinea pig atrial myocardium.     

Platelet aggregation inhibitory activity and vasodepressor activity of a series of bicyclo[3.2.0]hetp-6-ylidene iminoxy alkanoic acids with modified lower side chains

Prostacyclin analogues derived from modification of the lower side chain of the bicyclo[3.2.0]hept-6-ylidene iminoxyacetic acid ( ) were studied in inhibition of and platelet aggregation and in the spontaneously hypertensive rat. Iminoxyacetic acids ( ) and iminoxypropionic acid ) were 2.9, 3.0, 1.9 and 2.0 times respectively more potent than PGE₁ in inhibiting ADP-induced aggregation of human platelets . Following intravenous administration at a dose of 90–110 μg/kg in the guinea pig, iminoxyacetic acids ( ), ( ) and iminoxypropionic acid ( ) showed a maximum inhibition of 82–92% with a half life in the range of 14–22 min. Following oral administration at a dose of 1 mg/kg in the guinea pig, iminoxyacetic acids ( ) and ( ) inhibited heterologous platelet aggregation for 4.5 h. Following intravenous administration in spontaneously hypertensive rats, acids )-( ) lowered the mean blood pressure in a dose dependent manner. At a dose of 100 μg/kg, the effect lasted for 20–40 min.     

Toxoplasma gondii: trypsin treatment of trophozoites for enhanced immune cytolysis in the dye test

The Sabin-Feldman dye test for the detection of toxoplasma antibodies has been modified by the use of fresh guinea pig serum and of trypsinized toxoplasma trophozoites. Guinea pig serum at a final concentration as low as 1.0–1.5% has been found sufficient to give satisfactory results. The use of trypsinized parasites has been observed to enhance the sensitivity of the titer. It is suggested that the organisms are coated with a trypsin labile substance which may be responsible for preventing access of antibodies to the parasite cytoplasm. The complement system in the serum may be the only essential accessory factor in this respect.     

Prostaglandin release by slow reacting substance from guinea pig and human lung tissue

Slow reacting substance (SRS) injected into the pulmonary artery released prostaglandin E (PGE) and F_2α (PGF_2α) and the 15-keto-13, 14-dihydro PG metabolites from non-sensitized and ovalbumin sensitized, isolated, perfused guinea pig lungs. PGs were also released from lungs incubated with SRS. Sensitized lungs released more PGs in both types of preparations. Indomethacin inhibited the effect of SRS. Passively sensitized human lung fragments, in parallel to guinea pig lung, released PGE, PGF_2α and the metabolites when incubatted with SRS or antigen. In experiments, SRS and arachidonic acid given intravenously increased the airway insufflation pressure in anesthetized guinea pigs. These effects, but not the action of injected PGF_2α and histamine, were abolished by indomethacin. The results indicate that one of the modes of SRS action is by release of PGs, and are consistent with the hypothesis that PGs are predominantly “secondary” mediators (in the temporal sense) of the antigen-antibody reaction.     

Capsaicin-sensitive structures as potential target sites for neurotensin and bradykinin in guinea pig atria

We tested the influence of capsaicin (CAP) desensitization on the positive chronotropic and inotropic effects of neurotensin (NT), bradykinin (BK), calcitonin gene-related peptide (CGRP) and noradrenaline (NA) in guinea pig isolated atria. The positive chronotropic and inotropic effects of NT and BK were completely inhibited, whereas those elicited by CGRP and NA were either slightly reduced (CGRP) or unaffected (NA), in CAP-desensitized compared to control atria. Cross-desensitization studies using CAP, NT and BK showed that the positive chronotropic and inotropic effects of CAP are slightly affected, whereas those evoked by BK are markedly reduced in NT-desensitized atria. On the other hand, the positive chronotropic and inotropic effects of CAP and NT were similar in BK-desensitized and control atria. The results were interpreted as an indication that NT, BK and CAP produce their excitatory effects in guinea pig atria by interacting with a common population of CAP-sensitive sensory nerve fibers (presumably substance P (SP)- and CGRP-containing nerve fibers). The absence of cross-desensitization between NT or BK and CAP, or between NT and BK, suggests that the activation and desensitization of atrial, CAP-sensitive sensory nerve fibers by the latter agents involve different receptors and/or mechanisms.     

