Beta-adrenergic and antiadrenergic modulation of cardiac adenylyl cyclase is influenced by phosphorylation

Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A1 receptor (A1R). Because beta 1-adrenergic receptor (beta 1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the beta 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the beta 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140-120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51-54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29-50%, thereby yielding a total antiadrenergic effect of approximately 78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.     

Mechanism of self-regulation and the inotropic reaction of the rat myocardium during aging

The experiments on rat papillary muscles revealed that in ageing rats myocardium has a decreased distension ability. The curves of shortening value/length and force/velocity for the myocardium of old animals are shifted downwards. The alterations in isotonic contraction parameters had a distinct age differentiation, while the age did not affect the inotropic effects of increased frequency, paired stimulation and external calcium. It is suggested that changes in biomechanical properties of ageing myocardium are associated with alterations in calcium transport in sarcoplasmic reticulum.     

Characteristics of the frequency dependence of the contraction force in the hyperthyroid rat myocardium

We have studied the effect of increased contraction frequency (from 0.2 to 1.5 Hz) on developed tension (delta T) in thin papillary muscles of eu- and hyperthyroid rats. The results show that while increasing the contraction frequency, the delta T of euthyroid papillary muscles decreased at lower frequencies than in hyperthyroid group. Also, at the contraction frequencies above 1.0 Hz the absolute and relative levels of delta T of hyperthyroid myocardium were less decreased than in euthyroid preparations. In conclusion, the myocardium of hyperthyroid rat is characterized by a decreased sensitivity to negative inotropic effect of enhanced contraction frequency. In is probably due to the acceleration of the processes of intracellular Ca2+ recycling during diastole under the influence of hyperthyroidism.     

Characteristics of force-frequency relations in the myocardium of the squirrel Citellus undulatus

The force-frequency relationship (FFR) in papillary muscles of the heart of active ground squirrel in different seasons was studied. For comparison, similar preparations from rat and rabbit were used. It was shown that the FFR of papillary muscles of active ground squirrel undergo significant seasonal changes. In summer and a part of autumn squirrels, a negative staircase (a decrease in the isometric force with increasing stimulation frequency) similar to that in adult rat was revealed. The FFR of the majority of autumn, winter and spring squirrels were polyphasic and contained both positive and negative components. Changes in the force in response to the introduction of pauses at a constant stimulation frequency were recorded. Two types of the post-rest recovery pattern were revealed in the myocardium of ground squirrels. For frequencies range with the negative direction of FFR, a typical pattern of rest-potentiation similar to that in rat papillary muscles was observed. The amplitude of the first post-rest contraction (F1) was usually higher than that of the preceding steady-state contraction. In papillary muscles of autumn animals the F1 value was greater that in summer, which suggests an enhanced release of Ca2+ from the sarcoplasmic reticulum. There was no post-rest potentiation in the range of frequencies with positive direction of FFR, and the post-rest recovery pattern in these cases was principally different from those of rat and rabbit preparations. It was proposed that seasonal differences of the FFR of active ground squirrel heart are associated with changes in the ratio of activities of the calcium-transporting system in the hibernation period.     

Genistein attenuates postischemic depressed myocardial function by increasing myofilament Ca2+ sensitivity in rat myocardium

The present study investigated whether genistein, a broad-spectrum tyrosine kinase inhibitor, could increase the myofilament Ca(2+) sensitivity and partially reverse postischemic depressed myocardial function. Left ventricular papillary muscles were isolated from adult Wistar rats and loaded with the Ca2+ indicator, aequorin. The use of fluorocarbon immersion with hypoxia simulated a model of ischemia. Myofilament responsiveness to Ca2+ was evaluated from force-[Ca2+]i relationship recorded during tetani in papillary muscles. Protein levels of troponin I (TnI) were measured in postischemic papillary muscles with the Western blot technique. Isometric contraction was depressed during the period of ischemia and remained low after 60 min of reoxygenation without a corresponding significant change of peak [Ca2+]i in the control group (n = 7). In contrast, the depression of isometric contraction was ameliorated during ischemia in muscle preparations in the presence of genistein (2 micro M; n = 8), and postischemic depressed myocardial contractility partially recovered after a 60-min reperfusion. The myofilament Ca2+ responsiveness was significantly increased in papillary muscles in the presence of genistein. Protein levels of TnI were reduced in postischemic papillary muscles, whereas genistein partially restored decreased protein levels of TnI. Our results reveal that genistein produces an effective attenuation of postischemic depressed myocardial function and improves myofibrillar Ca2+ responsiveness in rat myocardium.     

