Effects of calcium antagonist, 6-(N, N-diethylamino) hexyl-3, 4, 5-trimethoxybenzoate, on digitalis-induced arrhythmias and cardiac contractions

6-(N, N-Diethylamino) hexyl-3, 4, 5-trimethylbenzoate (TMB-6) and lidocaine were equipotent (1 mg/kg) in the conversion of ectopic rhythms to normal rhythms in digoxin-toxic dogs. However, TMB-6 had fewer side effects on heart rates and dp/dt than lidocaine. TMB-6 inhibited the contractile force of electrically stimulated dog and guinea-pig atria and ventricles at concentrations ranging from 2.5 X 10(-5) to 1.7 X 10(-4) M. Elevation of extracellular Ca++ concentrations from 2.7 to 5.4 mM produced a significant increase in the ID50 of TMB-6 in atria (from 2.5 X 10(-5) to 5.0 X 10(-5) M in dogs and from 7.2 X 10(-5) to 1.0 X 10(-4) M in guinea pigs). TMB-6 (7.3 X 10(-5) to 2.4 X 10(-4) M) depressed the amplitude of Ca++-dependent action potentials in depolarized dog cardiac Purkinje fibers. These results are discussed with regard to the antagonism of TMB-6 on Ca++ availability in the myocardium which leads to the conversion of cardiac arrhythmias.     

Effects of diltiazem on electrical and mechanical activities of isolated guinea pig taenia coli

Effects of diltiazem on electrical and mechanical activities of isolated guinea pig taenia coli were studied by means of the double sucrose-gap method. In the spontaneously active preparations, diltiazem (2.2 X 10(-6) M) suppressed both electrical activity and isometric contraction, while electrical and mechanical activities evoked by the depolarizing current pulse were not affected at the concentration of 2.2 X 10(-6) M. In the presence of 2.2 X 10(-5) M diltiazem, the evoked contractile force and the number of repetitive firings during depolarization were reduced, whereas the single spike was almost unchanged or somewhat inhibited. At 2.2 X 10(-4) M diltiazem, both electrical and mechanical activities were almost abolished. The contractile force and single spike suppressed by diltiazem were partly reversed by the addition of 5 mM CaCl2. There was little significant change in membrane potential and membrane resistance. Similar but somewhat weaker effects were observed when NaCl was replaced with sucrose. In some preparations, 2.2 X 10(-4) M diltiazem reduced the contractile force without significant influence on the electrical activity in Na+-free Locke solution. CoCl2 (3 mM) inhibited the evoked activities in both normal and Na+-free solutions. Possible mechanisms for the relaxing effects of diltiazem on isolated guinea pig taenia coli were discussed.     

Effects of angiotensin II and its blockers Sar1-Ile8-angiotensin II and DuP 753 on drinking in ducks in relation to properties of subfornical organ neurons

Properties of systemically applied angiotensin II in stimulating water intake of normally hydrated ducks were studied and the results compared with properties of angiotensin II-responsive neurons of the subfornical organ which are considered as targets for circulating angiotensin II acting as a dipsogen. Following intravenous infusion of hypertonic saline (2000 mosmol.kg-1 at 0.3 ml.min-1 for 1 h), intravenous infusion of 0.3 ml.min-1 isotonic saline with angiotensin II (200 ng.min-1), starting 1 h later, stimulated drinking in each case at an angiotensin II plasma level of about 1400 pg.ml-1. Without hypertonic priming, the same angiotensin II infusion did not stimulate drinking in each experiment; however, if effective, repeated infusions of ANGII induced stable dipsogenic responses. Angiotensin II infusions did not alter plasma levels of antidiuretic hormone. Sar1-Ile8-angiotensin II, a non-selective angiotensin II antagonist, acted weakly as a partial agonist when infused at a dose 200-fold higher than angiotensin II and effectively blocked the dipsogenic action of angiotensin II; this corresponds to the inhibition of angiotensin II-induced excitation by Sar1-Ile8-angiotensin II observed in duck subfornical organ neurons. DuP 753 (losartan), an angiotensin II antagonist specifically blocking AT1 receptors in mammals, had equivocal effects on angiotensin II-induced drinking in ducks at rates 50- and 200-fold higher than angiotensin II, which corresponds to the weak inhibitory action of this compound on angiotensin II-induced neuronal excitation in the duck SFO. Blood pressure was only marginally elevated by the applied angiotensin II dose and Sar1-Ile8-angiotensin II had no effect.     

