The Calcitonin Gene-Related Peptide Activates Both cAMP and NO Pathways to Induce Relaxation of Circular Smooth Muscle Cells of Guinea-Pig Ileum

Rekik, M., M. Delvaux, J. Frexinos, and L. Bueno. Calcitonin gene-related peptide activates both cAMP and NO pathways to induce relaxation of circular smooth muscle cells of guinea-pig ileum. Peptides 18(10) 1517–1522, 1997.—The direct effects and the intracellular pathways of rCGRP were investigated on smooth muscle cells (SMC) isolated by enzymatic digestion from the circular and longitudinal layers of guinea-pig ileum. In circular SMC, rCGRP inhibited CCK8-induced contraction in a concentration-dependent manner (Cmax = 100 μM and EC₅₀ = 0.7 ± 0.4 nM). Preincubation of SMC with 1 μM Rp-cAMPs, a cAMP antagonist, abolished the relaxing effect of rCGRP; moreover, preincubation of SMC with 100 μM L-NAME, an inhibitor of NOS, inhibited the relaxing effect of rCGRP. hCGRP(8-37), a selective antagonist of rCGRP receptors, inhibited the rCGRP-induced relaxation in a concentration dependent manner whereas the vasoactive intestinal polypeptide (VIP) antagonist had no significant effect. In longitudinal SMC, rCGRP-induced relaxation was abolished by Rp-cAMPs, whereas L-NAME had no effect. In conclusion, rCGRP triggers different intracellular pathways to induce relaxation of circular or longitudinal intestinal SMC; cAMP is involved in cells from both layers while nitric oxide (NO) is involved only in relaxation of circular SMC.     

Cross Talk between β-Adrenergic and Bradykinin B2 Receptors Results in Cooperative Regulation of Cyclic AMP Accumulation and Mitogen-Activated Protein Kinase Activity

Costimulation of G protein-coupled receptors (GPCRs) may result in cross talk interactions between their downstream signaling pathways. Stimulation of GPCRs may also lead to cross talk regulation of receptor tyrosine kinase signaling and thereby to activation of mitogen-activated protein kinase (MAPK). In COS-7 cells, we investigated the interactions between two particular mitogenic receptor pathways, the endogenously expressed beta-adrenergic receptor (beta-AR) and the transiently transfected human bradykinin (BK) B(2) receptor (B(2)R). When beta-AR and B(2)R are costimulated, we found two different cross talk mechanisms. First, the predominantly G(q) protein-coupled B(2)R is enabled to activate a G(i) protein and, subsequently, type II adenylate cyclase. This results in augmentation of beta-AR-mediated cyclic AMP (cAMP) accumulation by BK, which alone is unable to increase the cAMP level. Second, independently of BK-induced superactivation of the cAMP system, costimulation of beta-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B(2)R to MAPK, which essentially involves protein kinase C activation, is selectively switched off. The MAPK activation in response to isoproterenol was not affected due to costimulation. Furthermore, in the presence of isoproterenol, BK lost its ability to stimulate DNA synthesis in COS-7 cells. Thus, our findings might establish a novel paradigm: cooperation between simultaneously activated mitogenic pathways may prevent multiple stimulation of MAPK activity and increased cell growth.     

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: Study of strain dependency and the role of protein kinase A and C signaling pathways

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0–7% center; 7–24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7–24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0–7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 μM Rp-cAMP) or PKC (with 5–20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses. J. Cell. Physiol. 170:228–234, 1997. © 1997 Wiley-Liss, Inc.     

beta-Adrenoceptor signaling in regenerating skeletal muscle after beta-agonist administration

Stimulating the beta-adrenoceptor (beta-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of beta-AR agonists causes beta-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine beta-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, beta-agonist administration does not cause beta-AR desensitization. Rats received either the beta-AR agonist fenoterol (1.4 mgxkg(-1)xday(-1) ip) or saline for 7 days postinjury. Fenoterol reduced beta-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in beta-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. beta-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and Mn(2+)). Although beta-agonist treatment reduces beta-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of beta-AR signaling in regenerating muscles is prevented by altered rates of beta-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating beta-AR signaling to enhance muscle repair after injury.     