Force-frequency-relation in human atrial and ventricular myocardium

In human heart failure, an increase in frequency of stimulation is followed by a reduced force of contractionin vivo andin vitro. The present study aimed to investigate whether a different origin of the myocardial sample or pretreatment with the cardioprotective agent 2,3-butanedione-monoxime (BDM) influences the force-frequency-relationship in electrically driven muscle strips taken from failing and nonfailing human myocardium. With as well as without pretreatment with BDM, the altered force-frequency-relationship in failing compared to nonfailing human ventricular myocardium can be observed. The effectiveness and the potency to increase force of contraction following an increase in frequency of stimulation was significantly higher in atrial than in ventricular myocardium in nonfailing and failing tissue. The different observations in atrial and ventricular myocardium provide evidence for functionally relevant differences in the electromechanical coupling between the human atrial and ventricular myocardium.     

Differential effects of Ro 20-1724 and isobutylmethylxanthine on the basal force of contraction and beta-adrenoceptor-mediated response in the rat ventricular myocardium.

Effects of Ro 20-1724, a selective inhibitor of soluble cGMP-insensitive type IV phosphodiesterase, on the force and cAMP levels were compared with those of 3-isobutyl-1-methylaxanthine, a non-selective inhibitor, in the rat ventricular myocardium. Ro 20-1724 scarcely affected the basal force of contraction and cAMP levels, whereas it enhanced the positive intropic effect and cAMP accumulation induced by isoproterenol more effectively than 3-isobutyl-1-methylxanthine. These results imply that inhibition of the soluble cGMP-insensitive type IV PDE by Ro 20-1724 may be crucially involved in the regulation of myocardial contractility through the interaction with cAMP generation in the rat ventricular myocardium.     

Endothelium-derived nitric oxide mediates the antiadrenergic effect of human vasostatin-1 in rat ventricular myocardium

Vasostatins (VSs) are vasoactive peptides derived from chromogranin A (CgA), a protein contained in secretory granules of chromaffin and other cells. The negative inotropic effect and the reduction of isoproterenol (Iso)-dependent inotropism induced by VSs in the heart suggest that they have an antiadrenergic function. However, further investigation of the mechanisms of action of VSs is needed. The aim of the present study was to define the signaling pathways activated by VS-1 in mammalian ventricular myocardium and cultured endothelial cells that lead to the modulation of cardiac contractility. Ca(2+) and nitric oxide (NO) fluorometric confocal imaging was used to study the effects induced by recombinant human VS-1 [STA-CgA-(1-76)] on contractile force, L-type Ca(2+) current, and Ca(2+) transients under basal conditions and after beta-adrenergic stimulation in rat papillary muscles and ventricular cells and the effects on intracellular Ca(2+) concentration and NO production in cultured bovine aortic endothelial (BAE-1) cells. VS-1 had no effect on basal contractility of papillary muscle, but the effect of Iso stimulation was reduced by 27%. Removal of endocardial endothelium and inhibition of NO synthesis and phosphatidylinositol 3-kinase (PI3K) activity abolished the antiadrenergic effect of VS-1 on papillary muscle. In cardiomyocytes, 10 nM VS-1 was ineffective on basal and Iso (1 microM)-stimulated L-type Ca(2+) current and Ca(2+) transients. In BAE-1 cells, VS-1 induced a Ca(2+)-independent increase in NO production that was blocked by the PI3K inhibitor wortmannin. Our results suggest that the antiadrenergic effect of VS-1 is mainly due to a PI3K-dependent NO release by endothelial cells, rather than a direct action on cardiomyocytes.     