The role of angiotensin AT1 receptors in the diuretic, natriuretic, kaliuretic and blood pressure responses induced by angiotensin II activation of the median preoptic nucleus in conscious rats

We determined the effects of two classical angiotensin II (ANG II) antagonists, [Sar1, Ala8]-ANG II and [Sar1, Thr8]-ANG II, and losartan (a nonpeptide and selective antagonist for the AT1 angiotensin receptors) on diuresis, natriuresis, kaliuresis and arterial blood pressure induced by ANG II administration into the median preoptic nucleus (MnPO) of male Holtzman rats weighing 250-300 g. Urine was collected in rats submitted to a water load (5% body weight) 1 h later. The volume of the drug solutions injected was 0.5 microliters over 10-15 s. Pre-treatment with [Sar1, Ala8]-ANG II (12 rats) and [Sar1, Thr8]-ANG II (9 rats), at the dose of 60 ng reduced (13.7 +/- 1.0 vs 11.0 +/0 1.0 and 10.7 +/0 1.2, respectively), whereas losartan (14 rats) at the dose of 160 ng totally blocked (13.7 +/- 1.0 vs 7.6 +/- 1.5) the urine excretion induced by injection o 12 ng of ANG II (14 rats). [Sar1, Ala8]-ANG II impaired Na+ excretion (193 +/- 16 vs 120 +/- 19), whereas [Sar1, Thr8]-ANG II and losartan block Na+ excretion (193 +/- 16 vs 77 +/- 15 and 100 +/- 12, respectively) induced by ANG II. Similar effects induced by ANG II on K+ excretion were observed with [Sar1, Ala8]-ANG II, [Sar1, Thr8]- ANG II, and losartan pretreatment (133 +/- 18 vs 108 +/- 11, 80 +/- 12, and 82 +/- 15, respectively). The same doses as above of [Sar1, Ala8]-ANG II (8 rats), [Sar1, Thr8]-ANG II (8 rats), and losartan (9 rats) blocked the increase in the arterial blood pressure induced by 12 ng of ANG II (12 rats) (32 +/- 4 vs 4 +/- 2, 3.5 +/- 1, and 2 +/- 1, respectively. The results indicate that the AT1 receptor subtype participates in the increases of diuresis, natriuresis, kaliuresis and arterial blood pressure induced by the administration of ANG II into the MnPO.     

Agonist and antagonist effects of Sar1-ala8--angiotensin II in salt-loaded and salt-depleted normal man

1 Three normal subjects were infused with Sar1-ala8-angiotensin II (Saralasin, P113) whilst on a high sodium (200 mEq + normal diet) and a low sodium (10 mEq diet) intake. 2 On the high sodium intake when angiotensin II and plasma renin activity (PRA) were suppressed, P113 infusion (5-10 mug kg-1 min-1) caused a slight rise in BP and a marked drop in urine flow and sodium excretion, with a fall in glomerular filtration rate, and effective renal plasma flow. 3 On the low sodium intake, when angiotensin II and PRA were increased, P113 infusion (5-10 mugkg-1 min-1) caused no change in blood pressure, urine flow or sodium excretion. However, when P113 was infused at an incremental rate starting at 0.25 mug kg-1 min-1 there was a fall in standing BP, which was maximal at an infusion rate of 1 mug kg-1 min-1, and this fall in standing BP was largely abolished as the rate of infusion was increased to 10 mug kg-1 min -1. 4 These results show firstly that angiotension II is involved in maintaning standing blood pressure during dietary sodium depletion in normal man and secondly that P113 does have agonist as well as antagonist activity in normal man, the effect depending on the level of angiotension II and sodium intake. When looking for angiotensin II mediated hypertension it may ne important to use an incremental rate of infusion of P113 as the agonist activity of larger doses may mask its hypotensive action.     