Regional differences in signalling transduction pathways among smooth muscle cells from rabbit colon

Smooth muscle cells (SMC) from the circular muscle layer of rabbit colon, taken from the proximal and distal regions that are known to have different physiological and motor activities, were used to highlight distinct regional intrinsic myogenic properties and to investigate the correlations between receptor and signalling transduction pathways. Contractile agonists were shown to be more potent on proximal than on distal SMC in inducing contraction and intracellular Ca(2+) increase. Concentration-response curves of agonists-induced Ca(2+) increase were constantly shifted to the right, though remaining parallel, with respect to contraction curves, independently of the region analysed. Using agents activating different steps of cAMP-or cGMP-mediated intracellular cascades, main regional differences were revealed as far as relaxation was concerned. Relaxation of proximal SMC was found to be essentially cGMP mediated, while that of distal SMC was cAMP mediated. In conclusion, the motor patterns of the two regions appear to be influenced by distinct regional biochemical characteristics that are intrinsic to colonic SMC.     

The mechanisms in NO-dependent regulation of electrical and mechanical activity in smooth muscles

The role of nitric oxide (NO) and its implication in intracellular and intercellular signaling pathways attract an attention of many research teams up to now. Away of its signaling functions. NO is considered as one of the key molecules in maintenance of balance between the physiological and pathological processes due to cytoprotective and cytotoxic functions of this molecule. In this regard, elucidation of the NO-dependent mechanisms, involved into the physiological processes and pathophysiological reactions, remains an urgent problem of conntemporary biology and medicine. Analysis of obtained results establishes a relative contribution of electro- and pharmaco-mechanical coupling mechanisms in NO-dependent regulation of smooth muscle cels (SMC) functions. The authors show that elevation of intracellular Ca2+ concentration by biologically active substances promotes relaxing effect of NO through both voltage-dependent and -independent intracellular mechanisms of calcium redistribution. Namely the peculiarities of considered mechanisms in each certain type of SMCs cause the final direction of alterations in contractility and membrane potential. It has been shown that voltage-dependent effects of NO are mediated by suppression of calcium and/or sodium components and modulation of Ca2+ -dependent and ATP-seisitive potassium components of SMC membrane permeability, Voltage-independent NO control of mechanical smooth muscles activity mainly is mediated by 1) modulation of protein kinase C (PK-C) branch of calcium signaling system, 2) ratio of cyclic nucleotides intracellular concentrations (cGMP/cAMP), and 3) directional mode of electrosilent Na+, K+, 2Cl- -cotransport. Our results show that the features of the myogenic effects of NO are caused by the peculiarities of PK-C operation in SMC.     

Multiple, PKA-dependent and PKA-independent, signals are involved in cAMP-induced PRL expression in the eosinophilic cell line Eol-1

Besides its pivotal role in reproduction, the polypeptide hormone prolactin (PRL) has been attributed an immunomodulatory function. Extrapituitary PRL expression is regulated differently from that in the pituitary, due to the use of an alternative promoter. In leukocytes, cAMP is an important regulator of PRL expression. We report that in the human eosinophilic cell line Eol-1, cAMP-induced PRL expression is partially abrogated by two protein kinase A (PKA) inhibitors (H89, PKI) and by the p38 inhibitor SB203580. Phosphorylation of p38 was PKA-independent and could be stimulated by a methylated cAMP analogue, which specifically activates the exchange factor directly activated by cAMP (EPAC). Furthermore, cAMP induced a PKA-dependent phosphorylation of cAMP-responsive element binding protein (CREB). We postulate that cAMP induces PRL expression via two different signalling pathways: a PKA-dependent pathway leading to the phosphorylation of CREB, and a PKA-independent pathway leading to the phosphorylation of p38.     