Homologous IRCM-Serine Protease 1 from pituitary,heart atrium and ventricle: A common pro-hormone maturation enzyme?

IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem. 261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem. 261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a similar enzyme was isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific for the same cleavage sites in POMC, as the porcine pituitary homologue. Furthermore, the rat and the porcine enzymes cleave rat pro-Atrial Natriuretic Factor (pro-ANF 1–126) to yield ANF 103–126, 102–126 and 99–126 in that order of preference. This suggests thatin vitro the cleavage sites preferred in pro-ANF resemble those found in brain and hypothalamus. The enzyme is nine times more abundant in atria versus ventricles/mg protein. It is concluded that IRCM-SP1, could well represent a common pro-hormone maturation enzyme for POMC and Pro-ANF and possibly many other pro-hormones.     

Angiotensin-(1–7) Does Not Affect Norepinephrine Neuronal Uptake or Catabolism in Rat Hypothalamus and Atria

SUMMARY 1. Since we previously reported that angiotensin-(1–7) [Ang-(1–7)] increases or inhibits norepinephrine (NE) release in rat atria or hypothalamus, respectively, the present work was undertaken to investigate the effect of the heptapeptide on NE neuronal uptake and metabolism in atria and hypothalamus isolated from rats.2. Ang II (1–10 M) caused a decrease in neuronal NE uptake in both atria and hypothalami isolated from rats. On the contrary, tissues incubated with [³H]NE in the presence of 0.1–10 M Ang-(1–7) showed no modification in [³H]NE content with respect to the control group, suggesting that the heptapeptide did not modify [³H]NE neuronal uptake.3. To study the effect of the heptapeptide on NE catabolism, monoamine-oxidase (MAO) and catechol-O-methyltransferase (COMT) activities were determined. Pretreatment of the tissue with Ang-(1–7) (0.1–1.0 M) showed a tendency to diminish MAO activity in rat atria, while no significant changes were observed in hypothalamic MAO activity. Moreover, the heptapeptide (0.1–1.0 M) did not affect central COMT activity with respect to the control group.emsp;4. Present results allow us to conclude that Ang-(1–7) interacts with noradrenergic neurotransmission by increasing or inhibiting NE release at the peripheral and central levels, respectively, without affecting either the neurotransmitter neuronal uptake or catabolism.     

Species comparison of adenosine A1 receptors in isolated mammalian atrial and ventricular myocardium

Various species have been used as models to study the effects of adenosine (ADO) on atrial and ventricular myocardium, but few direct tissue comparisons between species have been made. This study further characterizes adenosine A(1) receptor binding, adenylate cyclase activity and direct and indirect A(1) receptor-mediated functional activity in atrial and ventricular tissue from Sprague-Dawley rats and Hartley guinea pigs. Rat right atria (RA) were found to be significantly more sensitive to cyclopentyladenosine (CPA), while guinea pig left atria (LA) were more sensitive to CPA. After the addition of isoproterenol (ISO), the reduction of CPA response in rat RA was significantly greater than in guinea pig; however, after ISO treatment, the guinea pig LA was more sensitive to CPA than the rat. Adenylate cyclase inhibition by CPA was significantly greater in atria and ventricles obtained from guinea pig than rat. In competition binding experiments, guinea pig RA had significantly more high affinity sites than rat, but the K(i)s were not significantly different. There were no significant differences between guinea pig LA and rat LA. Guinea pig ventricular tissue had fewer high affinity sites than rat without any differences in their K(i) values. In antagonist saturation experiments, the density and affinity of A(1) receptors in guinea pig cardiac membranes were significantly greater than in rat. Our results indicate definite species differences as well as tissue differences between rat and guinea pig. These differences must be considered when interpreting studies using rat and guinea pig tissue as models for cardiac function.