Modification by temperature of the response of isolated aorta to stimulatory agents and transmural stimulation

Contractile responses of helically-cut strips of the rabbit aorta to drugs, ions and transmural electrical stimulation were compared at different temperatures of the bathing medium, the response at 37 degrees C being taken as control. The dose-response curve of norepinephrine was moved to the right and downward by lowering the temperature from 37 to 25 degrees C and by raising temperature to 40 degrees C. Responses to transmural neural stimulation at frequencies of 5 and 20/sec were attenuated at 25 degrees C, the attenuation being greater in the response at the lower frequency. Concentrations of exogenous norepinephrine needed to produce the same magnitude of contraction as that with transmural stimulation were markedly increased by lowering the temperature to 25 degrees C. Contractile responses to norepinephrine (2 X 10(-6) M), histamine (2 X 10(-5) M) and angiotensin II (10(-7) M) were attenuated by 32-44% at 25 degrees C, whereas the responses to K+ (25 mM) and Ba++ (2 mM) were dependent on temperatures between 25 and 37 degrees C and were attenuated by 69 and 92%, respectively, at 25 degrees C. Contractures induced by Ca++ in K+-depolarized preparations exposed to Ca++-free media and also by Ba++ in preparations exposed to Ca++-free media varied directly by raising temperatures. Interference with the influx of divalent cations, such as Ca++ and Ba++, may be involved in the cold inhibition of aortic contractility.     

Angiotensin antagonists and the adrenal cortex and medulla

Several analogs of angiotensin in which the phenylalanine in position 8 of the peptide chain was replaced by an aliphatic amino acid residue are specific antagonists of angiotensin in aorta, the adrenal medulla, and adrenal zona glomerulosa. In the adrenal cortex and medulla, all actapeptide analogs have more agonist activity than in aortic strips. In studies with N-terminally substituted analogs, it appears that adrenal degradation of the angiotensin molecule by aminopeptidase(s) does not occur or is not retarded by N-terminal mocifications such as sarcosine substitution. The decapeptide analog [Ile8]-angiotensin I and heptapeptide analog [des-Asp1, Ile8]-angiotensin II were excellent antagonists in the adrenal medulla and each peptide was devoid of intrinsic activity. These substituted homologs of angiotensin may offer a novel approach for the development of selective antagonists of angiotensin receptors. In the adrenal cotex, [des-Asp1, Ile8]-heptapeptide possessed greater receptor affinity than any of the angiotensin octapeptides studied. This C-terminally substituted heptapeptide does have significant intrinsic activity in the adrenal cortex which would limit the use of this compound as an antagonist of vascular responses to angiotensin II. In studies with [Ile8]-angiotensin II, [Sar1, Ile8]-angiotensin II, and [des-Asp1, Ile8]-angiotensin II, the pA2 values calculated indicate that the N-terminal residue is not important for receptor binding in the adrenal cortex but may be of significance in binding to adrenal medullary and aortic smooth muscle receptors. At the present time it appears unlikely that any single animal model or assay system can reliably predict the agoinst/antagonist activities of angiotensin analogs for all the various end organs which respond to the angiotensins.     

Defects of immediate memory in Broca''s and conduction aphasia

The effects of local anesthetic agents (lidocaine, procaine, cocaine) and diphenylhydantoin (DPH) were studied on the slow electrical responses induced by isoproterenol or caffeine in cardiac muscle preparations rendered inexcitable by tetrodotoxin (TTX) or by partial depolarization with elevated K+ (26 mM). In such inexcitable cells, we previously demonstrated that addition of some positive inotropic agents, such as catecholamines, histamine, and methylxanthines, rapidly increase the number of available slow Ca2+--Na+ channels, thus allowing slowly rising electrical responses resembling the plateau component of the cardiac action potential. In embryonic chick (16-20-day-old) myocardial cells (ventricular) studied as intact perfused hearts or as reaggregated cell cultures of trypsin-dispersed cells, high concentrations (10-(3) M) of all of the above drugs blocked the induced slow responses with their associated contractions; low concentrations (10-(5) M) of these agents reduced the maximal rate of rise (+Vmax) of the slow responses and depressed the contractions. For comparison with their effects on the slow response, the actions of these drugs on the normal action potential were also studied. As with the slow response, all of these drugs depressed the rate of rise of the action potential (10-(4) M) or blocked it at higher concentrations (10-(3) M); in contrast, low concentrations (10-(5) M) of lidocaine and DPH increased +V max. These findings suggest that local anesthetics, which interact with the lipid phase of the cell membrane, lead to blockade of the slow Ca2+--Na+ channels as well as of the fast Na+ channels in the myocardial sarcolemma.     