Signalling mechanisms regulating lipolysis

Adipose tissue plays an important role providing energy to other tissues and functioning as an energy reserve organ. The energy supply is produced by triglycerides stored in a large vacuole representing approximately 95% of adipocyte volume. In the fasting period, triglyceride hydrolysis produces glycerol and free fatty acids which are important oxidative fuels for other tissues such as liver, skeletal muscle, kidney and myocardium. Hormone-sensitive lipase (HSL) is the enzyme that hydrolyzes intracellular triacylglycerol and diacylglycerol, and is one of the key molecules controlling lipolysis. Hormones and physiological factors such as dieting, physical exercise and ageing regulate intensively the release of glycerol and free fatty acids from adipocytes. One of the best known mechanisms that activate lipolysis in the adipocyte is the cAMP dependent pathway. cAMP production is modulated by hormone receptors coupled to Gs/Gi family of GTP binding proteins, such as beta-adrenergic receptors, whereas cAMP degradation is controlled by modulation of phosphodiesterase activity, increased by insulin receptor signalling. cAMP activates PKA which activates HSL by promoting its phosphorylation. Hormonal control of lipolysis can also be achieved by receptors coupled G proteins of the Gq family, through molecular mechanisms that involve PKC and MAPK, which are currently under investigation. cGMP and PKG have also been found to activate lipolysis in adipocytes. In this review we have compiled data from literature reporting both the classical and the alternative mechanisms of lipolysis.     

Protein phosphorylation and control of tick salivary gland function

Tick salivary glands are controlled by nerves, dopamine being a neurotransmitter at the neuroeffector junction. Dopamine and cyclic AMP (cAMP) stimulate fluid secretion by isolated salivary glands. Dopamine activates an adenylate cyclase to increase intracellular cAMP within the female salivary glands. Phosphoproteins whose levels of phosphate are affected by cAMP-dependent protein kinase have been identified in subcellular fractions. Protein(s) phosphorylated by cAMP appears to activate protein phosphatase in the salivary glands.Another phosphorylation pathway appears to act through protein kinase C because of an ability of phorbol esters (known activators of protein kinase C) to stimulate the phosphorylation of proteins, and an ability of a peptide factor in tick brain to metabolize salivary-gland phosphoinositides, an event that often precedes activation of protein kinase C. Because cAMP modulates brain-factor-stimulated formation of inositol phosphates (products of phosphoinositide breakdown) an interrelationship between the two pathways seems likely.Evidence of regulatory processes, including protein phosphorylation'dephosphorylation reactions, will provide a basis for helping asses the physiological significance of secretory products and the role of the salivary glands in disease transmission.     

Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.     

Acidic heparin binding growth factor transiently activates adenylate cyclase activity in human adult arterial smooth muscle cells

Acidic heparin binding growth factor (HBGF-1) is a potent mitogen for human adult arterial smooth muscle cells (SMC) in culture. Exposure of quiescent SMC to HBGF-1 resulted in a rapid and transient stimulation of the adenylate cyclase activity. Maximal stimulation (2-fold) was observed at 60 sec after growth factor exposure and there was a concurrent elevation of intracellular cAMP levels. GTP analogue was required indicating that the activation of this enzyme is mediated through G-proteins. The results suggest that part of the signal transduction pathway of HBGF-1 might be mediated by cAMP dependent protein kinase.     

A novel signaling pathway for beta-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes

Stimulation of cardiac beta-adrenergic receptors (beta-AR) activates both the G(s)- and G(i)-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking beta-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute beta-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a beta(2)-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. beta-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gbetagamma sequestrant peptide derived from the COOH-terminus of beta-AR kinase-1 failed to activate PI3K after beta-AR stimulation, suggesting Gbetagamma is required for the transactivation. Furthermore, acute beta-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kalpha isoform activities but not the activities of other isoforms (PI3Kbeta, -delta, -gamma) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of beta-AR-mediated transactivation of cardiac PI3Kalpha via sequential involvement of Galpha(i)/Gbetagamma, Src, and PDGFR.     

