New Developments in Platelet Cyclic Nucleotide Signalling: Therapeutic Implications

Altered platelet physiology may contribute to the emergence of thrombosis in patients with many forms of cardiovascular disease. Excess platelet activation may reflect increased stimulation of pro-aggregatory pathways. There is, however, increasing evidence that excessive platelet response, due to impaired efficacy of anti-aggregatory autacoids such as nitric oxide (NO) and prostacyclin (PGI₂), may be just as important. For example, diminished platelet response to NO has been documented in acute and chronic myocardial ischaemia, heart failure, aortic valve disease and in the presence of hyperglycaemia. This “NO resistance” has been shown to reflect both the scavenging of NO by reactive oxygen species and dysfunction of its intracellular “receptor”, soluble guanylate cyclase. Importantly, these abnormalities of NO signalling are potentially reversible through judicious application of pharmacotherapy. The analogous condition of impaired PGI₂/adenylate cyclase (AC) signalling has received comparatively less attention to date. We have shown that platelet response to prostaglandin E₁ (PGE₁) is frequently impaired in patients with symptomatic myocardial ischaemia. Because the effects of ADP receptor antagonists such as clopidogrel and ticagrelor at the level of the P2Y₁₂ receptor are coupled with changes in activity of AC, impaired response to PGE₁ might imply both increased thrombotic risk and a reduced efficacy of anti-aggregatory drugs. Accordingly, patient response to treatment with clopidogrel is determined not only by variability of clopidogrel bio-activation, but also extensively by the integrity of platelet AC signalling. We here review these recent developments and their emerging therapeutic implications for thrombotic disorders.     

Competitive Inhibition of Beef Heart Cyclic AMP Phosphodiesterase by Cytokinins and Related Compounds

Two cytokinins and four related analogs, none of which is a cyclic ribonucleotide, have been shown to act as competitive inhibitors of the high K(m) cyclic-AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) activity from beef heart. Weak inhibition of the low K(m) cyclic AMP phosphodiesterase activity was also observed, suggesting a possible mechanism for regulation of intracellular cyclic AMP levels by the exogenously added compounds. In addition to the kinetic data, obtained on the six inhibitors in four different heterocyclic series, 15 other cytokinins and related compounds have been shown to inhibit the high K(m) cyclic AMP phosphodiesterase activity at single concentrations of substrate and inhibitor. Heterocycles such as adenosine and 7-amino-3-methylpyrazolo[4,3-d]pyrimidine, which lack the N-substituent, were inactive as cyclic AMP phosphodiesterase inhibitors. The observed inhibition of cyclic AMP phophodiesterase supports prior observations which implicate exogenously added cytokinins in cyclic AMP metabolism.     

Cilostazol (Pletal®): A Dual Inhibitor of Cyclic Nucleotide Phosphodiesterase Type 3 and Adenosine Uptake

Cilostazol (Pletal®), a quinolinone derivative, has been approved in the U.S. for the treatment of symptoms of intermittent claudication (IC) since 1999 and for related indications since 1988 in Japan and other Asian countries. The vasodilatory and antiplatelet actions of cilostazol are due mainly to the inhibition of phosphodiesterase 3 (PDE3) and subsequent elevation of intracellular cAMP levels. Recent preclinical studies have demonstrated that cilostazol also possesses the ability to inhibit adenosine uptake, a property that may distinguish it from other PDE3 inhibitors, such as milrinone. Elevation of interstitial and circulating adenosine levels by cilostazol has been found to potentiate the cAMP‐elevating effect of PDE3 inhibition in platelets and smooth muscle, thereby augmenting anti‐platelet and vasodilatory effects of the drug. In contrast, elevation of interstitial adenosine by cilostazol in the heart has been shown to reduce increases in cAMP caused by the PDE3‐inhibitory action of cilostazol, thus attenuating the cardiotonic effects. Cilostazol has also been reported to inhibit smooth muscle cell proliferation in vitro and has been demonstrated in a clinical study to favorably alter plasma lipids: to decrease triglyceride and to increase HDL‐cholesterol levels. One, or a combination of several of these effects may contribute to the clinical benefits and safety of this drug in IC and other disease conditions secondary to atherosclerosis. In eight double‐blind randomized placebo‐controlled trials, cilostazol significantly increased maximal walking distance, or absolute claudication distance on a treadmill. In addition, cilostazol improved quality of life indices as assessed by patient questionnaire. One large randomized, double‐blinded, placebo‐controlled, multicenter competitor trial demonstrated the superiority of cilostazol over pentoxifylline, the only other drug approved for IC. Cilostazol has been generally well‐tolerated, with the most common adverse events being headache, diarrhea, abnormal stools and dizziness. Studies involving off‐label use of cilostazol for prevention of coronary thrombosis/restenosis and stroke recurrence have also recently been reported.     