Calcium-dependent potassium exchange in human red cell ghosts

1. Ca buffers may be introduced into human red cells by reversible haemolysis. The resealed ghosts retain Ca and chelating anions in the same ratio as in the haemolysing solution, enabling the intracellular Ca2+ concentration to be calculated simply. 2. The passive permeability of the ghosts to Na and Cl is unaffected by intracellular Ca2+ concentrations in the 10(-8)-10(-4) M range, whereas the K permeability is greatly increased at concentrations above 10(-7) M. 3. These preparations enable Ca-dependent K movements to be studied under stable conditions. When the ghosts contain about 5 X 10(-6) M-Ca2+, over 96% of K transport occurs via the Ca-sensitive route.     

Angiotensin-(1-7) and the rat aorta: modulation by the endothelium

Peptide metabolites of angiotensin I and II are active components of the renin-angiotensin system. One such peptide is angiotensin-(1-7), which has been shown to be present in various tissues and has properties distinct from those of angiotensin II. We examined the effects of angiotensin-(1-7) on endothelium-intact and denuded rat aorta. Second, we evaluated whether an interaction occurred between angiotensin-(1-7) and angiotensin peptides, as well as noradrenaline. Finally, we addressed whether the responses to angiotensin-(1-7) were mediated by an AT1 receptor. Angiotensin-(1-7) produced concentration-dependent relaxations of the rat aorta that were significantly greater in endothelium-intact preparations (81.1 +/- 18.9% and 29.6 +/- 2.9% for intact and denuded, respectively). Angiotensin-(1-7) inhibited responses generated to angiotensin I, II, III, and noradrenaline. In endothelium-denuded preparations, angiotensin-(1-7) produced a rightward shift of the concentration-effect curves to angiotensin II and noradrenaline. In addition, the inhibition against angiotensin I and II was significantly greater in endothelium-intact preparations [mean median inhibitory concentration (IC50) values for endothelium-intact preparations, 1.25 x 10(-9) M and 1.57 x 10(-9) M for angiotensin I and II, respectively; and for endothelium-denuded preparations, 1.77 x 10(-8) M and 1.17 x 10(-8) M for angiotensin I and II, respectively). Losartan did not affect relaxations in endothelium-intact preparations but caused a significant potentiation of the relaxation by angiotensin-(1-7) in denuded preparations. We conclude that angiotensin-(1-7) is a component of the renin-angiotensin system that acts to modulate the pressor effects of angiotensin II and noradrenaline.     

Diagnosis of rheumatoid polyarthritis at onset. Manifestations, enigmas, hopes or solution

Quantitative nutrient requirements for unrestricted autotrophic growth of Alcaligenes eutrophus were determined. Minimum saturating concentrations of Mg2+, SO42-, PO43-, Fe3+, and Na2+ for an optical density increase of 2 were 10(-4) M 8 X10(-5) M, 5 X 10(-4) to 6 X 10(-4) M, 10(-5) M, and 10(-7) to 2 X 10(-7) M, respectively. Trace metal requirements for cobalt, chromium, and copper were also demonstrated, but minimum concentrations could not be determined because other reagents contributed a high background of these metals. Under certain conditions an apparent response to zinc was observed, although other experiments suggest the zinc salt contained another metal that was required for growth. Poly-beta-hydroxybutyrate biosynthesis was shown to be initiated by a magnesium or sulfate deficiency as well as by a nitrogen or phosphate deficiency.     

Effects of growth rate and cell density on nerve growth factor secretion in cultures of vascular and bladder smooth muscle cells from hypertensive and hyperactive rats

Changes in action potential parameters by and inotropic responses to nicardipine, verapamil, ryanodine and cyclopiazonic acid were examined in isolated ventricular myocardial preparations from neonatal and adult mice. The action potential of both neonatal and adult mice had a unique configuration with little evidence of a plateau at depolarized membrane potential; the action potential duration was significantly larger in neonatal preparations. Nicardipine had no effect on action potential parameters in the adult while it significantly shortened the action potential duration at 50% repolarization in the neonate. Ryanodine significantly shortened the action potential duration at 80% repolarization at both ages: the shortening was significantly larger in the adult when compared with the neonate. The contraction of ventricular preparations from adult mice were relatively resistant to nicardipine and verapamil. Nicardipine or verapamil, even at 10(-5) M, only decreased the contractile force to 70% of control values; the decrease was much less than that reported in other experimental species such as chick, guinea pig or rabbit. In the neonate, 10(-5) M nicardipine or verapamil decreased the contractile force to 30% of control values. Ryanodine had a potent negative inotropic effect both in the neonate and adult; the effect was significantly larger in the adult. Cyclopiazonic acid produced a decrease in contractile force and prolongation of the time required for relaxation; both effects were significantly larger in the adult. These results suggest that the contraction of the adult mouse myocardium is highly dependent on SR function and less dependent on transsarcolemmal Ca2+ influx when compared with the myocardium of the neonatal mouse and that of other species.     

