Effects of amrinone, a cardiotonic drug, on calcium movements in dog erythrocytes.

Dog erythrocytes (RBC) have a system for passive Ca and Na movements that resembles the Ca-Na exchanger first described in cardiac muscle. Amrinone, a new cardiotonic drug active in humans with congestive heart failure, is shown to stimulate net Ca uptake by dog RBC. Amrinone's action is on Ca influx rather than efflux. The influence of Amrinone on Ca uptake is enhanced when the cells are placed in low Na media; raising external Na or lowering intracellular Na both abolish the effect of the drug. The data suggest that amrinone potentiates passive Ca entry into the cells by a Na-dependent pathway. If Ca moves through myocardial sarcolemma as it does through dog RBC membranes, then the inotropic action of amrinone can be explained on the basis that the drug increases intracellular Ca levels.     

Differential effects of sulmazole (AR-L 115 BS) on contractile force and cyclic AMP levels in canine ventricular muscle: comparison with MDL 17,043

MDL 17,043 [1,3-dihydro-4-methyl-5-[4-(methylthio)-benzoyl]-2H-imidazol-2-one] and AR-L 115 BS [sulmazole, 2-[(2-methoxy-4-methylsulfinyl)phenyl]-1H-imidazo[4,5-b] pyridine] produced substantial positive inotropic effects in a concentration-dependent manner (10(-5) to 10(-3) M) in the isolated canine ventricular trabeculae in the presence of pindolol (3 X 10(-8) M). Both agents exhibited equal potency and intrinsic activity in this preparation. The time to peak tension, relaxation time and total duration of contraction were shortened by MDL 17,043 in a concentration-dependent manner up to the highest concentrations tested (10(-3) M). In contrast, AR-L 115 BS shortened these times in concentrations of 3 X 10(-4) M and lower, but prolonged them in higher concentrations. The positive inotropic effects of MDL 17,043 and AR-L 115 BS were associated with increases in the cyclic AMP levels of the muscles. The time course of the increase in cyclic AMP levels differed for the two drugs. The elevation in cyclic AMP and the positive inotropic effect produced by MDL 17,043 followed a similar time course. By contrast, the rise in the cyclic AMP level became significant only after the positive inotropic effect produced by AR-L 115 BS had reached a steady level. Carbachol, a muscarinic agonist, converted the positive inotropic effect of 10(-3) M MDL 17,043 into a negative inotropic effect, but did not alter the positive inotropic effect of 10(-3) M AR-L 115 BS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac drugs: new inotropes

Conventional pharmacologic therapy for the management of congestive heart failure includes inotropic, vasodilator, and diuretic agents. Phosphodiesterase inhibitors are a new class of inotropic agent that possesses both inotropic and vasodilating capability. Currently, only one of these agents, amrinone (Inocor), has been approved for clinical use in the United States. Nursing management of the patient receiving amrinone requires knowledge of the appropriate vehicle for administration, the recommended dose, and adverse reactions. A thorough understanding of the hemodynamic alterations in CHF, as well as the effect of amrinone on these hemodynamic parameters, is an essential component of nursing care. Amrinone is only available for intravenous administration. Clinical trials, involving several investigational phosphodiesterase inhibitors, are being conducted. These drugs are being administered orally and intravenously. The goal of research is to develop a potent positive inotrope that will be available for both oral and intravenous administration. The focus of current research involving phosphodiesterase inhibitors includes studying the effect of these agents on myocardial ischemia as well as the mortality and morbidity of CHF. Additional knowledge regarding these and other issues is needed before the new inotropes can become a routine component of the pharmacological armamentarium for treatment of CHF.     

Diminished inotropic response to amrinone in ventricular myocytes from myopathic hamsters is linked to depression of high-gain Ca2+-induced Ca2+ release