Cyclic Nucleotide Metabolism in Tumours

Summary: Many hormones act by combining with cell surface receptors and stimulating adenylate cyclase activity. The cyclic AMP generated is the mediator of a number of cellular metabolic processes. Other processes may be influenced by changes in cyclic GMP levels. Although much evidence from cultured cells suggested that low cellular levels of cyclic AMP and high levels of cyclic GMP are a feature of rapid cell growth and of malignant transformation, review of the data reveals many inconsistencies. Thus in established tumours growing in vivo, for example, cyclic AMP levels appear to be unrelated to tumour growth rates. It seems that tumour cell cyclic AMP is more likely concerned with the regulation of tumour cell function than of growth.
This would have implications for therapy, in that drugs which influence cyclic nucleotide metabolism could influence tumour cell function. The control of cyclic nucleotide production in normal and tumour cells is discussed, together with the possible ways in which abnormalities of this may occur.     

Cyclic AMP-Mediated Induction of the Cyclic AMP Phosphodiesterase of C-6 Glioma Cells

Long-term regulation of the cyclic nucleotide phosphodiesterase of the C-6 rat glioma cell line has been studied. Both the low K(m) and high K(m) activities can be induced by elevation of intracellular cyclic AMP levels following either dibutyryl cyclic AMP or norepinephrine treatment of the cells. The enzymes are maximally induced by 3-4 hr. The presence of either cycloheximide or actinomycin D prevents induction by either dibutyryl cyclic AMP or norepinephrine. Evidence is presented that the norepinephrine effect is mediated by the beta-catecholamine receptor. The increased phosphodiesterase activity causes a partial refractoriness to a second challenge with norepinephrine, which can be overcome by blockade of the induction with cycloheximide. The results suggest that just as short-term regulation of cyclic AMP levels occurs via changes in the rates of synthesis or degradation, long-term alterations of the system may also involve either the adenylate cyclase or the phosphodiesterase.     

Role of Phosphodiesterase 5 and Cyclic GMP in Hypertension

Cyclic GMP (cGMP) is a ubiquitous intracellular second messenger that mediates a wide spectrum of physiologic processes in multiple cell types within the cardiovascular and nervous systems. Synthesis of cGMP occurs either by NO-sensitive guanylyl cyclases in response to nitric oxide or by membrane-bound guanylyl cyclases in response to natriuretic peptides and has been shown to regulate blood pressure homeostasis by influencing vascular tone, sympathetic nervous system, and sodium and water handling in the kidney. Several cGMPs degrading phosphodiesterases (PDEs), including PDE1 and PDE5, play an important role in the regulation of cGMP signaling. Recent findings revealed that increased activity of cGMP-hydrolyzing PDEs contribute to the development of hypertension. In this review, we will summarize recent research findings regarding the cGMP/PDE signaling in the vasculature, the central nervous system, and the kidney which are associated with the development and maintenance of hypertension.     