HR 720, a novel angiotensin receptor antagonist inhibits the angiotensin II-induced trophic effects, fibronectin release and fibronectin-EIIIA+ expression in rat aortic vascular smooth muscle cells in vitro

The aim of this study was to evaluate the direct trophic effects of angiotensin II (AII) on rat vascular smooth muscle cells obtained from a single cellular isolate. Cell volume, protein synthesis, fibronectin (FN) release and FN-EIIIA+ mRNA isoform expression were analyzed in parallel. The effects of HR 720, a novel AT1 angiotensin receptor antagonist with some AT2 receptor affinity, were compared with those of selective AT1 antagonist EXP 3174. Both HR 720 and EXP 3174 inhibited in a concentration-dependent manner the maximum increase in cell volume induced by 10(-9) M Sar1-All (IC50 = 0.49 x 10(-9) M and 0.79 x 10(-9) M, respectively). Maximum [3H]leucine incorporation was also achieved at 10(-9) M All. HR 720 blocked the increase in protein synthesis with potency similar to EXP 3174; the respective IC50 values were 1.04 x 10(-9) M and 1.36 x 10(-9) M. All dose-dependently increased FN release, which was also equally inhibited by about 50% with both compounds at 10(-6) M. Furthermore, All enhanced FN-EIIIA+ mRNA in rat vascular smooth muscle cells (VSMC), which indicated a modulation of FN isoform expression which was inhibited by angiotensin II antagonists. In conclusion, All induced parallel and concentration-dependent increases in cell volume, protein synthesis, FN release and FN-EIIIA+ mRNA expression in vascular smooth muscle cells. These effects appeared to be essentially mediated by AT1 receptor stimulation as indicated by the equal inhibitory effects of HR 720 and EXP 3174.     

Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors

The uterotonic potencies of the naturally occurring mammalian tachykinins and the synthetic subtype-selective agonist analogues of these agents [Lys5,MeLeu9,Nlel0]neurokinin A-(4-10) and [Nle10]neurokinin A-(4-10) (tachykinin NK2 receptor-selective), [Sar9,Met(O2)11]substance P (tachykinin NK1 receptor-selective) and senktide (tachykinin NK3 receptor-selective) were determined using preparations from oestradiol-treated rats. The endopeptidase 24.11 inhibitor, N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl-(S)-isoserine+ ++ (SCH 39370), potentiated responses to neurokinin A, neurokinin B and substance P, but not to [Lys5,MeLeu9,Nle10)]neurokinin A-(4-10) or senktide. [Nle10]neurokinin A-(4-10) effects were potentiated by SCH 39370 with amastatin and those to [Sar9,Met(O2)11]substance P were potentiated by SCH 39370 and captopril in combination. In the presence of optimal concentrations of peptidase inhibitors the relative order of agonist potency was: neurokinin A > substance P > neurokinin B for the naturally occurring mammalian tachykinins and [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) > [Nle10]neurokinin A-(4-10) > [Sar9,Met(O2)11]substance P > senktide for the synthetic tachykinin analogues. Thus, while a tachykinin NK2 receptor predominates in the oestrogen-primed uterus, a tachykinin NK1 receptor may also be present. The non-peptide tachykinin NK3 receptor antagonist, SR 142801, did not antagonise the effects of senktide suggesting that tachykinin NK3 receptors do not mediate its relatively minor effect on the uterus of the oestrogen-primed rat.     