This study investigates whether amrinone (100-1000 microM), a phosphodiesterase-III inhibitor, can alleviate depression of contractions in ventricular myocytes from prefailure cardiomyopathic (CM) hamsters (80-100 days). Cell shortening and ion currents were measured in voltage-clamped cells at 37 degrees C. Normal myocytes exhibited low-gain Ca(2+)-induced Ca(2+) release (CICR) initiated by test steps from -40 mV and high-gain CICR initiated from more negative potentials. In normal myocytes, amrinone selectively increased contractions initiated by high-gain CICR (fractional shortening increased from 3.6 +/- 0.5% to 5.3 +/- 0.6%, 300 microM amrinone) but had no effect on low-gain CICR. Amrinone decreased L-type Ca(2+) current (I(Ca-L); -5.5 +/- 0.8 to -3.7 +/- 0.5 picoAmp/picoFarad, 300 microM amrinone). In contrast, in CM myocytes, high-gain CICR was virtually absent, and amrinone had no inotropic effect. Amrinone inhibited I(Ca-L) less in CM than normal myocytes. Sarcoplasmic reticulum (SR) Ca(2+) stores, assessed by caffeine, were significantly increased by amrinone in normal but not CM myocytes. Thus, the positive inotropic effect of amrinone in normal hamster myocytes was mediated by selective enhancement of high-gain CICR. This effect was not mediated by stimulation of I(Ca-L) because I(Ca-L) is inhibited by this drug in hamster. High-gain CICR, which is depressed in CM myocytes, cannot be restored by amrinone. However, minimal stimulation of adenylyl cyclase with forskolin restored the positive inotropic effect of amrinone in CM cells. This positive inotropic effect of amrinone may reflect increased SR Ca(2+) stores because increased stores accompanied the positive inotropic effect in normal myocytes but were absent in CM myocytes.     

Developmental changes in the interactions of amrinone and ouabain in canine ventricular muscle

We studied the effects of amrinone on contractility and aftercontractions of ouabain-superfused ventricular muscle from neonatal and 3-month-old dogs. In 3-month ventricular muscle, amrinone, 5.3 X 10(-4) M, alone, increased active tension by 150%. When amrinone was superfused over ouabain-treated ventricular muscle, the increment in contraction was greatest for those muscles in which ouabain had induced a small positive inotropic effect and diminished as the positive inotropic effect of ouabain increased. Amrinone alone did not induce aftercontractions but increased the amplitude of those induced by ouabain by 92 +/- 6%. In neonatal ventricular muscle, amrinone alone was negatively inotropic and it decreased the increment in active tension induced by ouabain.     

In contrast to forskolin and 3-isobutyl-1-methylxanthine, amrinone stimulates the cardiac voltage-sensitive release mechanism without increasing calcium-induced calcium release

The objective of this study was to determine whether the voltage-sensitive release mechanism (VSRM) can be stimulated independently from Ca(2+)-induced Ca(2+) release (CICR) by drugs that elevate intracellular cAMP. Contractions were measured in voltage-clamped guinea pig ventricular myocytes at 37 degrees C. Na(+) current was blocked. We compared effects of agents that elevate cAMP through activation of adenylyl cyclase (1 microM forskolin), nonspecific inhibition of phosphodiesterases (PDEs) [100 microM 3-isobutyl-1-methylxanthine (IBMX)], and selective inhibition of PDE III (100-500 microM amrinone) on contractions initiated by the VSRM and CICR. Forskolin and IBMX significantly increased peak Ca(2+) current and CICR. In addition, these agents also markedly increased contractions elicited by test steps from -65 to -40 mV, which activate the VSRM. However, because these steps also induced inward current in the presence of forskolin or IBMX, CICR could not be excluded. In contrast, amrinone caused a large, concentration-dependent increase in VSRM contractions but had no effect on CICR contractions or Ca(2+) current. Sarcoplasmic reticulum Ca(2+), assessed by rapid application of caffeine (10 mM), was increased only modestly by all three drugs. Normalization of contractions to caffeine contractures indicated that amrinone increased fractional release by the VSRM, but not CICR. Forskolin and IBMX increased fractional release elicited by steps to -40 mV. Increases in CICR induced by forskolin and IBMX were proportional to caffeine contractures. Thus, positive inotropic effects of cAMP on VSRM contractions may be compartmentalized separately from effects on Ca(2+) current and CICR.     

The effects of amrinone on transport and cyclic AMP metabolism in toad urinary bladder

Amrinone is a new noncatechol, nonglycoside agent with cardiotonic and vasodilator properties. This paper examines the effects of amrinone in the toad urinary bladder, a tissue whose function may be altered by many factors which also change cardiovascular activity. Amrinone enhanced the effect of vasopressin and cyclic AMP on water and urea permeabilities, as well as the effect of vasopressin on sodium transport. Consistent with these actions, amrinone inhibited cyclic AMP phosphodiesterase activity in epithelial homogenates and increased both cyclic AMP content and the protein kinase activity ratio measured in intact epithelial cells. The inhibitory effect of amrinone on phosphodiesterase may be relevant to its cardiostimulatory and vasodilator activities.     