The proinflammatory cytokines TNF-alpha and IL-1 beta impair economy of contraction in human myocardium

Considerable experimental evidence has accumulated over the past years that proinflammatory cytokines, especially TNF-alpha and IL-1beta, impair myocardial function in different animal species. On the other hand, several prospective clinical trials studying TNF-alpha antagonist in patients with chronic heart failure were not able to demonstrate a benefit. As there might be a relevant species-related discrepancy, we intended to prove our previous results demonstrating impaired myocardial economy after exogenous administration of recombinant TNF-alpha in rat myocardium. In the present study, both TNF-alpha and IL-1beta not only revealed an immediate negative inotropic effect but also increased specific oxygen demand in human right-atrial myocardium. Enhanced oxygen consumption was not caused by an elevated basal metabolism but an impaired economy of contraction. Our results suggest that proinflammatory cytokines have a considerable effect on myocardial mechano-energetic parameters in human myocardium as well.     

Gene expression of adrenomedullin in failing myocardium: comparison to atrial natriuretic peptide.

The expression of adrenomedullin (AM) and atrial natriuretic factor (ANF) were investigated in the myocardium of a rat model of chronic ischemic heart failure (CHF) compared with sham-operated controls. In addition, human myocardium of patients with end-stage heart failure due to idiopathic dilated cardiomyopathy compared with myocardium of normal subjects (NF) was studied. In CHF, similar AM levels but increased ANF expression were observed in left ventricular myocardium, as assessed by semiquantitative PCR. Functional experiments with freshly excised papillary muscles showed no influence of AM on myocardial contractility. In NF human myocardium, the expression of AM mRNA was threefold higher in atrial compared with ventricular tissue. In analogy, ANF mRNA was increased by approximately 15-fold in atrial tissue. In dilated cardiomyopathy, the expression of AM was significantly increased in right and left ventricles compared with NF. In parallel, ventricular ANF expression was enhanced.     

Mechanical, biochemical, and morphological changes in the heart from chronic food-restricted rats

Food restriction (FR) has been shown to induce important morphological changes in rat myocardium. However, its influence on myocardial performance is not completely defined. We examined the effects of chronic FR on cardiac muscle function and morphology. Sixty-day-old Wistar-Kyoto rats were fed a control (C) or a restricted diet (daily intake reduced to 50% of the amount of food consumed by the control group) for 90 days. Myocardial performance was studied in isolated left ventricular (LV) papillary muscle. Fragments of the LV free wall were analysed by light microscopy, and the ultrastructure of the myocardium was examined in the LV papillary muscle. The myocardial collagen concentration was also evaluated. FR decreased body weight (BW) and LV weight (LVW); the LVW/BW ratio was higher in the restricted group (C, 1.86+/-0.17 mg/g; FR, 2.19+/-0.31 mg/g; p < 0.01). In the FR animals, the cardiac fibers were polymorphic, some of them were of small diameter and others presented lateral infoldings; the ultrastructural alterations were focal and included reduction of sarcoplasmic content, absence and (or) disorganization of myofilaments and Z line, numerous electron dense and polymorphic mitochondria, and deep infoldings of the plasma membrane. The hydroxyproline concentration was higher in the FR animals (p < 0.01). FR prolonged the contraction and relaxation time of the papillary muscle and did not change its ability to contract and shorten. In conclusion, although a 90-day period of FR caused striking myocardial ultrastructural alterations and increased the collagen concentration, it only minimally affected the mechanical function.     