Transmural care. A new approach in the care for terminal cancer patients: its effects on re-hospitalization and quality of life

The pharmacological properties of non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission were investigated in the rabbit choledocho-duodenal junction (CDJ), using the microelectrode and tension recording methods. L-NAME (10(-4) M) and apamin (5 X 10 (-6) M) suppressed NANC relaxation evoked by electrical field stimulation (EFS) in the presence of atropine and guanethidine (each 10(-6) M) to a similar extent (to about 40% of the initial control). However, combined application of L-NAME (10(-4) M) and apamin (5 X 10(-6) M) did not abolish it. EFS also evoked biphasic inhibitory junction potentials (IJPs) consisting of initial fast and slow sustained components in the presence of atropine and guanethidine (each 10(-6) M). Apamin (5 X 10(-8)-5 X 10(-6) M) dose-dependently suppressed the initial fast component by about 70%. In contrast, L-NAME (10(-4) M) did not affect either the amplitude of IJP or the resting membrane potential. PACAP-38 (> 10(-8) M) dose-dependently hyperpolarized the smooth muscle membrane of rabbit CDJ followed by a slow repolarization to the original level. After pretreatment with apamin (5 X 10(-7) M), PACAP-38 (10(-6) M) failed to evoke membrane hyperpolarization. During repolarization in the continued presence of PACAP-38, the amplitude of initial fast component of IJP was reduced to about 40-60% of control value, while that of the slow one was unaffected. A similar suppression of initial fast component of IJP (about 40% of the control value) also occurred after application of PACAP (6-38), a PACAP antagonist, or prolonged treatment with monoclonal antibodies to PACAP-27 or PACAP-38. Furthermore, the substantial part of residual fast IJP in the presence of PACAP (6-38) was suppressed by desensitization to alpha,beta-methylene ATP (10(-3) M). These results indicate that in rabbit CDJ NANC relaxation consists mainly of apamin- and L-NAME-sensitive components, which occur in a membrane potential dependent (through membrane hyperpolarization) and independent fashion, respectively. It has further been suggested that PACAP, together with a smaller contribution of ATP, may be involved as the principal apamin-sensitive transmitter in NANC relaxation of this muscle.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of 125I-labeled alpha-bungarotoxin to a 34800 X g pellet of a whole rat brain homogenate has been obtained at levels of 2 pmol toxin per g of whole brain with a Kd of 8-10(-9) M. Binding is reduced 90% by 10(-5) M (+)-tubocurarine chloride and 10(-4) M nicotine, whereas concentrations of 10(-4) M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with 125I-labeled alpha-bungarotoxin and soluble acetylcholine receptor protein preparations from Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character. Extraction of the 34800 X g pellet with 1% Emulphogene yields a soluble fraction with specifically binds 125I-labeled alpha-bungarotoxin with a Kd of 5-10(-9) M. Nicotine and alpha-bungarotoxin at concentrations of 10(-5) M abolish toxin-receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35-40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10(-5) M.     

Positive inotropic responses in cardiac muscles: influence of stimulation frequency and species

Inotropic responses to cumulative additions of methoxamine (10(-7) to 3 X 10(-4) M), isoproterenol (10(-9) to 10(-5) M), or calcium (2 to 32 mM) were measured in isolated rat left atria and papillary muscles and rabbit right ventricular papillary muscles at three stimulation frequencies. Cardiac muscles were incubated in oxygenated Chenoweth-Koelle solution (2 mM calcium) at 37 degrees C. The basal developed force (BDF) before and maximum developed force (MDF) after challenge with methoxamine and isoproterenol were inversely related to stimulation frequency in rat preparations. BDF was directly related to stimulation rate in rabbit papillary muscles while MDF was independent of the rate. Drug-induced increases in force (MDF - BDF) were independent of stimulation frequency in rat and inversely related to stimulation frequency in rabbit. Responses to calcium were similar to the observed adrenergic responses. Also, force-frequency relationships of the rat and rabbit preparations were not similar in the absence and presence of these agonists. These data show that inotropic responses by rat and rabbit hearts are not affected similarly by stimulation frequency and this may reflect a species difference in the utilization of extracellular calcium for contraction.     

Generation of chromophore Tyr-containing mutants of the ribosomal protein L7/L12 from Escherichia coli by site-directed mutagenesis and characterization