Cardiotonic action of [8]-gingerol, an activator of the Ca++-pumping adenosine triphosphatase of sarcoplasmic reticulum, in guinea pig atrial muscle

[8]-Gingerol (gingerol), a component of ginger, produced a concentration-dependent positive inotropic effect on guinea pig isolated left atria at concentrations of 1 X 10(-6) to 3 X 10(-5) M. Gingerol also exhibited positive inotropic and chronotropic effects on guinea pig right atria. The gingerol-induced inotropic effect was abolished by ryanodine, but was little affected by propranolol, chlorpheniramine, cimetidine, tetrodotoxin, diltiazem or reserpine. The time to peak tension and relaxation time within a single contraction were shortened by gingerol (1 X 10(-5) M) as well as isoproterenol, whereas they were prolonged by BAY K 8644. In guinea pig isolated atrial cells, gingerol (3 X 10(-6) M) caused an increase in the degree and the rate of longitudinal contractions. In guinea pig left atria, gingerol (1 X 10(-6) to 3 X 10(-5) M) gave little influence on the action potential, although it increased the contractile force of the atria. The whole-cell patch-clamp experiments showed that the slow inward current was little affected by gingerol (1 X 10(-6) to 3 X 10(-5) M) in voltage-clamped guinea pig cardiac myocytes. The measurement of extravesicular Ca++ concentration using a Ca++ electrode indicated that gingerol (3 X 10(-6) to 3 X 10(-5) M) accelerated the Ca++ uptake of fragmented sarcoplasmic reticulum (SR) prepared from canine cardiac muscle in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles for Ca++ and cyclic AMP in mediating the cardiotonic actions of isomazole (LY175326)

The contractile state of cat papillary muscles was increased by isomazole in a concentration-dependent manner; inotropic effects of the drug were not altered by either prazosin, propranolol or cimetidine. Isomazole inhibited the peak III isozyme of dog heart phosphodiesterase with an IC50 of 100 microM; effects on isozymes I and II were less pronounced. In cat papillary muscles, carbachol (10(-5) M) shifted the relationship between contractility and concentration of isomazole to the right. These data suggest cyclic AMP (cAMP) is involved in the actions of isomazole. In order to assess the relative effects of isomazole on intracellular cAMP and Ca++, cAMP-dependent protein kinase and glycogen phosphorylase, respectively, were used as reporters of these two second messengers. The source of enzymes was either cultured cardiomyocytes or right ventricular biopsies obtained from anesthetized dogs. In the latter case, biopsies were obtained after i.v. administration of isomazole; the pure beta agonist, isoproterenol, was included for comparative purposes. A submaximal inotropic dose of isomazole (0.1 mg/kg i.v.) in dogs resulted in a pronounced increase in contractility that was associated with a 3-fold increase in phosphorylase activity (0.15 +/- 0.01 to 0.46 +/- 0.06, -5'-AMP: +5'-AMP, P less than .05); the activation state of protein kinase was not altered. By contrast, a comparably effective inotropic dose of isoproterenol (0.1 microgram/kg) caused less than a 2-fold increase in phosphorylase activity (0.15 +/- 0.01 to 0.26 +/- 0.02, -5'-AMP: +5'-AMP, P less than .05) and this was associated with a significant increase in the protein kinase activity ratio (0.36 +/- 0.01 to 0.51 +/- 0.04, -cAMP: +cAMP, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Does the positive inotropic action of a novel cardiotonic agent, MCI-154, involve mechanisms other than cyclic AMP?