Modulation of monocarboxylic acid transporter-1 kinetic function by the cAMP signaling pathway in rat brain endothelial cells

MCT1 (monocarboxylic acid transporter 1) facilitates bidirectional monocarboxylic acid transport across membranes. MCT1 function and regulation have not been characterized previously in cerebral endothelial cells but may be important during normal cerebral energy metabolism and during brain diseases such as stroke. Here, by using the cytoplasmic pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester, the initial rates of monocarboxylate-dependent cytoplasmic acidification were measured as an indication of MCT1 kinetic function in vitro using the rat brain endothelial cell (RBE4) model of blood-brain transport. The initial rate of L-lactate-dependent acidification was significantly inhibited by 5-10-min incubations with agonists of intracellular cAMP-dependent cell signaling pathways as follows: dibutyryl cAMP, forskolin, and isoproterenol. Isoproterenol reduced V(max) but did not affect K(m) values. The effects of forskolin were completely reversed by the protein kinase A inhibitor H89, whereas H89 alone increased transport rates. Cytoplasmic cAMP levels, measured by radioimmunoassay, were increased by forskolin or isoproterenol, and the effect of isoproterenol was inhibited by propranolol. MCT1-independent intracellular pH control mechanisms did not contribute to the forskolin or H89 effects on MCT1 kinetic function as determined with amiloride, monocarboxylate-independent acid loading, or the transport inhibitor alpha-cyano-4-hydroxycinnamate. The data demonstrate the direct modulation of MCT1 kinetic function in cerebral endothelial cells by agents known to affect the beta-adrenergic receptor/adenylyl cyclase/cAMP/protein kinase A intracellular signaling pathway.     

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells

The effect of neuropeptide Y (NPY), a co-transmitter with noradrenaline in peripheral sympathetic nerve fibers, on the osteoclastogenesis in mouse bone marrow cell cultures treated with isoprenaline, a beta-adrenergic receptor (beta-AR) agonist, was examined. The mouse bone marrow cells constitutively expressed mRNAs for the NPY-Y1 receptor and beta2-AR. NPY inhibited the formation of osteoclast-like cells induced by isoprenaline but not that by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) or soluble receptor activator of nuclear factor-kappaB ligand (RANKL); and it suppressed the production of RANKL and cyclic AMP (cAMP) increased by isoprenaline but not those increased by 1alpha,25(OH)2D3. NPY also inhibited osteoclastogenesis induced by forskolin, an activator of adenylate cyclase; however, it did not inhibit that induced by exogenously supplied dibutyryl cAMP, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase. These results demonstrate that NPY inhibited the isoprenaline-induced osteoclastogenesis by blocking the agonist-elicited increases in the production of cAMP and RANKL in mouse bone marrow cells, suggesting an interaction between NPY and beta-AR agonist in bone resorption.     

Diabetes-related changes in cAMP response element-binding protein content enhance smooth muscle cell proliferation and migration

We hypothesized that diabetes and glucose-induced reactive oxygen species lead to depletion of cAMP response element-binding protein (CREB) content in the vasculature. In primary cultures of smooth muscle cells (SMC) high medium glucose decreased CREB function but increased SMC chemokinesis and entry into the cell cycle. These effects were blocked by pretreatment with the antioxidants. High glucose increased intracellular reactive oxygen species detected by CM-H(2)DCFA. SMC exposed to oxidative stress (H(2)O(2)) demonstrated a 3.5-fold increase in chemokinesis (p < 0.05) and accelerated entry into cell cycle, accompanied by a significant decrease in CREB content. Chronic oxidative challenge similar to the microenvironment in diabetes (glucose oxidase treatment) decreases CREB content (40-50%). Adenoviral-mediated expression of constitutively active CREB abolished the increase in chemokinesis and cell cycle progression induced by either high glucose or oxidative stress. Analysis of vessels from insulin resistant or diabetic animals indicates that CREB content is decreased in the vascular stroma. Treatment of insulin-resistant animals with the insulin sensitizer rosiglitazone restores vessel wall CREB content toward that observed in normal animals. In summary, high glucose and oxidative stress decrease SMC CREB content increase chemokinesis and entry into the cell cycle, which is blocked by antioxidants or restoration of CREB content. Thus, decreased vascular CREB content could be one of the molecular mechanisms leading to increased atherosclerosis in diabetes.     