Coronary perfusion and muscle lengthening increase cardiac contraction: different stretch-triggered mechanisms

An increase in coronary perfusion, transversal stretch of the myocardium, increases developed force (F(dev)) (Gregg effect) through activation of stretch-activated ion channels (SACs). Lengthening of the muscle, longitudinal stretch of the myocardium, causes an immediate increase in F(dev) followed by a slow F(dev) increase (Anrep effect). In isometrically contracting perfused papillary muscles of Wistar rats, we investigated whether both effects were based on similar stretch-induced mechanisms by measuring F(dev) and intracellular Ca(2+) concentration ([Ca(2+)](i)) after a muscle length increase from 85% to 95% L(max) (length at which maximal isometric force develops) at low and high coronary perfusion before and after inhibition of SACs with gadolinium (10 micromol/l Gd(3+)). The increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect was of similar magnitude as the Anrep effect (from 3.5 +/- 0.8 to 3.9 +/- 1.2 mN/mm(2) and from 3.0 +/- 0.7% to 3.8 +/- 0.9% normalized [Ca(2+)](i), means +/- SE). SAC blockade completely blunted the increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect; however, it did not affect the Anrep effect. The slow force response, but not the calcium response, was augmented by an increase in coronary perfusion. Therefore, increased coronary perfusion, transversal stretch of the myocardium, and muscle lengthening, longitudinal stretch of the myocardium, increase myocardial contraction in the rat through different stretch-triggered mechanisms.     

Differences between mouse and rat myocardial contractile responsiveness to calcium

Genetically altered mice have become an increasingly important tool for the study of mechanisms of cardiac function, and therefore it is vital to characterize the basic contractile properties of the mouse heart. As a first approach to this goal, we first optimized perfusion conditions and characterized the effect of incremental left ventricular balloon inflation on end-diastolic, systolic and developed pressures in the isovolumically-contracting mouse heart. Under constant loading conditions, we determined developed pressure in response to changing perfusate calcium (1.25, 2.5, 3.75 and 5.0 mM) and perfusate temperature (30 and 37 degrees C). We then compared the intrinsic inotropic responsiveness to changes in extracellular calcium of left ventricular myocardium from mouse to that from the rat. In the baseline state (1.25 mM extracellular calcium; [Ca2+]o), both isometric contraction duration and normalized active force at the peak of the active force-length relationship (Lmax) were less in mouse than in rat myocardium. Under isotonic conditions, temporal parameters of shortening and the relative shortening were less in mouse vs rat myocardium. Increasing [Ca2+]o from 1.25 to 2.5 mM markedly increased active isometric force and rate of force development (+dF/dt) in the mouse. However, rat myocardium responded to a lesser extent. Under isotonic conditions, peak shortening and the rate of shortening also increased to a greater extent in mouse relative to rat myocardium. Increasing the bath calcium concentration to 5.0 mM increased isometric force and +dF/dt further in the rat but not the mouse, suggesting that two species operate at different points on the force vs [Ca2+]o relationship. We conclude that mouse myocardium exhibits increased sensitivity to changes in [Ca2+]o within the physiologic range in comparison to rat. These differences do not appear to be due to differences in loading conditions. The data suggest that differences in inotropic responsiveness to calcium may reflect intrinsic differences in myocardial calcium sensitivity between species.     

Evidence for adenylate cyclase-coupled A1-adenosine receptors on ventricular cardiomyocytes from adult rat and dog heart

Isolated metabolically stable cardiomyocytes from adult rats and mongrel dogs were used to characterize the mechanism underlying the antiadrenergic effect of adenosine. In a system not affected by cellular heterogeneity, isoproterenol (3 x 10(-9) M - 10(-5) M) in the presence of adenosine deaminase (5U/ml) dose dependently increased cellular cAMP (5-80 pmol/mg). The effect of isoproterenol (0.1 microM) was inhibited by various adenosine derivatives, the rank order of potency being in the rat: (-)-N6-(R-phenyl-isopropyl)-adenosine (R-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) greater than S-PIA, and in the dog NECA greater than R-PIA greater than S-PIA. The cAMP increase induced by forskolin (1 microM) was attenuated in the rat by R-PIA. 8-phenyltheophylline (3 microM) antagonized the effect of R-PIA on isoproterenol-stimulated cAMP formation. Basal cAMP content was not influenced by R-PIA or NECA. Omission of adenosine deaminase from the incubation medium attenuated the isoproterenol-induced cAMP increase in the rat by about 30%. Our findings provide evidence for the presence of adenylate cyclase-coupled A1-adenosine receptors on cardiomyocytes which may mediate the antiadrenergic effect of adenosine in the heart.     