MCI-154 is a new positive inotropic agent with vasodilating property. Experiments were carried out in the canine isolated right ventricular muscle in order to elucidate whether or not cyclic AMP is involved in the positive inotropic effect (PIE) of MCI-154. MCI-154 (10(-7) to 10(-4) M) produced a concentration-dependent PIE amounting to 75% of the maximal effect of isoproterenol. MCI-154 did not affect the time to peak tension and had a tendency to shorten the relaxation time and total duration of contraction. Pindolol, reserpine-pretreatment or tetrodotoxin did not modify the PIE of MCI-154. MCI-154 increased the cyclic AMP levels only at 3 X 10(-4) M, whereas CI-914, of which chemical structure is similar to that of MCI-154, elevated definitely the cyclic AMP at the lower concentrations (10(-5) to 10(-4) M). Carbachol at a concentration known to decrease markedly the PIE of amrinone, milrinone and papaverine, did not affect the PIE of MCI-154. MCI-154 inhibited the activity of a crude phosphodiesterase (PDE) from the canine ventricular muscle and it enhanced the PIE of isoproterenol, which implied the involvement of cyclic AMP. However, the maximal inhibition of PDE by MCI-154 remained less than 18%. Amrinone, milrinone and papaverine inhibited more potently the PDE activity than MCI-154. These results suggest that the elevation of cyclic AMP levels is only partially involved in the PIE of MCI-154 in the canine right ventricular muscle, and that MCI-154 may have novel mechanisms of action different from those of amrinone, milrinone and CI-914 that are largely cyclic AMP-dependent.     

Electrophysiological actions of the pimobendan metabolite, UD-CG 212 Cl, in guinea pig myocardium.

Pimobendan (UD-CG 115 BS), an inotropic agent and inhibitor of type III phosphodiesterase activity, is demethylated in vivo to form UD-CG 212 Cl, which is a more potent type III phosphodiesterase inhibitor. This study examined cyclic AMP (cAMP)-mediated actions of UD-CG 212 Cl. In guinea pig papillary muscles, UD-CG 212 Cl increased cAMP and stimulated Ca(++)-dependent slow action potentials (APs) in a dose-dependent manner. When compared to previous studies using pimobendan, UD-CG 212 Cl was approximately 100-fold more potent. UD-CG 212 Cl had no additional effects on slow APs in the presence of a maximal dose of isoproterenol (1 microM). Propranolol had little effect on UD-CG 212 Cl-induced slow APs. These results, along with previous studies, indicate that slow AP induction by UD-CG 212 Cl was cAMP-dependent, and the increase in cAMP levels was most likely due to phosphodiesterase inhibition and not beta receptor stimulation. Experiments with tetraethylammonium.Cl suggested that UD-CG 212 Cl probably did not induce slow APs by blocking K+ channels. In voltage-clamped ventricular myocytes UD-CG 212 Cl (100 microM) could stimulate Ca++ current (+21 +/- 5%) when basal cAMP levels were enhanced with a submaximal dose of isoproterenol (10(-9)-10(-8) M). Isoproterenol was not required to observe the stimulating effect of UD-CG 212 Cl on Ca++ current in intact, nondialyzed cells prepared using the nystatin-perforated patch method. Studies with the stereoisomers of UD-CG 212 Cl showed that the D-isomer was more potent than the L-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibitory effects of amrinone on isolated rat uterus

Amrinone, a new cardiotonic drug, has received attention as a better therapeutic agent than the cardiac glycosides in the treatment of congestive heart failure. In this study, the effects of amrinone on isolated rat uterus and its probable mechanism of action were investigated. At two different concentrations (0.1 and 0.5 mM) the inhibitory effects of amrinone on the spontaneous contractions of rat uterus were noted. In addition, amrinone (0.5 mM) was found to inhibit the tonic contractions induced by potassium sulphate (K₂SO₄)-Ringer solution (91.74%) and calcium chloride (CaCl₂) (93.04%). These inhibitory effects were compared with regulators of the phosphodiesterase enzyme (PDE). It was concluded that amrinone could behave as a calcium antagonist and PDE inhibitor.     

Effects of anagrelide on platelet cAMP levels, cAMP-dependent protein kinase and thrombin-induced Ca++ fluxes