Cyclic Nucleotide Metabolism in Pineal Homogenates

Adenylate cyclase (AC) in pineal particulate fractions from rabbit, rat, cow, and the vole Microtus montanus was stimulated by L-norepinephrine (NE) and L-isoproterenol (ISO). NE stimulation of rabbit and bovine pineal AC was biphasic, with a plateau between 0.01 microM and 1.0 microM and additional stimulation by NE above 1.0 microM. Stimulation by different ISO concentrations gave a typical hyperbolic curve, and optimal stimulation by ISO exceeded that by NE. Melatonin decreased ISO and NE stimulation of AC 10-20%. Although, alpha-adrenergic agonists increase beta-agonist-mediated adenosine-3',5'-cyclic monophosphate (cyclic AMP) accumulation in intact pinealocytes, similar amplification of AC stimulation was not seen with broken-cell preparations. Most (60-70%) pineal guanylate cyclase (GC) was recovered in supernatant fractions after centrifugation of homogenates at 110,000 x g; this soluble GC was unaffected by potential agonists. Low concentrations (0.01-1 nM) of NE, ISO, and phenylephrine (PE) stimulated GC in impure and purified membrane fractions, but each inhibited at concentrations above 10 microM. All concentrations of ISO and NE inhibited GC in the presence of the alpha-agonist PE. Melatonin alone did not affect particulate GC, but L-ISO stimulation was not seen in the presence of equivalent concentrations of melatonin. The in vitro data are consistent with both alpha- and beta-receptor regulation of cyclic nucleotide metabolism in pinealocytes. Endogenous NE may differentially regulate cyclic AMP and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in pineal; low NE concentrations that stimulate GC have only a slight effect on AC, but higher NE concentrations that inhibit GC maximally stimulate AC. Particulate GC and AC also were resolved by equilibrium centrifugation, to give several discrete peaks of enzyme activity. The results support the existence of several forms of AC and GC, which have different responses to adrenergic agonists.     

Effect of Sildenafil on Reperfusion Function,Infarct Size,and Cyclic Nucleotide Levels in the Isolated Rat Heart Model

We have previously shown that NO-donor induced elevation in myocardial cGMP levels is associated with improved reperfusion function of the isolated rat heart. The phosphodiesterase 5 (PDE 5) inhibitor, sildenafil could potentially increase myocardial cGMP levels and thus protect the heart against ischaemic/reperfusion injury.Methods: To test our hypothesis we treated the isolated working rat heart with vehicle, OR sildenafil (10, 20, 50, 100, 200 nM), OR sildenafil (50 nM) plus a sarcolemmal (HMR 1098) or a mitochondrial (5-Hydroxydecanoate (5-HD)) K_ATP channel blocker. Hearts were then subjected to 20 min global, or 35 min regional ischaemia at 37C before reperfusion function (aortic output, coronary flow and aortic pressure) and infarct size were documented. Pre-ischaemic, ischaemic and reperfusion myocardial cAMP and cGMP concentrations were determined.Results: Low concentrations of sildenafil (10, 20 and 50 nM) improved reperfusion aortic output (AO) recovery (61.4± 4.5%, 64.8 ± 5.2% and 62.3 ± 5.0% vs. 45.4 ± 3.8% for controls (p < 0.05)) and infarct size, while high concentrations (200 nM) worsened AO recovery (24.9 ± 4.9.0%, p < 0.05). Myocardial cGMP levels of ischaemic tissue were elevated (34.7 ± 2.4 vs. 27.3 ± 2.2 pmol/g ww) and cAMP levels were suppressed (0.59 ± 0.03 vs. 0.87 ± 0.06 nmol/g ww) in the sildenafil (50 nM) treated hearts. Co-perfusion with sildenafil plus HMR 1098 decreased AO recovery (21.7 ± 7.6% vs. 62.3 ± 5.0% for sildenafil alone, p < 0.05) and increased infarct size (29.7 ± 2.04% vs. 8.6 ± 2.39% for sildenafil alone, p < 0.05).Similarly, sildenafil plus 5-HD decreased reperfusion AO recovery (44.4 ± 6.0% vs. 62.3 ± 5.0% for sildenafil alone, p < 0.05) and increased infarct size (33.8 ± 1.62% vs. 8.6 ± 2.39% for sildenafil alone, p < 0.05).Conclusions: (1) Pretreatment with low concentrations of sildenafil (20–50 nM) improves, while higher concentrations (200 nM) worsen reperfusion function in this model. (2) Low concentrations of sildenafil (20–50 nM) decrease infarct size while the higher concentrations had no effect. (3) These protective properties of low concentrations of sildenafil may be related to its cGMP elevating and cAMP suppressing effects in the ischaemic heart. (4) Possible end-effectors for sildenafil in the ischaemic heart include the mitochondrial and sarcolemmal K_ATP channel.     

Intravenous Iron in Heart Failure: Beyond Targeting Anemia

Iron deficiency is commonly seen in congestive heart failure (CHF) in both anemic and nonanemic patients. In six studies in which these iron-deficient patients with CHF were treated with intravenous (IV) iron, five found an improvement in the hemoglobin. In uncontrolled and controlled studies, the New York Heart Association (NYHA) class, quality of life, and exercise capacity were improved consistently with IV iron. In some studies, cardiac function also was improved. In one large, double-blind, placebo-controlled study of IV iron, the patient global assessment, quality of life, and NYHA class improved rapidly in both those who were anemic or not anemic. In contrast to these studies, another controlled study of anemia in CHF showed no effect of oral iron on hemoglobin or on any cardiac parameters over 1 year. These studies suggest that CHF in both anemic and nonanemic iron-deficient patients may benefit from a course of IV iron, but not oral iron.     