Changes in the contractile state, fine structure and metabolism of cardiac muscle cells during the development of rigor mortis

Transmural slices from the left anterior papillary muscle of dog hearts were maintained for 120 min in a moist atmosphere at 37 degrees C. At 15-min intervals tissue samples were taken for estimation of adenosine triphosphate (ATP) and glucose-6-phosphate (G6P) and for electron microscopic examination. At the same time the deformability under standard load of comparable regions of an adjacent slice of tissue was measured. ATP levels fell rapidly during the first 45 to 75 min after excision of the heart. During a subsequent further decline in ATP, the mean deformability of myocardium fell from 30 to 12% indicating the development of rigor mortis. Conversely, G6P levels increased during the first decline in adenosine triphosphate but remained relatively steady thereafter. Whereas many of the myocardial cells fixed after 5 min contracted on contact with glutaraldehyde, all cells examined after 15 to 40 min were relaxed. A progressive increase in the proportion of contracted cells was observed during the rapid increase in myocardial rigidity. During this late contraction the cells showed morphological evidence of irreversible injury. These findings suggest that ischaemic myocytes contract just before actin and myosin become strongly linked to maintain the state of rigor mortis.     

The role of calcium at the sarcolemma in the control of myocardial contractility

The importance of sarcolemmal-bound calcium (Ca) in the control of contraction in mammalian myocardium is indicated by the following results. The curve that relates [Ca]o (from 50 microM to 10 mM) to force development and that which relates [Ca]o to Ca bound to a highly purified sarcolemmal fraction are superimposable. The ability of a series of cations to uncouple excitation from contraction is the same as their relative ability to displace Ca from the sarcolemma. Dimethonium, which specifically displaces cation from the diffuse double layer of the cellular surface, has little effect on contractile force. This indicates that the Ca actually bound to the sarcolemma is the surface Ca important in contractile control. Polymyxin B, a highly charged cationic amphiphilic peptidolipid, specifically competes for Ca-binding sites on anionic and zwitterionic phospholipid. It is a potent displacer of Ca from myocardial cells and purified sarcolemma and a potent uncoupler. Phospholipase D cleaves the nitrogenous base from sarcolemmal phospholipid with production of anionic phosphatidic acid. Phospholipase D treatment increases Ca bound to cells and purified sarcolemma and increases force development of ventricular tissue from both neonatal rat and adult rabbit. Insertion of charged amphiphiles in the sarcolemma as phospholipid analogues modulate interaction of Ca with the sarcolemma, e.g., anionic dodecylsulfate increases Ca bound to sarcolemmal vesicles by more than 80% and increases force development in rabbit papillary muscle by 100%. The effect of pH variation on Ca binding to phospholipid extracted from sarcolemma indicates that phospholipid accounts for at least 75% of the binding. The current model proposes a two-site control of Ca binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmentation of the inotropic response to insulin in diabetic rat hearts.

Insulin participates in the modulation of myocardial function, but its inotropic action in diabetes mellitus is not fully clear. In the present study, we examined contractile responses to insulin in left-ventricular papillary muscles and ventricular myocytes isolated from hearts of normal or short-term (5-7 days) streptozotocin-induced (65 mg/kg) diabetic rats. Mechanical properties of papillary muscles and ventricular myocytes were evaluated using a force transducer and an edge-detector, respectively. Contractile properties of papillary muscles or cardiac myocytes, electrically stimulated at 0.5 Hz, were analyzed in terms of peak tension development (PTD) or peak twitch amplitude (PTA), time-to-peak contraction (TPT) and time-to-90% relaxation (RT90). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity change (deltaFFI). Insulin (1-500 nM) had no effect on PTD in normal myocardium, whereas it produced a positive inotropic response in preparations from diabetic animals, with a maximal increase of 11%. Insulin did not modify TPT or RT90 in either group. Further studies revealed that insulin enhanced cell shortening in diabetic but not normal myocytes, with a maximal increase of 21%. Consistent with its action on the mechanical properties of papillary muscles and cardiac myocytes, insulin also induced a dose-dependent increase in the intracellular Ca2+ transient in diabetic but not normal myocytes. Collectively, these data suggest that the myocardial contractile response to insulin may be altered in diabetes.     