Anagrelide (BL-4162A, 6,7-dichloro-1,5-dihydroimidazo[2, 1-6] quinazolin-2[3H]one monohydrochloride hydrate) is a potent and broad spectrum inhibitor of platelet aggregation. Prior studies showed that anagrelide inhibited platelet cyclic AMP (cAMP) phosphodiesterase activity but did not appreciably elevate platelet cAMP levels. We examined the effects of anagrelide on washed human platelets and found that anagrelide caused significant elevation of cAMP levels. Anagrelide treatment also resulted in activation of the platelet cAMP-dependent protein kinase at anagrelide concentrations of 0.1 to 1 microgram/ml, which inhibited platelet aggregation but caused only small increases in platelet cAMP content. When whole platelets were incubated with radiolabeled phosphate, anagrelide increased phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. The pattern of protein phosphorylation stimulated by anagrelide treatment was similar to that observed when the platelets were treated with forskolin. Anagrelide also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets. The inhibition of increased intracellular Ca++ resulted from block of thrombin-induced mobilization of intracellular Ca++, as well as prevention of Ca++ influx through the plasma membrane. Anagrelide itself had no influence on inositol 1,4,5-trisphosphate-induced Caz5++ release from isolated platelet membrane vesicles. These studies suggest that anagrelide inhibits platelet phosphodiesterase activity in intact platelets resulting in an elevation in cAMP levels sufficient to activate the cAMP-dependent protein kinase and inhibit agonist-activated Ca++ fluxes.     

Enkephalins increase cyclic adenosine monophosphate content, calcium uptake, and contractile state in cultured chick embryo heart cells.

Peripheral vascular effects of opioid peptides are well known, but direct myocardial effects have not been established. We studied the inotropic response of spontaneously beating cultured chick embryo ventricular cells to the enkephalin analogue [D-Ala2]-enkephalin. Amplitude of cell motion increased in a concentration-dependent manner with 0.53 microM [D-Ala2]-enkephalin producing half-maximal response. The mechanism of this positive inotropic effect was investigated by examining alterations in 45Ca influx, cyclic AMP accumulation and adenylate cyclase activity in response to [D-Ala2]-enkephalin. At maximally inotropic concentrations, the 45Ca influx rate increased 39%, adenylate cyclase was stimulated by 30%, and cyclic AMP content rose more than twofold. Thus, in contrast to neural tissue, receptors for enkephalin in cultured heart cells are coupled to adenylate cyclase in a stimulatory manner. Occupancy of these receptors produces an increase in cyclic AMP levels and exerts a positive inotropic effect via a verapamil-sensitive enhancement of Ca influx.     

Sarcoplasmic reticulum-associated cyclic adenosine 5''-monophosphate phosphodiesterase activity in normal and failing human hearts.

Sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was examined in microsomes prepared from the left ventricular myocardium of eight heart transplant recipients with end-stage idiopathic dilated cardiomyopathy and six unmatched organ donors with normal cardiac function. At cAMP concentrations less than or equal to 1.0 microM, sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was functionally homogeneous. cAMP phosphodiesterase activity was inhibited competitively by cGMP (Ki = 0.031 +/- 0.008 microM) and the cilostamide derivative OPC 3911 (Ki = 0.018 +/- 0.004 microM), but was essentially insensitive to rolipram. Vmax and Km were 781.7 +/- 109.2 nmol/mg per min and 0.188 +/- 0.031 microM, respectively, in microsomes prepared from nonfailing hearts and 793.9 +/- 68.9 nmol/mg per min and 0.150 +/- 0.027 microM in microsomes prepared from failing hearts. Microsomes prepared from nonfailing and failing hearts did not differ with respect to either the ratio of cAMP phosphodiesterase activity to ATP-dependent Ca2+ accumulation activity or the sensitivity of cAMP phosphodiesterase activity to inhibition by OPC 3911. These data suggest that the diminished inotropic efficacy of phosphodiesterase inhibitors in failing human hearts does not result from changes in the level, kinetic properties, or pharmacologic sensitivity of sarcoplasmic reticulum-associated cAMP phosphodiesterase activity.     

MCI-154, a novel cardiotonic agent, reverses the acidic pH-induced decrease in responses of cardiac myofilaments to Ca++: comparison with sulmazole and pimobendan

In the present study, we have examined the effects of decreasing pH from 7.0 to 6.6 on the tension developed by direct activation of the myofilaments in chemically skinned fibers from guinea pig papillary muscles. We then compared the effects of the novel inotropic agents MCI-154, pimobendan and sulmazole, which have direct action on cardiac myofilaments, on the acidic pH-induced changes in responses of the contractile system to Ca++. The reduction of pH from 7.0 to 6.8 shifted the pCa (-log[Ca++] M)-tension relation curve to the right with no change in maximum tension. However, the reduction of pH from 7.0 to 6.6 shifted the pCa tension relation curve to the right and also depressed maximum force development. These effects were reversible by returning to neutral pH (pH 7.0), but were not overcome by increasing the free [Ca++] (decreasing pCa from 4.4 to 4.0). The amplitude of pMg-ATP (-log[MgATP]M)-tension curve in the absence of free Ca++ (Ca++ less than 1 nM, bell-shaped curve) was shifted downward by reducing pH from 7.0 to 6.6. MCI-154 (1-100 microM) reversed the acidic pH-induced decrease of tension development which was activated by pCa 5.8 in a concentration-dependent manner. Moreover, the acidosis induced reductions of maximum tension (pCa, 4.4) and pMgATP 6.0-activated tension (Ca++ less than 1 nM) were also reversed by MCI-154 (1-100 microM) in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency modulation of the inotropic action of isoproterenol in mouse heart