Apoptotic Signaling in Multiple Myeloma: Therapeutic Implications

Fifteen thousand new cases of multiple myeloma (MM) will occur in the United States in 2003, and the disease remains incurable. Diverse classes of chemotherapeutic agents induce cell death or apoptosis in MM cells; however, prolonged drug exposures ultimately induce chemoresistance. The mechanisms whereby MM cells resist drugs include alterations in intracellular signaling as well as adherence and cytokines in the bone marrow (BM) microenvironment. Novel agents that target the MM cell in its BM microenvironment are needed to both enhance anti-MM activity and prevent development of drug resistance. Delineation of cellular growth and apoptotic signaling pathways in MM cells may identify molecules that serve as novel therapeutic targets on the basis of interruption of MM cell growth or triggering of MM cell death.     

Alterations in Brain Cannabinoid Receptor Levels Are Associated with HIV-Associated Neurocognitive Disorders in the ART Era: Implications for Therapeutic Strategies Targeting the Endocannabinoid System

HIV-associated neurocognitive disorders (HAND) persist despite the advent of antiretroviral therapy (ART), suggesting underlying systemic and central nervous system (CNS) inflammatory mechanisms. The endogenous cannabinoid receptors 1 and 2 (CB₁ and CB₂) modulate inflammatory gene expression and play an important role in maintaining neuronal homeostasis. Cannabis use is disproportionately high among people with HIV (PWH) and may provide a neuroprotective effect for those on ART due to its anti-inflammatory properties. However, expression profiles of CB₁ and CB₂ in the brains of PWH on ART with HAND have not been reported. In this study, biochemical and immunohistochemical analyses were performed to determine CB₁ and CB₂ expression in the brain specimens of HAND donors. Immunoblot revealed that CB₁ and CB₂ were differentially expressed in the frontal cortices of HAND brains compared to neurocognitively unimpaired (NUI) brains of PWH. CB₁ expression levels negatively correlated with memory and information processing speed. CB₁ was primarily localized to neuronal soma in HAND brains versus a more punctate distribution of neuronal processes in NUI brains. CB₁ expression was increased in cells with glial morphology and showed increased colocalization with an astroglial marker. These results suggest that targeting the endocannabinoid system may be a potential therapeutic strategy for HAND.     

Targeting Atrio-Atrial Conduction in the Post-Orthotopic Heart Transplant Patient

The ICE CHIP study is a sequential Phase I and Phase II pilot study comparing the cardiac imaging capabilities of intracardiac echocardiography (ICE) with with transesophageal echocardiography (TEE) followed by a randomized comparison of ICE guided cardioversion with a conventional cardioversion strategy in patients with atrial fibrillation. It is a prospective open label randomized multi-center investigation performed in two phases designed to initially compare two distinct imaging modalities (Phase 1) and subsequently two different strategies (ICE guided Cardioversion and Conventional) in the management of AF in patients undergoing invasive cardiac procedures in whom electrical cardioversion is indicated (Phase 2). This study will examines two hypotheses in AF patients undergoing invasive cardiac procedures: (1) ICE has comparable efficacy to TEE in visualization of left atrial pathology including thrombi or interatrial septal defects. This will be evaluated during the Phase I component of the study. (2) ICE can identify low risk patients in whom immediate cardioversion during the procedure is safe and comparably effective to electrical cardioversion performed based on a conventional strategy of a minimum of 3 weeks of preceding anticoagulation therapy. Phase 1 will enroll 100 patients at 12 centers, who will undergo a clinically indicated TEE procedure and cardiac catheterization procedure. Each patient will be imaged by TEE &; ICE and a core echo laboratory will perform a blinded comparison of the two imaging modalities. In Phase 2, a total of 300 patients (3:2 randomization) will be enrolled in the study at up to 15 investigational sites in USA and Europe. The composite incidence rate of major cardiac and bleeding complications (stroke, TIA, peripheral embolism, major hemorrhagic event) will be compared between the two treatment groups over the duration of the study.