Intensification of human myocardial contractile activity as affected by blood serum

The effect of human serum diluted in Tyrode solution (1:1) on the cardiac contractility has been studied. Fragments of the right auricle myocardium from patients with congenital and acquired heart disease have been used to study serum effect on the contraction force. Myocardial strips were repeatedly stimulated with electrical impulses at a frequency of 0.1 to 1.5 Hz. All the perfusion solutions were maintained at 31-33 degrees C and Ca++ ion concentration was 2 +/- 0.2 mM/l. The serum increased the contraction force in the isolated myocardial fibers from patients with congenital and acquired heart disease. Positive inotropic serum action on the myocardium of patients with acquired heart disease was accompanied by a marked diminution of mechanical oscillations. At the same time human serum does not affect the form of force-frequency curves in both types of the preparations. The results suggest that human serum increases calcium ion homeostasis in the cardiac cells of patients during contraction-relaxation cycle.     

Modulation of stimulation frequency responses and calcium dependency of functional parameters in hyperthyroid rat ventricular papillary muscles

The effects of stimulation frequency (0.2-1.5 Hz) and extracellular calcium concentration ([Ca2+]o) (0.6-15.0 mM) on the contractile function of thin papillary muscles of euthyroid and hyperthyroid rats were studied. Hyperthyroidism led to a decrease in developed tension (DT) and time to peak tension (TPT), but it exhibited no influence on the maximal rates of contraction (+dT/dt) and relaxation (-dT/dt). Also, the mean rates of contraction were similar in euthyroid and hyperthyroid muscle groups. The increase in stimulation frequency brought about a marked decrease in DT, +dT/dt, and -dT/dt of euthyroid papillary muscles at lower frequencies in comparison to papillary muscles in the hyperthyroid group. At stimulation frequencies above 1.0 Hz, the absolute and relative levels of DT and -dT/dt of hyperthyroid myocardium were elevated over euthyroid preparations. At the same time, TPT was unchanged in any of the muscle groups. Hyperthyroidism modulated the relationships between contractile parameters and [Ca2+]o. At a [Ca2+]o of 1.0-4.0 mM, the DT of hyperthyroid papillary muscles was lower than in euthyroid muscle. At 4.0 and 8.0 mM of [Ca2+]o, the equal values of maximal DT were registered for euthyroid and hyperthyroid papillary muscles, respectively. An increase in the [Ca2+]o in the range of 1.0-15.0 mM was accompanied by an increase in TPT of both muscle groups, but to a greater extent in hyperthyroid myocardium. In conclusion, the myocardium of hyperthyroid rat appeared to exhibit decreased sensitivity to calcium as well as to the negative inotropic effect of enhanced stimulation frequency. Alterations of the processes of transsarcolemmal movement and intracellular recycling of Ca2 may be implicated.     

In vitro study of rat uterus after chronic formaldehyde exposure.

To measure cholinergic, adrenergic and tryptaminergic receptor activity of formaldehyde (HCHO) in rat uterus, albino rats were treated with 5 and 10 mg/kg, ip HCHO for 30 days. Acetylcholine (ACh) in doses 1.33, 2 and 3 micrograms/ml produced mild to moderate contraction of isolated rat uterus in control group. HCHO had no effect on isolated rat uterus per se, however it reduced ACh and carbachol induced contraction and presence of adrenaline influences in respect of ACh and carbachol activity. Adrenaline per se had no effect in control preparations, but reduced carbachol induced contraction. Propranolol had no effect on rat uterus; but its presence in the bathing medium increased activity of adrenaline. 5-Hydroxytryptamine (5-HT) had no effect of its own on isolated rat uterus but its presence in the bathing medium enhanced contractions of carbachol and oxytocin.