In mouse right ventricular strips, field-stimulated to contract isometrically in an oxygenated bicarbonate-buffered physiological salt solution at 22--24 degrees C, the EC50 for the inotropic action of isoproterenol decreased from 37 nM in muscles stimulated at 0.2 Hz to 5 nM in muscles stimulated at 3.3 Hz. At higher rates of contraction, there was also an increased sensitivity to the inotropic actions of norepinephrine and epinephrine but not to those of Ca++ and N6,O2'-dibutyryl cyclic AMP. Increasing the Ca++ concentration further decreased the EC50 for isoproterenol at 3.3 Hz but had no effect on the EC50 at 0.2 Hz. The leftward shift of the contractile response curve at 3.3 Hz was inhibited by verapamil (0.6 microM) and Mn++ (0.25 mM). The stimulation of cyclic AMP accumulation was approximately 6-fold more sensitive to isoproterenol at 3.3 than at 0.2 Hz, but isoproterenol increased contractile force at concentrations two orders of magnitude lower than those that significantly increased cyclic AMP accumulation. Inhibition of cyclic AMP phosphodiesterase activity further increased the sensitivity to the inotropic actions of isoproterenol but did not attenuate the frequency difference. The results indicate that isoproterenol-stimulated Ca++ influx through the slow channel plays an important role in the mechanism of the increased sensitivity to the inotropic action of isoproterenol found at higher frequencies of contraction. Although cyclic AMP accumulation was also frequency dependent, its role in the inotropic action of isoproterenol in mouse heart is not clear.     

Histamine modulates contraction and cyclic nucleotides in cultured rat mesangial cells. Differential effects mediated by histamine H1 and H2 receptors.

Histamine influences the glomerular microcirculation and modulates immune-inflammatory responses. In the rat kidney, histamine is synthesized by glomeruli and stimulates cyclic nucleotide production specifically in glomeruli. We investigated the in vitro effect of histamine on cyclic nucleotide accumulation in rat cultured glomerular mesangial and epithelial cells. Histamine stimulated cyclic AMP (cAMP) accumulation in cultured mesangial cells (64.0 +/- 22.1 to 511.4 +/- 86.6 pmol/mg protein, n = 9) but had no effect on cAMP accumulation in epithelial cells. This effect was dose-dependent and time-dependent. Stimulation of cAMP accumulation occurred in the range of 5 X 10(-6) M-10(-4) M histamine with a half maximal stimulatory effect of 2 X 10(-5) M. Initial stimulation was noted by 30 s, and maximum stimulation was observed at 5 min. The H2 antagonist cimetidine (10(-4) M) abolished the stimulatory effect of histamine (10(-4) M), while equimolar concentrations of the H1 antagonist diphenhydramine had no significant effect on cAMP accumulation. Moreover, the specific H2 agonist dimaprit, but not the H1 agonist 2-pyridylethylamine, stimulated cAMP accumulation. Histamine had no effect on cAMP accumulation in epithelial cells or on cyclic guanosine monophosphate accumulation in epithelial or mesangial cells. Since the in vivo infusion of histamine reduces ultrafiltration coefficient and since mesangial cell contraction is thought to be responsible for the reduction in the ultrafiltration coefficient, we examined the effect of histamine on the contractile property of mesangial cells. Histamine (5 X 10(-6)-10(-4) M) contracted mesangial cells, and the H1 antagonist diphenhydramine (10(-4) M) but not the H2 antagonist cimetidine (10(-4) M) prevented histamine (10(-4) M) induced contraction. In addition, the H1 agonist 2-pyridylethylamine, but not the H2 agonist dimaprit, contracted mesangial cells. Histamine and its specific agonists and antagonists induced contraction of isolated glomeruli as assessed by glomerular planar surface area in a manner parallel to their effect on mesangial cells. Cinnarizine (10(-5) M), a Ca++ channel blocker, or Ca++, Mg++-free medium prevented histamine (10(-4) M) induced mesangial cell and glomerular contraction. Thus, histamine enhances cAMP accumulation specifically in mesangial cells via an H2 receptor. In contrast, histamine contracts mesangial cells and glomeruli via an H1 receptor, an effect that is dependent on extracellular Ca++ entry. These findings show that histamine potentially influences intraglomerular hemodynamics via effects on mesangial cell contraction. Moreover, our findings considered with the in vivo observation that histamine reduces kf via and H1 receptor provide further support of the hypothesis that mesangial cell contraction regulates the glomerular capillary surface area available for filtration. Our studies also show that this contractile effect of histamine is dependent on extracellular calcium. The presence of a cAMP system sensitive to histamine may have major implications in the pathogenesis of inflammatory glomerulopathies. Mesangial cells possess characteristics similar to circulating and tissue immune effector cells, including lysosomal enzyme release, oxygen radical production, and release of a number of immunomodulatory factors. Histamine and cAMP have been shown to modulate such characteristics of inflammatory cells. It is therefore conceivable that histamine, via its interaction with H2 receptors and subsequent generation cAMP, may have profound effects on such properties of mesangial cells, suggesting that this autacoid may modulate not only glomerular hemodynamics but also immune, inflammatory responses within the glomerulus.     