1. The effects of tachykinins and capsaicin were studied by means of intracellular membrane potential and isometric tension recordings in the isolated trachea of the guinea-pig. 2. The basal membrane potential averaged -51 mV, and most preparations demonstrated spontaneous slow waves. Tetraethylammonium (TEA), a potassium channel blocker (8 x 10(-3) M), depolarized the membrane potential to -44 mV and induced a rhythmic activity. 3. In control solution, substance P (10(-8)-10(-6) M), [Nle10]-neurokinin A(4-10) (10(-8)-10(-6) M) and capsaicin (10(-7)-10(-6) M) induced concentration-dependent depolarizations which were statistically significant at the highest concentration tested (depolarization by 10(-6) M: 8, 11 and 16 mV for the NK1 agonist, the NK2 agonist and capsaicin, respectively). 4. In the presence of TEA (8 x 10(-3) M), the three substances induced depolarizations which were statistically significant at the highest concentration tested for substance P (10(-6) M) and at 10(-7) and 10(-6) M for both [Nle10]-neurokinin A(4-10) and capsaicin (depolarization by 10(-6) M: 11, 17 and 10 mV for substance P, [Nle10]neurokinin A(4-10) and capsaicin, respectively). 5. In the presence or absence of tetraethylammonium, [MePhe7]-neurokinin B (10(-8)-10(-6) M) did not induce any significant changes in membrane potential. 6. The depolarizing effects of substance P (10(-6) M) and [Nle10]-neurokinin A(4-10) (10(-6) M) were blocked only by the specific antagonists for NK1 and NK2 receptors, SR 140333 (10(-7) M) and SR 48968 (10(-7) M), respectively. The effects of capsaicin (10(-6) M) were partially inhibited by each antagonist and fully blocked by their combination. 7. Substance P (10(-9) to 10(-4) M), [Nle10]-neurokinin A(4-10) (10(-10) to 10(-5) M), [MePhe7]-neurokinin B and capsaicin (10(-7) to 10(-5) M) evoked concentration-dependent contractions. 8. The contractions to substance P were significantly inhibited by SR 140333 (10(-8) to 10(-6) M) but unaffected by SR 48968 (10(-8) to 10(-6) M). Furthermore, the response to [Nle10]-neurokinin A(4-10) was significantly inhibited by SR 48968 and unaffected by SR 140333 at the same concentrations. Although SR 48968 (10(-7) M) alone did not influence the effects of substance P, it potentiated the inhibitory effect of SR 140333 (10(-7) M). A similar synergetic effect of these two compounds was observed in the inhibition of the contractile response to [Nle10]-neurokinin A(4-10). 9. Neither SR 140333 (10(-7) M) nor SR 48968 (10(-7) M) alone influenced the contractions to [MePhe7]-neurokinin B and capsaicin. However, the combination of the two antagonists abolished the contractions to either peptide. 10. These results demonstrate that the stimulation of both NK1 and NK2 tachykinin-receptors induced contraction and depolarization of the guinea-pig tracheal smooth muscle and that both receptors were stimulated during the endogenous release of tachykinins by capsaicin. There was no evidence for a major role of NK3 receptors in the contractile and electrical activity of the guinea-pig isolated trachea.     

Reciprocal modulation of the binding of angiotensin agonists and antagonists to angiotensin receptors in smooth muscle

1. Direct ligand binding studies have shown that the agonist 125I-[Sar1]Ang II and the antagonist 125I-[Sar1Ile8]Ang II bind to bovine uterus smooth muscle membranes in a time-dependent, reversible and saturable manner; both ligands had the same number of high affinity sites. 2. [Sar1Ile8]Ang II inhibited the binding of 125I-[Sar1]Ang II in a non-competitive manner by decreasing the number of high affinity sites without changing the binding affinity of the radioligand. 3. [Sar1]Ang II also inhibited the binding of 125I-[Sar1Ile8]Ang II in a non-competitive manner. 4. Dissociation of both radioligands from their receptor sites was fast enough that pseudo irreversible occupancy of the binding sites could not account for the observed non-competitive inhibition. 5. Displacement studies using 125I-[Sar1Ile8]Ang II as the radioligand provided evidence for the existence of two binding sites when the displacing ligand was [Sar1]Ang II but not when the displacing ligand was [Sar1Ile8]Ang II. 6. GTPS gamma S had no discernible effect on the binding of either 125I-[Sar1]Ang II or 125I-[Sar1Ile8]Ang II to bovine uterine membranes. 7. The present findings are consistent with an allosteric mechanism of antagonism for [Sar1Ile8]Ang II. The data are also consistent with a mechanism wherein agonist and antagonist ligands occupy different binding modes at the same receptor site and induce long-term conformational changes in the receptor which are idiosyncratic with respect to the nature of the ligand. An emerging relationship between the actions of angiotensin peptides and non-peptide mimetics of angiotensin is presented.