Parathyroid hormone-related protein-mediated responses in pulmonary arteries and veins of newborn lambs

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 10(-4) M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 10(-4) M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.     

Is Ca2+ the second messenger in the response to melatonin in cuckoo wrasse melanophores?

Pigment aggregation in melanophores of Labrus ossifagus is controlled by an alpha2-adrenoceptor and is somehow modulated by melatonin. The signal transduction mechanisms seem to involve both an attenuation of cAMP and an increase in intracellular Ca2+, inhibiting protein kinase A or activating a phosphatase, respectively. These effects result in dephosphorylation, which in turn induces aggregation. Various alpha2-adrenoceptor agonists attenuate cAMP levels or increase the concentration of intracellular Ca2+. Noradrenaline, for example, lowers cAMP but does not affect the calcium signal whereas B-HT 920, an alpha2-adrenoceptor specific agonist, does not induce a cAMP decrease but does appear to induce an increase in intracellular Ca2+. This later inference is drawn from experiments with BAPTA/AM, an intracellular calcium chelator, which counteracts the aggregation induced by B-HT 920. Interestingly, the very potent alpha2-adrenoceptor agonist medetomidine apparently activates both signal transduction pathways, which could explain its high efficacy in producing aggregation. Melatonin itself does not cause pigment aggregation, but it potentiates noradrenaline-induced aggregation. It has been suggested that melatonin receptors and alpha2-adrenoceptors follow the same signal transduction pathway, i.e. an attenuation of cAMP. In our experiments, melatonin did not reduce cAMP levels; instead it appears to increase Ca2+ concentration, since melatonin-potentiated aggregation was inhibited by BAPTA/AM. Thus, aggregation amplified by melatonin is probably not mediated by a further decrease in cAMP, but by the same signal transduction mechanism as B-HT 920, i.e. an increase in Ca2+. This further strengthens the suggestion that melatonin and B-HT 920 bind to the same site, but it is unclear if that particular site is on the melatonin receptor or the alpha2-adrenoceptor.     

Functional desensitization to isoproterenol without reducing cAMP production in canine failing cardiocytes

To corroborate alterations in the functional responses to beta-adrenergic receptor (beta-AR) stimulation with changes in beta-AR signaling in failing cardiomyocytes, contractile and L-type Ca(2+) current responses to isoproterenol along with stimulated cAMP generation were compared among cardiomyocytes isolated from canines with tachycardia-induced heart failure or healthy hearts. The magnitude of shortening of failing cardiomyocytes was significantly depressed (by 22 +/- 4.4%) under basal conditions, and the maximal response to isoproterenol was significantly reduced (by 45 +/- 18%). Similar results were obtained when the responses in the rate of contraction and rate of relaxation to isoproterenol were considered. The L-type Ca(2+) current amplitude measured in failing cardiomyocytes under basal conditions was unchanged, but the responses to isoproterenol were significantly reduced compared with healthy cells. Isoproterenol-stimulated cAMP generation was similar in sarcolemmal membranes derived from the homogenates of failing (45 +/- 6.8) and healthy cardiomyocytes (52 +/- 8.5 pmol cAMP. mg protein(-1). min(-1)). However, stimulated cAMP generation was found to be significantly reduced when the membranes were derived from the homogenates of whole tissue (failing: 67 +/- 8.1 vs. healthy: 140 +/- 27.8 pmol cAMP. mg protein(-1). min(-1)). Total beta-AR density was not reduced in membranes derived from either whole tissue or isolated cardiomyocyte homogenates, but the beta(1)/beta(2) ratio was significantly reduced in the former (failing: 45/55 vs. healthy: 72/28) without being altered in the latter (failing: 72/28, healthy: 77/23). We thus conclude that, in tachycardia-induced heart failure, reduction in the functional responses of isolated cardiomyocytes to beta-AR stimulation may be attributed to alterations in the excitation-contraction machinery rather than to limitation of cAMP generation.