Ca++ inhibition of isoproterenol responses in mammalian skeletal muscle

In noncontracting mouse hemidiaphragms incubated in modified Krebs-Ringer--bicarbonate buffer with 10 mM Ca++, isoproterenol-stimulated phosphorylase a formation, conversion of phosphorylase kinase to the activated form, elevation of cyclic AMP-dependent protein kinase activity ratios and increase in cyclic AMP concentrations were reduced 35 to 50% over the responses in buffer with 2.5 mM Ca++. In buffer with 10 mM Ca++, the initial rate of isoproterenol-stimulated cyclic AMP accumulation was 59% of that in buffer with 2.5 mM Ca++. The inhibitory action of Ca++ on cyclic AMP accumulation was antagonized by verapamil, but not by inhibitors of cyclic nucleotide phosphodiesterase activity. In buffer with 2.5 mM Ca++, isoproterenol-stimulated cyclic AMP accumulation was inhibited by A23187 and caffeine, agents that can increase intracellular Ca++ concentrations. In addition to Ca++, high concentrations of Co++, Ni++, Mn++ and, to a lesser extent, Sr++ inhibited the isoproterenol response. The results of these studies indicate that high buffer Ca++ concentrations inhibit the response of the glycogenolytic pathway to isoproterenol by an action on cyclic AMP formation. We propose that the site of the inhibitory action of Ca++ is the divalent metal activator site associated with hormone-stimulated adenylate cyclase activity.     

Gentamicin blockade of slow Ca++ channels in atrial myocardium of guinea pigs.

Cardiac dysfunction is occasionally detected in patients undergoing treatment with amino-glycoside antibiotics, however, the mechanism responsible for the negative inotropic effect of these agents has not been identified. In the present investigation electrically driven left atria of guinea pigs were used to study the effects of gentamicin on calcium ion (Ca++)-dependent contractile events in heart muscle isolated from in vivo influences. When atria were first inactivated by excess potassium ion (K+; 22mM) and contractions were then restored by isoproterenol (an experimental model that accentuates the contractile dependence of myocardial fibers on influx of Ca++ through specific "slow channels" of the sarcolemma), the cardiac depressant activity of gentamicin (0.1 mM) was profoundly augmented. Conversely, the negative inotropic effect of tetrodotoxin (23.5 micron) was abolished by the same experimental conditions. Also, gentamicin (1 mM) and La+++ (0.5 mM) markedly decreased the positive inotropic response to increased frequency of stimulation; whereas, D600 (1.05 micron) converted the positive frequency-force relationship to a negative relationship. Present data indicate a direct cardiac depressant action of gentamicin, and suggest that this antibiotic adversely affects either the transport system responsible for Ca++ movement through slow channels of the sarcolemma, the availability of Ca++ for translocation to these sites, or both.